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THE GENETICS

of the Horse

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THE GENETICS

of the Horse

Edited by

A.T. BOWLING Veterinary Genetics Laboratory School of Veterinary Medicine University of California Davis, California USA and

A. RUVINSKY Animal Sciences, SRSNR University of New England Armidale, NSW Australia

CABI Publishing

250

A3759:AMA:First Revise: 20 January 1999

CHAP-

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CABI Publishing is a division of CAB International CABI Publishing CAB International Wallingford Oxon OX10 8DE UK Tel: +44 (0)1491 832111 Fax: +44 (0)1491 833508 Email: [email protected] Web site: http://www.cabi.org

CABI Publishing 10 E 40th Street Suite 3203 New York, NY 10016 USA Tel: +1 212 481 7018 Fax: +1 212 686 7993 Email: [email protected]

© CAB International 2000. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. Library of Congress Cataloging-in-Publication Data The genetics of the horse / edited by A.T. Bowling and A. Ruvinsky. p. cm. Includes bibliographical references and index. ISBN 0-85199-429-6 (alk.) 1. Horses--Genetics. 2. Horses--Breeding. I. Bowling, Ann T. II. Ruvinsky, Anatoly. SF201.G36 2000 636.1′0821--dc21

98-057248 CIP

ISBN 0 85199 429 6 Typeset in Garamond by AMA DataSet Ltd Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn

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Contents

Contents Preface Ann Bowling and Anatoly Ruvinsky 1.

Systematics and Phylogeny of the Horse C.P. Groves and O.A. Ryder 2. Genetic Aspects of Domestication, Breeds and Their Origins A.T. Bowling and A. Ruvinsky 3. Genetics of Colour Variation A.T. Bowling 4. Genetics of Morphological Traits and Inherited Disorders F.W. Nicholas 5. Blood Groups and Biochemical Polymorphisms K. Sandberg and E.G. Cothran 6. Molecular Genetics of the Horse M. Binns, J.E. Swinburne and M. Breen 7. Immunogenetics of the Horse E. Bailey, E. Marti, D.G. Fraser, D.F. Antczak and S. Lazary 8. Genetic Aspects of Disease in Horses E. Collinder and M. Rasmuson 9. Cytogenetics and Physical Gene Maps B.P. Chowdhary and T. Raudsepp 10. Linkage and Comparative Maps for the Horse (Equus caballus) J.D. Murray and A.T. Bowling 11. Genetics of Behaviour K.A. Houpt and R. Kusunose 12. Reproduction and Modern Breeding Technologies in the Mare W.R. Allen and D.F. Antczak 13. Developmental Genetics A. Ruvinsky and F. Stewart 14. Genetic Resources and Their Conservation D.P. Sponenberg

vii 1 25 53 71 85 109 123 157 171 243 281 307 343 387 v

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vi

Contents

15. 16. 17. 18.

Genetics of Performance Traits A. Ricard, E. Bruns and E.P. Cunningham Genetics of Conformation, Locomotion and Physiological Traits M.T. Saastamoinen and E. Barrey Genetic Improvement of the Horse T. Arnason and L.D. Van Vleck Standardized Genetic Nomenclature for the Horse C.H.S. Dolling

411 439 473 499

Glossary

507

Index

517

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Preface Preface

The horse was domesticated about 6000 years ago somewhere in the steppes area of a region now known as modern southern Russia and the Ukraine. Since then, horses have played essential roles in the development of many civilizations. The destiny of the horse was quite different from that of other domestic animals. No other animal played such a special role in the acceleration of social processes and political development as the horse. The rise and fall of empires, conquest of whole continents, great battles, development of transport systems, mail, agricultural progress and sport were carried out on the horse’s back. The 20th century put an end to many ways in which horses traditionally had been used throughout history. However, recent trends have shown that humans need the horse even in the epoch of modern cars, tractors and planes. In developed countries, the population of horses has started to increase again after a dramatic decrease 50–70 years ago. The current number of horses worldwide, according to FAOSTAT Database 1998, is about 62 million, excluding 14.6 million mules and 43 million asses. Tremendous progress in mammalian genetics during the last decade has not by-passed horse genetics. This knowledge is important from many points of view, including breeding, selection and health and also for better understanding the history of horse domestication. This history is still full of gaps and questions. Hopefully, this book will have a positive impact on further investigations in the field of horse genetics and domestication. Scientific, practical and cultural value of such information should not be underestimated. Genomic revolution and biotechnology, which changed animal genetics, promise further progress. Just a few years ago the locations of only a few genes on the horse chromosomes were known. At the time of publication of this book, this number has reached about 500 loci. Previously separated, quantitative and molecular genetics are now taking a united approach toward identification of loci underlying important traits in domestic and laboratory animals. Investigation of quantitative trait loci (QTL) and implementation of marker-assisted selection (MAS) are expected to provide new tools for animal breeding and will probably be utilized in horse selection in the near future. vii

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Preface

Cloning and other new reproductive technologies may also benefit the horse industry significantly in the years to come. This could be particularly important because one of the horse’s most distinctive traits – speed – has hardly improved under traditional selection methods since the 19th century. It is quite possible that this trait has reached physiological limits. Alternatively, the problem could be that closed breeding structures produced by stud book controls have effectively limited the material available for selection. Combining the technologies of molecular genetics and assisted reproduction will provide the means to look at some of the questions of breed improvement and will be among the most significant innovations available for the horse breeding industry in the 21st century. The main purpose of this book is to collect available data concerning horse genetics and bring together previously separate areas of research. The book covers all major directions in horse genetics. The first four chapters discuss systematics and phylogeny of the horse, domestication, breeds and single-gene traits. Chapters 5–10 present information about biochemical polymorphisms, immunogenetics and disease resistance, genome structure and gene mapping. The third section covers genetic aspects of reproduction, development and behaviour. Finally, chapters 14–17 are devoted to genetics applied to horse improvement. Standard genetic nomenclature is presented in Chapter 18. The authors of this book have made every attempt to highlight the most important publications in the area of horse genetics for the last few decades with emphasis on the most recent papers, reviews and books. However we realize that omissions and errors are unavoidable and apologize for possible mistakes. This book is addressed to a broad audience, which includes researchers, lecturers, students, farmers and specialists working in the industry. The Genetics of the Horse is the fourth publication in the series of monographs on mammalian genetics published by CAB International. Three previous books, The Genetics of Sheep (1997), The Genetics of the Pig (1998) and The Genetics of Cattle (1999) are based on similar ideas and structure. The Genetics of the Dog will continue this series. This book is a result of truly international cooperation. Scientists from several European countries, the USA, Australia and Japan contributed to this project. The editors are very grateful to all of them. It is our pleasure and debt to thank many people who helped tremendously in reviewing the book: S. Adalsteinsson, D. Bernoco, M. Bowling, W.D. Flood, R. Fries, K. Johnson, B. Langlois, P. McGreevy, J.-C. Meriaux, T.H.E. Meuwissen, C. Moran, S.J. Valberg, J. van der Werf and W. Wong. The editors are thankful to Ms G. Kaercher (SORREL, Germany) for providing a photograph of tarpan. It is our hope that this book will be useful for many people who are interested in horse genetics. Perhaps it will support consolidation and further progress in this field of science. Ann Bowling Anatoly Ruvinsky

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Systematics and Phylogeny of the Horse 1 and O.A. Ryder2 C.P. Groves 1Systematics C.P. and and Groves Phylogeny O.A. Ryder 1Department of Biological Anthropology, Australian National University, Canberra, ACT 0200, Australia; 2Center for Reproduction of Endangered Species, Zoological Society of San Diego, PO Box 551, San Diego, CA 92112–0551, USA

The Order Perissodactyla The Family Equidae Genetics of the Equidae Generic Limits Early History of the Genus Equus The species question The subspecies question Nomenclature of Domestic Animals A Taxonomy of the Genus Equus Subgenus Equus: horses Subgenus Asinus: ass, onager and kiang Subgenus Hippotigris: zebras References

1 2 4 5 6 7 8 9 9 11 13 18 22

The Order Perissodactyla The order Perissodactyla, known as odd-toed hoofed mammals, are characterized by the relative enlargement of digit III on each extremity. Other characteristics include, in the skull, the persistence of the tuber maxillare, and, in the dentition, the basic π-shape of the occlusal pattern of the maxillary molars (modified in living Equidae, but extremely useful for recognizing primitive fossil perissodactyls). The order contains three living groups, the horses, tapirs and rhinos, and two major extinct groups, the brontotheres (or titanotheres) and the ancylopods (or chalicotheres). The brontotheres arose in the Early Eocene and survived into the Oligocene but no longer; the ancyclopods arose in the late Eocene and survived into the Pleistocene. Tapirs and rhinos differentiated in the Late Eocene. ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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The following outline classification of the Perissodactyla is based on Prothero and Schoch (1989), but excludes the hyraxes which (contrary to the classification of Prothero and Schoch) are not perissodactyls but are related to the elephants and sirenians. Order Perissodactyla Suborder Titanotheriomorpha – brontotheres Suborder Hippomorpha Superfamily Pachynolophoidea Superfamily Equioidea Family Palaeotheriidae Family Equidae Suborder Moropomorpha Parvorder Ancylopoda – chalicotheres Parvorder Ceratomorpha – tapirs and rhinos The Pachynolophoidea, restricted to the Eocene, were small perissodactyls with simple teeth, but with some development of the complexity of the cheek teeth which characterizes equids; in particular, the premolars were somewhat molarized (meaning that they had come to resemble the molars to some degree). The Palaeotheriidae, from the Late Eocene and Early Oligocene, were larger (some of them tapir-sized) and longer necked, but still extremely primitive; they include Palaeotherium, which was described by Georges Cuvier in 1804, and was one of the very first fossil mammals to be discovered.

The Family Equidae The Equidae are known from the earliest Eocene. The famous Hyracotherium, described by Richard Owen in 1841, is very primitive, and the various species which have been included within it, and which differ only very slightly from each other, are now regarded as probable ancestors to the Pachynolophoidea, the Palaeotheriidae and probably the Moropomorpha as well as the Equidae, so many authors now place them on cladistic grounds into different genera. The species described by Owen, Hipparion leporinum, is thought by Hooker (1984) to be the ancestor of the Palaeotheriidae. The ancestor of the Equidae, according to Hooker, is the species formerly known as Hyracotherium cuniculum, which he places in a new genus Cymbalophus. The general outline of the evolution of the Equidae is summarized by Evander (1989). After Cymbalophus of the earliest Eocene (54 million years ago (mya)) of Europe, the line moves to North America, and goes more or less straight through a series of genera which (except for the last two, which do overlap) simply mark points on the tree which are represented by good fossil material: Orohippus (Early Eocene, 50–47 mya), Epihippus (Middle and Late Eocene, 47–40 mya), Mesohippus (Late Eocene and Early Oligocene, 40–30 mya), and Miohippus (Latest Eocene and Oligocene, 37–25 mya). The line leading to modern horses had thus gone through more than half of its

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evolutionary history with hardly any branching until the latest stages. The narrow basicranium so characteristic of modern horses was absent in the primitive stages but had been developed by Mesohippus, and the dentition of each is advanced over its predecessor(s): more complex, with more cusp development, but still without any uniting of the cusps into ridges. The line then, at the beginning of the Oligocene some 25 mya branched into the Anchitheriinae and the Equinae. Both these lines were advanced in having molar ridges, unlike their predecessors; the Anchitheriinae, which lasted until about 12 mya in North America and 7 mya in Asia, lost some features of the molars and the foot skeleton which the Equinae retained, while the Equinae developed a close-packed foot skeleton suitable for running in open grasslands. The line of the Equinae continued through to the Middle Miocene (15 mya) via Kalobatippus, Archaeohippus and Parahippus, with increasingly complex molars. From Archaeohippus on, the skull developed a post-orbital bar, a complete strut of bone behind the orbit, separating it from the temporal fossa. From the later species of Parahippus on, the crowns of the molar and premolars had become high, covered with cementum, and suitable for shearing silica-rich grasses, and the radius and ulna had become fused. The line then, about 15 mya, split into three branches: the Protohippini, Hipparionini and Equini, though Hulbert (1989) considers that the Protohippini are actually a composite, made up of stem forms of the other two tribes and of their common ancestors, and includes Protohippus itself in the Equini; all these late groups are difficult to work out, and there was a good deal of parallel evolution in such features as large size, development of a pre-orbital fossa and retraction of the nasal notch. However, the Equini were the only horses to reduce their toes to one on each foot (with the laterals, represented by metapodials II and IV, retained as ‘splint bones’), whereas the Hipparionini developed their lateral toes into support digits, perhaps for marshy country. The Hipparionini, the last of the three-toed horses, lived in North America until the beginning of the Pliocene, 5 mya, but survived in the Old World until about 1 mya, disappearing last from Africa. Evander’s (1989) classification of the family is (abbreviated and slightly modified) as follows: Family Equidae Cymbalophus Orohippus Epihippus Mesohippus Miohippus Subfamily Anchitheriinae Subfamily Equinae Kalobatippus Archaeohippus Parahippus

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Tribe Protohippini Tribe Hipparionini Tribe Equini Dinohippus Hippidion Onohippidion Astrohippus Pliohippus Equus In this classification, the five unranked genera coordinate with the two subfamilies, and the three coordinate with the three tribes of Equinae, are given the status of Plesion: a category which means that they are of limited diversity (one or a few species in each), and primitive for that group (and very likely ancestral to the remainder). The genus Dinohippus, which lived in North America between about 8 and 5 mya, is the stem genus of Equus, and Hulbert (1989) even includes it in Equus. It emerged gradually from Pliohippus, which lived in North America from about 14.5 to 6 mya; Astrohippus lived in the same region from about 6 to 4.5 mya. Hippidion and Onohippidion were large single-toed horses that lived in South America from the Early Pliocene until the end of the Pleistocene; their earliest species lived in North America about 5 mya.

Genetics of the Equidae Studies of chromosomes and DNA have provided a rich source of information for interpretation of morphological and palaeontological data (George and Ryder, 1986; Ryder et al., 1978; Oakenfull et al., unpublished observations). The phylogenetic analysis of DNA sequence data sheds new light on the systematics and taxonomy of Equus and is highly relevant for conservation action plans for equid taxa (Oakenfull, equid action plan). The first DNA-based studies of Equus involved the analysis of mitochondrial DNA restriction maps and required purification of mitochondrial DNA (George and Ryder, 1986). With the advent of the polymerase chain reaction (PCR), DNA sequence data have become the accepted standard in studies of molecular evolution; PCR also allows a wide variety of samples previously unusable for genetic analysis to be utilized in genetic studies. The complete 16,660 nucleotide sequence of a domestic horse mitochondrial DNA has been published (Xu and Arnason, 1994). The first extinct organism to have its DNA cloned was the quagga, Equus (burchelli) quagga, an event of technical wizardry made even more remarkable for its accomplishment before PCR was invented (Higuchi et al., 1984). Samples of dried tissue, a residue of a roughshod taxidermy, provided sufficient DNA for the construction of a library that included quagga DNA sequences.

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Today, bone and tissue fragments, dried blood and dung samples provide routine sources of DNA for comparative genetic studies. Despite these advances, it is still the case that DNA sequence data are lacking for some equid populations and named taxa that confound the assembly of a clear picture of the systematic relationships of extant and recently extinct taxa. The Somali wild ass, Syrian, Indian and Mongolian wild asses, and the kiang have yet to be the subject of reports utilizing molecular methods on wild populations. Although most equid taxa have been the subject of at least preliminary DNA sequencing studies, the amount of data is still rather small, may not include nuclear loci and may incorporate limited sampling of the potential variability within extant wild populations. The investigation of molecular genetics of captive and wild equids is an area of great current interest. Thus, we can anticipate that new findings will soon become available (Oakenfull et al., unpublished observations) and that as more intensive analysis incorporating additional loci and more extensive sampling of extant populations is carried out, a more refined picture of evolutionary relationships and the resultant taxonomy will emerge. The most definitive genetic data pertinent to equid evolution collected to date involve sequence analysis of mitochondrial DNA. The region of the circular mitochondrial DNA at which strand displacement for the initiation of DNA replication takes place (the d-loop) is thought to be the most rapidly evolving portion of the approximately 16,500 bp molecule. Accordingly, this region can identify recent divergences due to mutations. Other portions of the mitochondrial DNA, such as the 12S region and the cytochrome b gene, accumulate mutations at a slower rate and there is a proportionally smaller chance that the same sequence of nucleotides is present as a result of two mutations (a ‘forward’ mutation and a ‘back’ mutation). For this reason, evidence of the divergence of lineages at the base of the phylogenetic tree may be derived less ambiguously from more slowly evolving sequences.

Generic Limits While most specialists have been content to include all living equids in a single genus, Equus, from time to time different authors have proposed to set aside one or more species into separate genera, on the general grounds that they were ‘different enough’. Part of the philosophy was no doubt that horses, asses, onagers and zebras are all the living species that we have in the family Equidae, and there is sufficient ‘taxonomic room’ for several genera. The wish to divide up the genus in this way persists in the modern era: Trumler (1961), Groves and Mazák (1967) and Bennett (1980) are examples of this. A different philosophy is behind Quinn’s (1957) multigeneric scheme: that author – incorrectly, as most specialists now concur – saw the different modern groups as the end points of lineages which could be traced back deep into the Miocene, and had even achieved monodactyly independently.

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Although proposals have been made to link taxonomic ranking to time depth, these have not achieved general acceptance, and the only widely acknowledged criterion for taxonomic categories (above the species level, at least) is monophyly. The single versus multigeneric schemes are essentially a matter of taste. The fossil record of the Equidae is one of the best known among mammals, and is replete at every level with genera, leaving much less ‘room’ for multiple genera among the living fauna. There is also some doubt what these genera would actually be: will Equus, Asinus and Hippotigris suffice, or must we have Hemionus, Dolichohippus and Quagga as well? What, really, are the interrelationships of the living species? We are of the opinion, in accord with most other equid specialists, that the less formal category of subgenus will have to do; but the question remains, what are they? The question in general narrows down to asking, what are the monophyletic groups and how well-supported are they? Below, we argue that three subgenera, Equus (horses), Asinus (asses and hemiones) and Hippotigris (zebras), adequately represent the major monophyletic groupings among living species, although the diversity of species makes it necessary to recognize species-groups within the latter two.

Early History of the Genus Equus Before examining the living members of Equus, a brief survey of fossil forms is in order. Like the family as a whole, the genus Equus evolved in North America, and spread from there into the Old World. It is likely that horses, asses and zebras all evolved in North America, and separately spread into the Old World when the Bering land bridge was open, from the mid-Pliocene onward. Azzaroli (1998) has reviewed the North American species. The earliest, Equus simplicidens, which lived from 4 to 2.5 mya, was very primitive, with many Dinohippus-like features, and was probably ancestral to all subsequent species; it is conventionally placed in a separate subgenus, Allohippus. The subgenus Equus was represented by an indigenous species, E. niobrarensis, in the Middle Pleistocene, and the immigrant E. ferus in the Late Pleistocene, surviving into the Holocene in Alaska. The subgenus Asinus had emerged by 2 mya, and was well known by fossils by 1.2 mya (E. semiplicatus) and survived into the Middle Pleistocene (E. francisci). Possible representatives of the subgenus Hippotigris were E. idahoensis (Late Pliocene to Early Pleistocene, 2.6–1.0 mya), E. excelsus and E. occidentalis (Middle and Late Pleistocene, respectively). A further subgenus, Amerhippus, is represented by early remains at 2 mya, by a Middle Pleistocene species (E. fraternus) and one or two Late Pleistocene species (E. conversidens and, possibly, E. mexicanus). Why equids eventually became extinct in North America is unknown, but the last of them, E. occidentalis (known from many skeletons from the tar pits at Rancho La Brea, Los Angeles, California), was perhaps exterminated by the first human inhabitants in the Early Holocene.

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It is worth noting, too, that one branch of Equus entered South America in the Late Pliocene. This is the zebra-like subgenus Amerhippus, which diversified into a variety of pampas and even Andean species (seven in number, according to Azzaroli, 1992a), which survived alongside Hippidion and Onohippidion until the Late Pleistocene, and finally died out for reasons that are unclear; however, Azzaroli (1992b) has argued that it was a case of ‘prehistoric overkill’, i.e. overhunting by early humans, perhaps abetted by climatic change. In both Europe and Asia, the earliest certain appearance of monodactyl equids is at about 2.5 mya, apparently part of a major faunal turnover as forest mammals disappeared and open-country fauna took their place (Azzaroli, 1990); they entered Africa around 2 mya (Azzaroli, 1998). The earliest species (E. stenonis and its relatives) were nearly identical to North American E. simplicidens and are placed in the subgenus Allohippus. After 1 mya, they began to be replaced by species related to modern forms: by the Middle Pleistocene, both the subgenus Equus (E. mosbachensis and later species) and the subgenus Asinus (E. altidens, a member of the E. hemionus group) had entered Eurasia; and in the Late Pleistocene, E. graziosii, a member of the E. asinus group known only from Italy, and the widespread but still very poorly known E. hydruntinus, which may be either a hemione or a zebra. The African fossil representatives of Equus are almost entirely zebras. From 2 mya on, one or several large species related to E. grevyi were widespread throughout east and south Africa (E. capensis, E. oldowayensis, E. koobiforensis). Remains of other zebra groups are rare, becoming common only in the Late Pleistocene. A fossil ass is known from Olduvai Bed II (~2 mya) and from the Middle (E. tabeti) and Late (E. melkiensis) Pleistocene of the Maghreb. Fragmentary horse remains (E. algericus) also occur in the Maghreb in the Late Pleistocene.

The species question Most non-taxonomists still operate on a misunderstanding of the biological species concept (BSC) of Mayr (1942). The popular misapprehension is that different species cannot interbreed; some go a step further, believing that species are sometimes able to interbreed, but hybrids between them are sterile. The six universally recognized ‘species’ (here, species-groups) of Equus therefore qualify under any rubric as valid species: hybrids between horses and asses, for example, are (almost always) sterile. Many commentators would go on to say that if hybrids between Indian and Persian wild asses prove to be fertile, this would indicate that they, by contrast, are not valid species. This is not in fact what Mayr said: he proposed that species ‘do not interbreed under natural conditions’, and emphasized that this reproductive isolation might be the result of either pre- or post-mating isolating mechanisms. The post-mating mechanisms are those which cause hybrids to be sterile; the pre-mating ones include such things as ethological mechanisms (different

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courtship displays, for example), which can be broken down under unnatural conditions, such as captivity. If the pre-mating mechanisms break down, we may well discover that post-mating mechanisms are not in place – and perfectly fertile hybrids will result. Clearly, sympatry between two taxa is prima facie evidence for the existence of reproductive isolating mechanisms between them; but, under the BSC, allopatric taxa are simply not amenable to being assessed objectively, unless we are able to conduct breeding experiments in captivity. Even then, if crossing them is unsuccessful, or hybrids between them are sterile, we can say that they are reproductively isolated and so are distinct species; but if they interbreed to give fertile hybrids, we are not at liberty to claim that they are members of the same species. All too often, of course, breeding experiments are simply not feasible, which means in practice that the majority of sexually reproducing species cannot even be tested. This is the case with living equids: hybrids are known in captivity between (for example) Indian and Persian wild asses, but their fertility has not been tested; and, today, the concern has always been to preserve the pure gene pool, and multigeneration crossing is of low priority. Several variants of the BSC have been proposed, but when all is said and done the biologist needs an operational definition of this concept, the species, which we all agree is so basic. Cracraft (1983) gave voice to such thoughts when he pointed out what an arbitrary decision it could be to assess whether two taxa might or might not interbreed were their ranges to meet, and proposed the phylogenetic species concept (PSC), whose operational criterion is simply the diagnosability (the consistency) of the candidates for species status. This is the attitude we take here: where two taxa are consistently different, given the available evidence, we rank them as separate species. This is why we recognize four species of the E. hemionus group, and two of the E. zebra group, and why we do not recognize more than one species in the E. quagga group, whose subspecies merge insensibly into each other. The potential importance of the reproductive factor is unquestioned, but it can never in itself be a criterion. We usually do not know whether there is interbreeding between two species, or we may even know that there actually is; the significant factor is that, if there is interbreeding, any resulting gene flow does not affect the character discontinuity between them.

The subspecies question Conceptually, there is no disagreement that subspecies are geographic segments of a species which are morphologically differentiated to some extent. They are geographic: by definition, they can never be sympatric. They are morphologically differentiated, but not consistently, not 100%: this would, under the PSC, make them different species. Mayr’s 75% rule, i.e. that three-quarters of individuals of one subspecies must be distinguishable from all individuals of all the others, is usually upheld, but this is a rule-of-thumb.

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The subspecies is just a point on the continuum of degrees of differentiation at which it becomes helpful (or desirable, or simply convenient) to recognize that differentiation with a trinomial. Subspecies are often the steps on a stepped cline. The subspecies of E. quagga are an example here. On a stepped cline (as within E. africanus), one can say, within limits, where subspecies A ends and subspecies B begins, and between the two points is a more rapid change in gene frequencies, metrical averages, whatever one is talking about. If the clinal gradient is insensibly smooth, obviously to dignify its two ends as subspecies is rather arbitrary: and this is why we recognize a single living subspecies of E. quagga south of the Zambezi. Note that either a species has at least two subspecies, or it has none. Subspecies exist in relation to one another: there is no such thing as a species having one single subspecies. One of the subspecies is called the nominotypical subspecies, and its subspecific name repeats its specific name; this will be the subspecies that exists at the species’ own type locality. There is no assumption that one subspecies is more ‘typical’ of the species than another.

Nomenclature of Domestic Animals Groves (1995) has argued that there is, and can be, no definitive answer to the question of whether domestic animals are the same species as their wild relatives, or different species. They are not subspecies because they are sympatric; they are not, or generally not, different species because except in a few outstanding cases they are not diagnosably different. Groves (1995) has called them paraspecies. Corbet and Clutton-Brock (1984) argued that, in most cases, it is convenient to maintain the fiction that, for nomenclatural purposes at least, they are different species. The wild species may have subspecies; the domestic species does not. Domestic breeds, however distinct they are, are sympatric; they arise and merge within very short periods of time.

A Taxonomy of the Genus Equus C.P.G. collected a number of characters from the literature (mainly Bennett, 1980; Groves and Willoughby, 1981; Eisenmann, 1986), but found that most of the unique states are clearly autapomorphic: the elongated metapodials of Hemionus, and so on. As in Bennett (1980), Dinohippus is taken as the outgroup. In the end, 26 characters seem to yield phylogenetically informative information (Table 1.1). The data matrix is given in Table 1.2. The resulting cladogram (Fig. 1.1) separates the living equids into three clades: horses, zebras, and asses plus hemiones. These same three clades are found by studies of mitochondrial DNA (George and Ryder, 1986), whereas a repetitive DNA sequence placed ass, hemione and the zebra group as three

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C.P. Groves and O.A. Ryder Table 1.1. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Morphological interrelationships within living Equus.

Presence and development of isthmus on lower molars (B, no. 6) Presence and development of infundibulum on third lower incisor (B, no. 11) Presence of ‘cranial broadening complex’ (B, no. 12) Degree of cranial flexion (B, no. 13) Relationships of mastoid, paramastoid and mastoid temporal (B, no. 14) Orientation of post-orbital bar relative to horizontal plane (B, no. 15) Presence of frontal doming (B, no. 18) Relative skull breadth (B, no. 19) Scapula length > 102% of radius, humerus length < 86% of scapula (GW) – polarity unknown Forefoot terminal phalanx breadth > 125% of length (GW) Forefoot terminal phalanx length > 55% of proximal phalanx, > 14% of radius (GW) Forefoot terminal phalanx plantar length > 125% of height (GW) Proximal humerus bicipital grooves deep (GW) – polarity unknown Skull vertex length > 560 where basal length standardized = 500 mm (GW) Palate–vomer length < vomer–basion (GW, E) Tooth row length > 170 where basal length standardized = 500 mm (GW) Occipital height > 298 (state 1), > 320 (state 2) where basal length standardized = 500 mm (GW) Nasal end of pre-maxilla narrowed, insinuated between nasal and maxilla (GW) Choanal opening horseshoe-shaped (GW) – polarity unknown Hypostylid present on third lower deciduous molar (GW) – polarity unknown Shape of wolf tooth (GW) – polarity unknown Ectoloph styles angle into walls (GW) Palate length < 52% of basal length (E) Muzzle length > 50% of palate length (E) Choanal length > 15% of basal length (E) Choanal breadth > 8.5% of basal length (E)

Morphological characters used in the cladistic analysis of species-groups of living equids (B = Bennett, 1980; GW = Groves and Willoughby, 1981; E = Eisenmann, 1986; in general, the derived states are described). Table 1.2.

Matrix of character states.

Horse Hemione Ass Mountain zebra Quagga Grévy’s zebra

12111100111110110010100001 21000001011001010010001001 20000000010001001100111001 00111100000001002001011110 00111111011001102111100100 00111111111111000001111110

The characters are listed in rows 1–26 as per Table 1.1. State 0 denotes the primitive state, state 1 the derived, state 2 (where relevant) most derived.

equal branches of a non-horse clade (Sakagami et al., 1999). A striking point of agreement is that the zebras form a monophyletic group; the common ancestor of the three extant species was probably a striped animal and each taxon of

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Fig. 1.1. Cladogram of living species-groups of Equus, based on the matrix of Table 1.2. The single most parsimonious tree has length 48, consistency index 0.604, retention index 0.486. The numbers above the lines are bootstrap values based on 1000 replicates. The horse/zebra clade is defined by derived states of characters 3, 4, 5 and 6; the hemione/ass clade by the derived state of character 1; the zebra clade by derived states of characters 7, 20 and 24; and the mountain/Grévy’s clade by derived states of characters 22, 23, 25 and 26.

zebra is more closely related to other zebras than it is to any other equid. A minor difference is that the ass/hemione clade separates first in the morphological tree, the horse clade in the genetic tree. Another possible area of disagreement is that asses and hemiones are associated unequivocally in the morphological tree, but less definitely in the genetic tree: whether hemiones and African (‘true’) wild asses are more closely related to horses or whether they are most closely related to each other and form a separate lineage has yet to be resolved unequivocally by the DNA data, and note that in the morphological data they are united by only a single derived condition (Fig. 1.1). In what follows, we have adopted the cladistic results (above), and lumped all zebras into one subgenus, all ‘asses’ into another, with the horses in a third. We briefly survey the evidence for species within each, giving less attention to subspecies.

Subgenus Equus: horses Externally, horses are recognizable by the long-haired tail; the mane that is both long and thick and tends, at least in winter and/or with increasing age, to fall to one side; the rounded croup; the usual presence of chestnuts on hindlimbs as well as forelimbs; the broad, rounded hooves; and the poor countershading, weak dorsal stripe and dark lower limbs with general traces of stripes on carpus and tarsus. Cranially, they have a relatively small skull, reduced cranial flexion, long diastema (the gap between the incisors and the cheek teeth), large pterygopalatine fossa (behind the hard palate) and a long, rounded occipital crest. The nasal end of the pre-maxilla is truncated or rounded, so that the nasal bone forms the angle of the narial notch. Postcranially, they have a long scapula and stout metapodials and phalanges. The metacarpus is short compared with the metatarsus, so that their hindlegs are

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longer than their forelegs. The pelvis is broad and splayed compared with other subgenera; the biischial breadth is high compared with the biacetabular (i.e. it is flared at the haunch compared with the hip joint); the height of the pelvic inlet is strongly sexually dimorphic, increasing the width of the female’s birth canal. Groves (1986, 1993) argued that all horses that survived into historic times belonged to one species, Equus ferus Boddaert, 1785, with three subspecies: E. f. ferus (Steppe tarpan), E. f. sylvestris (Forest tarpan) and E. f. przewalskii (Przewalski horse). The evidence that the Przewalski horse is the eastern representative of the species that contained the European tarpan is admittedly inferential: an apparent gradation of colours from west to east, and apparently the occasional appearance of Przewalski-like colours in Europe (including, as many people have noticed, on the walls of terminal Pleistocene caves such as Lascaux). This needs to be tested further (if possible!), but it does seem at the moment as if there was a continuum across Eurasia. A large number of species of wild horse have been described from the Late Pleistocene and Early Holocene of Europe, Siberia and China (see Forsten, 1988, and, for a recent survey, see Azzaroli, 1998). Eisenmann (1996) examined characters that have commonly been used to differentiate wild horse species; very few actually seem to characterize credible taxonomic units. Przewalski horses have a short vomer compared with other horses, both wild and domestic, including tarpans, and differ in the metacarpal proportions. On the other hand Przewalski horses and tarpans have shorter proximal phalanges compared with the metapodials (i.e. their feet are short compared with their lower limbs) than do the Late Pleistocene wild horses of France, Ukraine and Russia; however, the Portugese wild horses and the European Bronze Age (presumably domestic) horses resemble Przewalski horse and the tarpan. Przewalski’s horses have 2n = 66 chromosomes, while normal domestic horses possess 2n = 64 chromosomes. There is no evidence that there were differentiated subspecies within what conventionally has been considered the range of E. f. przewalskii. Analysis of the stud book of the Przewalski’s horses identifies that just four mitochondrial DNA haplotypes may descend from the 13 founders. However, DNA sequence analysis of the control region of these four matrilines suggests that only two distinct haplotypes survive (Oakenfull and Ryder, 1998). Previous studies of a single mitochondrial control region haplotype of Przewalski’s horses in comparison with domestic horses resulted in the suggestion that Przewalski’s horses may have been derived from domestic horses (Ishida et al., 1995), but more recent studies involving all surviving mitochondrial DNA control region haplotypes suggest that insufficient information exists to identify the phylogenetic relationships of Przewalski’s horses based on mitochondrial DNA. Erect mane, lack of forelock and moulting of the hair at the base of the tail are all primitive characteristics noted in wild, but not domestic, equids that are typical features for Przewalski’s horses. Studies of nuclear genetic variation in domestic and Przewalski’s horses may shed additional light on to the phylogeny of caballine horses. Of particular interest will be studies of

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domestic horses in Mongolia whose mitochondrial and nuclear diversity may provide otherwise unavailable insights into the relationship of Przewalski’s and domestic horses.

Subgenus Asinus: ass, onager and kiang These are characterized externally by having a tufted tail, chestnuts on forelimbs only and at least some degree of countershading. Cranially they are characterized by the squared, upturned occipital crest and wide external auditory meatus. Post-cranially, they have a short scapula; and, like horses, the metacarpus is short compared with the metatarsus, the biischial breadth is high compared with the biacetabular, and the height of the pelvic inlet is strongly sexually dimorphic. We can divide this subgenus into two certainly monophyletic speciesgroups, the E. hemionus group and the E. asinus group. This is necessary because of the existence of more than one living species of ‘hemiones’, and of multiple fossil species of ‘true asses’. Equus hemionus group The E. hemionus group (onagers or hemiones or Asian wild asses) are characterized externally by the short, clipped-appearing mane, short erect hair forming a broad dorsal stripe 50–100 mm broad, and white underparts and lower limbs. Cranially, they are characterized by the very narrow squared upturned occipital crest, shortened brain case, long vomer, short diastema and short pterygopalatine fossa. The nasal end of the pre-maxilla is truncated or rounded, so that nasal bone forms the angle of the narial notch. The tuber maxillae extend back, hiding the pterygopalatine fossa in the ventral view, like quaggas and unlike other equids. Post-cranially, they are distinguished by the short humerus and femur, the elongated, slender metapodials (so that the lower limb segments are long and fine) and the elongated distal phalanx. Groves and Mazák (1967) argued that the kiang, of the Tibetan plateau, is a distinct species (E. kiang) from the onagers (E. hemionus) of the more low-lying Asian deserts. The differences are very striking, and this separation has been widely followed. The subspecies onager, kulan, castaneus and hemionus form an intergrading series, overlapping (at the extremes) in their traits but each strongly distinct as a unit. The two other taxa usually placed as subspecies of E. hemionus, i.e. hemippus and khur, are in fact diagnosable taxa (in the PSC sense) and are here regarded as distinct species. The number of chromosomes in Asiatic wild asses varies from 2n = 56 to 2n = 50. This is a large amount of numerical and structural chromosomal polymorphism to be segregating within a single species. Tibetan kiangs, Equus kiang holdereri, apparently have fixed chromosomal differences from other subspecies of Asiatic wild asses, based on the available chromosomal data (Ryder et al., 1978; Ryder, 1986; Ryder and Chemnick, 1990). Mitochondrial DNA studies, including restriction fragment length polymorphism (RFLP)

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analysis (Ryder and Chemnick, 1990) and control region and 12S RNA sequence data (Oakenfull et al., unpublished observations), find that named E. hemionus subspecies are paraphyletic. Phylogenetic analysis identifies mitochondrial lineages encompassing the examined diversity of Asiatic wild asses that are present in multiple subspecies. Based on available data, extant mitochondrial DNA variation appears to reside largely within the named subspecies and not to be partitioned between onagers, kulans and kiangs. Subspecies of E. hemionus may hybridize, and be fertile, in spite of the chromosomal polymorphism (Pohle, stud book), providing evidence that reproductive isolation has not yet developed. The implication from mitochondrial DNA analyses is that the extant subspecies share a common pool of mitochondrial variation; divergence and genetic isolation are minimal, implying that these processes have begun only recently. The most divergent of the Asiatic wild asses based upon genetic analysis is the kiang, supporting their status as a separate species. Significant gaps exist, though, in the genetic studies of the hemione group. In particular, the khur of the Little Rann of Kutch has not been the subject of published molecular studies. Mongolian wild asses and the western and southern kiang similarly are absent from the literature on molecular evolution in Equus. Accordingly, there are opportunities for additional studies and a need to provide a more detailed view of nuclear and mitochondrial variation in Asiatic wild asses for conservation assessments, monitoring and management. Equus kiang Moorcroft, 1841: Kiang The kiang or Tibetan wild ass is of large size; it has a large head and thick muzzle, a relatively long mane and long hairs not restricted to the tail tuft but extending some way up either side of the tail. The pattern of contrasting dark (reddish) body blocks and white underside is characteristic: the demarcation between them on the flank is oblique from stifle to croup, and the white rump patch is infused with the reddish tone of the haunch. The dorsal stripe is thin and never bordered with white; it extends to the tail tuft. There is a dark ring round the hoof. The ear is 165–178 mm long. The skull resembles that of E. hemionus, except that the incisors tend to sit more vertically in the jaws (except in aged individuals, in which alveolar recession tends to reveal the oblique roots), and the highest point on the cranial profile is often directly above the posterior rim of the orbit, instead of behind it. There are three subspecies, which differ (as a whole, but not absolutely) as follows: 1. Equus kiang kiang Moorcroft, 1841. Western kiang. Dark red-brown colour in summer, dark brown in winter; the legs have brown tones. Size large; nasal bones short; tooth row relatively short. The southwestern corner of Tibet, into India (Ladakh) and Pakistan. 2. Equus kiang holdereri Matschie, 1911. Eastern kiang. Colour not so dark: strong red in summer, darker red-brown in winter; the legs are pure white. Size large; nasals very long; tooth row long. Eastern Tibet (Lhasa district) to Chinghai, Ganssu and the Seshu region of Sichuan.

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3. Equus kiang polyodon (Hodgson, 1847). Southern kiang. Colour dark as in E.k. kiang; size very small; nasals and tooth row long as in E. k. holdereri. The Gayam Tso region of Tibet, into Sikkim. Equus hemionus Pallas, 1775: Onager The onager varies from kiang-sized to khur-sized. The mane is very short and ‘clipped’; the dorsal stripe is thick, often bordered with a white line on either side; the white of the rump is not infused; the demarcation between the reddish flank block and whitish underside runs parallel to the body outline, before turning sharply up towards the dorsal stripe. The dorsal stripe extends to the tail tuft. There is a dark ring round the hoof. The nasal bones are relatively straight, the skull resembles that of E. kiang. The number and definition of the subspecies are disputed, but the following seem recognizable: 1. Equus hemionus hemionus Pallas, 1775. Mongolian wild ass. This is the least endangered subspecies, still found throughout the Mongolian desert regions, and formerly into Transbaikalia. Height 110–130 cm; poor demarcation of dark and light (off-white) areas; the only subspecies usually lacking a white border to the dorsal stripe. The concept that Groves and Mazák (1967) and Groves (1986) used was: (i) a disruptively coloured subspecies, occupying a northerly distribution extending from Transbaikalia to Kazakhstan, and (ii) a grading-toned one, called E. h. luteus, restricted to the Gobi desert. However, Denzau and Denzau (1999) have shown that the type illustration of Pallas’s E. hemionus, which was from Transbaikalia, in fact corresponds to the grading subspecies. The disruptively patterned ‘northern’ subspecies therefore never extended to Transbaikalia but was purely western in range, and must be called: Equus hemionus castaneus (Lydekker, 1905). Probably now extinct: formerly from Dzungaria through Kazakhstan to Uzbekistan. Also large; clear demarcation of coloured and white areas; much white on body and head. 2. Equus hemionus kulan (Groves and Mazák, 1967). Turkmenian wild ass or kulan. Badkhyz Reserve, Turkmenia. Smaller: height 108–120 cm. Coloured and white areas strongly demarcated like castaneus. Relatively larger teeth than the large Mongolian/Kazakhstan forms. 3. Equus hemionus onager Boddaert, 1785. Persian wild ass. Iran, east of the Zagros Range. Also small; demarcation between coloured and white areas less strong than in kulan, from which it differs in pattern details. Broader occipital crest; even larger teeth. This still occurs in the Bahram-e-Gur and Touran Reserves in Iran; according to Denzau and Denzau (1999), these two remnant populations differ in size. 4. Equus hemionus blanfordi Pocock, 1947. Probably now extinct; formerly, known only from Sham Plains (Pakistan) and Kandahar (Afghanistan). Also small, and large-toothed; with relatively narrow occipital crest and long nasals; extensive dark areas on flanks; the dorsal stripe fades out halfway down the tail. Equus khur Lesson, 1827; Khur (Indian wild ass) The Indian wild ass or khur is sharply distinct from E. hemionus, and because it can always be distinguished from other forms we recognize it as a full species. The coloured

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blocks on the flank and haunch are very small, so the predominant colour is white, and the lower 45% or more of flank is whitish; the demarcation on the lower haunch slants upward from the front (stifle) to the back. There is a dorsal stripe with a clear white border on either side. The white wedge between the haunch and flank blocks nearly or fully reaches the spine. The legs are pure white. The dorsal stripe fades out halfway down the tail. There is no dark ring round the hoof. The facial profile is concave; the nasal bones are raised (making the whole facial profile strongly concave), and comparatively short (Groves, 1986, Figure 1); and the teeth are small. The skull is noticeably high crowned. The choanae are small and the orbits are high. Height at withers is 110–130 cm. The metapodials are less elongated than in E. hemionus. The ear is very long: 187–210 mm. The ass survives in the Rann of Kutch, Gujarat, India; formerly known from the Sind deserts, Pakistan. Equus hemippus I. Geoffroy St Hilaire, 1855: Syrian wild ass The extinct Syrian wild ass or achdari was likewise diagnostically different from other hemiones. It was very small in size; the evidence suggests that this difference has come about since the end of the Pleistocene (Turnbull, 1986). The height at the withers was about 1 m. Colour was very grading; a sandy-brown flank patch extended well down, grading into off-white on the underside; only the lower 20% or less of flank was whitish. There was a dorsal stripe with a clear white border on either side; this became obfuscated with age, and eventually disappeared. The white wedge between the haunch and flank blocks was vague, strongly infused with body tone. The legs were strongly infused with body tone. The dorsal stripe faded out halfway down the tail. There was no dark ring round the hoof. The nasal bones were raised, and relatively longer than in other onagers (Groves, 1986, Fig. 1.1); the teeth were relatively large. Otherwise the skull, with its concave profile, high-placed orbit, and high crown, resembled a small E. khur. The metapodials were more elongated than those of other species; the terminal phalanges were shortened. It was known from Syria and northern Iraq.

Equus asinus group The Equus asinus group (true asses or donkeys) are characterized externally by a long, thin ‘scruffy’ mane, thin dorsal stripe, usually traces of stripes on the legs (at least fetlocks) and a restricted white (or pale) region on the underside. Cranially they have a very long cranium, short palate, long diastema, large pterygopalatine fossa and a squared, upturned occipital crest. The nasal end of the pre-maxilla is narrow, insinuated into a corner of the narial notch. The orbit is high, rounded and not anteroposteriorly elongated like other equids except mountain zebras. Post-cranially, there are very short, narrow distal phalanges, translating externally to small, narrow hooves.

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Eisenmann (1995) reviewed recently the palaeontological evidence for the evolution of this species group, and the evidence for extinct Early Holocene populations. There is one wild species: Equus africanus Heuglin and Fitzinger, 1866: African wild ass All African wild asses tend to be more reddish in summer, greyer in winter, with contrastingly white legs and a less contrastingly whitish underside; the white wedges behind the shoulder and in front of the haunch, so prominent in hemiones, are evanescent. Groves (1986) showed that, to some degree, there is clinal change from the Atbara population via the Red Sea coastal populations to Somalia. There is quite a marked change, a distinct step in the cline, between northern and southern Eritrea, perhaps representing a bottleneck to gene flow in the Massawa region, where the Highlands approach the sea, and this justifies the recognition of at least two subspecies. A supposed wild ass was described as Asinus taeniopus Heuglin (type locality: Little Dobar, south of Berbera), and even today this is sometimes recognized as a subspecies, but no such wild ass actually exists: it was based on a cross between a Somali wild ass and a domestic ass. A north African wild ass, with strong, often doubled, shoulder-cross and well-marked leg stripes was depicted in both rock art and Roman era mosaics, and was stated to survive at Siwa, on the Libyan–Egyptian border, by Hufnagel (1965). It is often called Equus africanus atlanticus, but should not be, as that name was first given to a fossil north African zebra. Eisenmann (1995) discusses whether the name Equus melkiensis (described from a genuine fossil ass, from the Late Pleistocene of Allobroges, Algeria) might apply to this form, though one ought to be rather cautious in associating Pleistocene fossils with Holocene rock depictions. In the earliest Holocene, wild asses were also present in northern Arabia (Ducos, 1986; Groves, 1986); a subspecies Equus africanus mureybeti Ducos, 1986 has been described from pre-pottery levels in Iraq, but Eisenmann (1995) is not convinced that the remains are ass rather than onager. Only the Somali wild ass has been studied genetically, and there are significant doubts that any other forms of African wild ass survive. Chromosomal polymorphisms in Somali wild asses have been described (Houck et al., 1998). Mitochondrial DNA analyses place Somali wild ass as a sister group to domestic donkey but, without additional study, the phylogenetics and systematics of African wild asses remain tentative. There are several subspecies: 1. Equus africanus africanus Heuglin and Fitzinger, 1866. Nubian wild ass. From Atbara River to Red Sea coast and northernmost Eritrea. May be extinct. Shoulder height 115–121 cm. A dorsal stripe is always present, and nearly always complete from mane to tail tuft; crossed by an usually thin, fairly short shoulder stripe. Leg stripes, where present are restricted to a few bands at the fetlocks. The diastema is relatively short, and the post-orbital constriction well

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marked. The nasal process of the pre-maxilla ends bluntly. There is never a ‘bridge’ between metaconid and metastylid in the lower premolars and molars. Groves (1986) found that specimens from the Atbara differ on average from those from the Red Sea Hills (Sudan) and Eritrea, and they could be subspecifically distinct. The Nubian wild ass is probably not, contrary to ‘received opinion’, the ancestor of the domestic donkey. 2. Equus africanus somaliensis Noack, 1884. Somali wild ass. Ears shorter, 187–200 mm; shoulder height 120–125 cm; the dorsal stripe is often absent, and when present is often incomplete, broken at some point along the dorsum. Shoulder-cross often absent. Leg stripes are present from hooves to above the carpus and tarsus. Diastema is relatively long, and the post-orbital constriction less marked. There is a thickened bar of bone behind the orbits, marking the highest point on the profile. The nasal process of the pre-maxilla is thin and pointed. There is always at least a trace of a ‘bridge’ between metaconid and metastylid in the lower premolars and molars. Somali asses seem to be longer legged, and shorter bodied than Nubian ones. Differences between Somalian and Danakil plus Djibouti populations exist, but are less marked than those between the two populations of E. a. africanus. Somali wild asses still exist in two small population nuclei: in the Afar (Danakil) country of southern Eritrea, and in the Nogal valey, northern Somalia. 3. Equus africanus subsp. Saharan wild ass. Still more a rumour than a fact, but best authenticated for Ahaggar, Tibesti and Fezzan; the appearance of the (apparently indigenous) wild ass of the Sahara was reconstructed by Groves (1986) as closest to E. a. africanus but smaller and greyer, and with a long, thin shoulder-cross.

Subgenus Hippotigris : zebras These are characterized externally by the long, rather thick upright mane, tufted tail, chestnuts on forelimbs only, and striking black and white stripes. Cranially, the occiput is high and raised; the post-orbital constriction is deep; the muzzle long; and the vomer long. Post-cranially, the metacarpus is long compared with the metatarsus, so that the forelimbs are longer than the hindlimbs. The biischial breadth is low compared with the biacetabular, so that the hip joints are further apart, giving zebras a fat-rumped appearance; the height of the pelvic inlet is not strongly sexually dimorphic, so that males, as well as females, have a large, high space corresponding to the birth canal. Chromosomal and molecular data, as well as the morphological analysis presented here, all support the position of monophyly for zebras – that all extant zebras share a recent common ancestor with each other. Mitochondrial DNA RFLP study suggested that plains zebra and Grévy’s zebra are sister taxa, as did study of a repeated sequence (Sakagami et al., 1999), but in another study involving control region and 12S gene sequencing, the mountain zebra and plains zebra were identified as sister taxa (Oakenfull and Ryder, 1998). The morphological tree, however, places mountain and Grévy’s zebras closer.

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Equus zebra group The E. zebra group (mountain zebras) is characterized externally by the long, rather thick upright mane, small dewlap, and stripes absent from venter, forming what has been called a ‘gridiron’ pattern (short transverse stripes meeting the uppermost of a long, thick, oblique/longitudinal series) on the croup. Cranially, the nasal end of the pre-maxilla is truncated or rounded, so that the nasal bone forms the angle of the narial notch. The external auditory meatus is wide, and (uniquely) directed horizontally instead of upward and backward, probably connected with the large external ears. The ventral bar of the orbit is slender and the orbit is high, rounded like that of asses. Postcranially, the scapula is short; the distal phalanges are short and narrow, so that the hoof is small and fine like that of a donkey. The skull is relatively large compared with the rest of the skeleton. The two forms usually ranked as subspecies of a single species, E. zebra, should certainly be regarded as distinct species. They are strikingly different externally, and some of the skull measurements do not overlap, although note that sample sizes are small. For example, occipital crest breadth is 63–71 mm in male zebra, 74–78 mm in male hartmannae (n = 5 of each); in females, 63–68 mm in zebra, 70–86 mm in hartmannae (n = 3 and 6, respectively). In the present sample, there is a sexual size difference in zebra (mean skull length 516.0 mm in males, 530.8 mm in females), but not in hartmannae (548.0 and 549.9 mm, respectively). Equus zebra Linnaeus, 1758. Cape mountain zebra. Mountains of the southern Cape (Skead, 1980). It was nearly extinct in the 1950s but has been very successfully preserved and reintroduced over much of its former range. Size smaller; black stripes broader than white interspaces. Occipital crest narrow; females apparently larger than males. Equus hartmannae Matschie, 1898. Hartmann’s mountain zebra Arid, hilly regions from southern Angola formerly to northwestern Cape. It still occurs in Angola and northern Namibia, but is very much less numerous than formerly. Size much larger; black stripes narrower than white interspaces. Occipital crest broad. Sexes equal in size. Equus quagga group The E. quagga group (plains zebras) have a mane varying from long, thick, neat to shorter, thinner, and even absent altogether; striping varies from dark brown and white on head and neck only to striking black and white over the whole body, including the venter, and a simple oblique/longitudinal pattern on the croup and haunch. The post-orbital constriction is relatively narrow, the vomer long, the diastema long, the teeth relatively small, and the occiput extremely raised. The narial notch is less deep than that of other zebras. The interorbital part of the frontal bone is strongly convex, so that these zebras have a forehead that is rounded and convex from side to side. The bizygomatic width (cheekbones

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behind eyes) generally exceeds the biorbital width, so that the eyes do not protrude like those of other equids. The nasal end of the pre-maxilla is narrow, insinuated into the corner of the narial notch. Pre-maxillae, unlike those of other zebras, are curved downward below the level of the alveolar line of the cheek teeth, so that the upper lip is unusually deep. The tuber maxillae extends back, hiding the pterygopalatine fossa in the ventral view, like Hemionus and unlike other equines. The foramen magnum (the hole through which the spinal cord enters the brain case) is an unusual, uniquely rectangular shape. The metapodials are somewhat lengthened, so that the lower legs are relatively long. Distal phalanges are less reduced than in the E. zebra group, translating to larger, broader hooves. The cranial differences between plains and mountain zebras are given by Eisenmann and de Giuli (1974) and Smuts and Penzhorn (1988), who agree in most respects, though the latter add a few characters, notably the important difference in the foramen magnum, in which this species group is unlike any other subgenus. Although in this species group two species, E. quagga and E. burchelli, are often recognized, they grade insensibly into each other; some of the skins described and illustrated by Rau (1974), especially the Mainz female ‘true quagga’ and another specimen in Mainz, the type of paucistriatus Hilzheimer, are difficult to allocate to one or the other. There seems no prospect of breaking the cline from the Cape to Sudan and Somalia, into species; though, the cline being stepped, the subspecies seem clear enough. The quagga was in fact the first extinct organism from which DNA was extracted (Higuchi et al., 1984); the result showed clearly its close affiliation to living plains zebras. The plains zebra is the most abundant wild equid and, though it can be divided into several fairly clear-cut subspecies, there is no clear picture to present from the genetic perspective until nuclear and mitochondrial DNA studies of populations across the range of the species has been undertaken. Equus quagga quagga Gmelin, 1788 The extinct true quagga lived west of the Drakensberg and south of the Vaal-Orange system (Skead, 1980). It had head and neck stripes, but on the body the stripes were incomplete at best; body colour was fawn, but legs were white. The available museum material was fully discussed by Rau (1974, 1978), who showed that there are some specimens that are so intermediate between ‘true quagga’ and burchelli that we cannot recognize any species distinction. The physical geographic barriers between the two are, however, sufficient to make a strong step in the cline, and so confirm their subspecific distinction. I have measured only three adult male skulls from the wild (Leiden, Berlin and Frankfurt). They range from 485 to 528 mm in length, and so average smaller than other subspecies apart from boehmi. Equus quagga burchellii Gray, 1824 Known in different regions as Burchell’s, Chapman’s, Wahlberg’s and Damara zebra. From Kwazulu-Natal and the Vaal-Orange system north to the Zambezi and Namibia. They are large in size. Three or four stripes (very rarely two or five) meet (sometimes they do not

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quite meet) the median ventral line between the elbow and the stripe that bends back to form the ‘saddle’ of the lumbar region. Colour is ochrey or off-white, never pure white; shadow stripes are usually well marked; leg stripes are absent or poor, almost never complete to the hooves. The infundibulum (‘cup’) on the lower incisors is better expressed than in other subspecies. The mane is well developed. Northerly populations (Zimbabwe, northern Mozambique) are paler, with less strikingly marked shadow stripes and more complete leg stripes than other populations. Those from Kwazulu-Natal and also northern Namibia (Etosha and Kaokoveld) are more ochrey with stronger shadow stripes and fewer leg stripes. There is simply no prospect of breaking up this subspecies as has usually been done. The least striped phenotype, which even lacks stripes on the lower haunches, occurs in both Etosha and Kwazulu-Natal and formerly occurred in the Free State; it has sometimes been called ‘true Burchell’s zebra’, but there was never a population even predominantly characterized by this type, and ‘true Burchell’s zebra’ has never existed as a discrete population. Equus quagga crawshayi de Winton, 1896 From north of the Zambezi and east of the Luangwa: so, in easternmost Zambia, Malawi, northern Mozambique and southern Tanzania. They are of large size. Stripes are numerous and narrow; there are always at least five stripes (often six to eight) meeting the median ventral line between the elbow and ‘saddle’ stripe; body tone is white or off-white; almost never even traces of shadow stripes; leg stripes complete to hooves. Equus quagga zambeziensis Prazak, 1898 From Angola and Zambia east to the Luangwa, and perhaps into Shaba. Large in size. Stripes are broad; only three or four meet the median ventral line between the elbow and ‘saddle’ stripe; colour varies from ochrey through off-white to white; shadow stripes vary from fairly prominent to absent; leg stripes are usually complete, or nearly so. Equus quagga boehmi Matschie, 1892: Grant’s or Boehm’s zebra Small in size. Externally it resembles the previous form closely, but is separated from it by E. q. crawshayi and by Lake Tanganyika. The infundibulum is generally absent. It intergrades with crawshayi in southern Tanzania, and then ranges north through Tanzania and Kenya into southern Somalia and Ethiopia, Karamoja (Uganda) and southeast Sudan, east of the Nile. Most northerly populations of this subspecies have high frequencies of manelessness, and the ears are either very vaguely striped or completely white; they could perhaps be separated as a different subspecies but there are differences among them: (i) manelessness appears to be fixed in the northeastern ones (northeast of Mount Kenya, into Somalia), but merely high frequency (especially frequent in males) in the northwestern ones (Baringo, Karamoja and Sudan), and apparently does not occur at all in Ethiopia or around Lake Turkana, and (ii) whereas northeastern zebras are as small as other boehmi or even slightly smaller, northwestern ones are larger, as big as the more southerly subspecies such as zambeziensis. If it is felt that they should be recognized as different subspecies: the name for the northeastern one would be granti, for the northwestern, borensis.

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Equus grevyi group The E. grevyi group, Grévy’s zebra, has a long thick upright mane, very small chestnuts, short erect black hairs along the dorsal stripe and narrow, almost dazzling, black and white stripes on the whole body except for the venter and croup, forming a complex triradial pattern on the haunch. The skull is very elongated, exceeding the cervical spine in length. The nasal end of the pre-maxilla is rounded, wedged into the nasal bone. Post-cranially, the scapula is lengthened, the metapodials somewhat lengthened, and distal phalanges small. There is only one living species, i.e. Equus grevyi Oustalet, 1882. Grévy’s zebra. Provisionally, two subspecies can be recognized, based entirely on size, although they need to be tested on larger samples: (i) Equus grevyi grevyi Oustalet, 1882. Greatest skull length 529–615 mm. From the Somali Arid zone proper: Harar region to northernmost Kenya; and (ii) Equus grevyi subsp. Larger: greatest skull length 608–639 mm. From the Guaso Nyiro district, Kenya.

References Azzaroli, A. (1990) The genus Equus in Europe. In Lindsay, E.H. (ed.), European Neogene Mammal Chronology. Plenum Press, New York, pp. 339–356. Azzaroli, A. (1992a) The present state of knowledge on the Ecuatorian species of the genus Equus. Bollettino della Società Paleontologica Italiana 31, 133–139. Azzaroli, A. (1992b) Ascent and decline of monodactyl equids: a case for prehistoric overkill. Annali Zoologici Fennici 28, 151–163. Azzaroli, A. (1998) The genus Equus in North America – the Pleistocene species. Palaeontographia Italica 85, 1–60. Bennett, D.K. (1980) Stripes do not a zebra make, part I: a cladistic analysis. Systematic Zoology 47, 272–287. Corbet, G.B. and Clutton-Brock, J. (1984) Appendix: taxonomy and nomenclature. In: Mason, I.L. (ed.), Evolution of Domesticated Animals. Longman, London, pp. 434–438. Cracraft, J. (1983) Species concepts and speciation analysis. In: Johnston, R.F. (ed.) Current Ornithology 1, 159–187. Denzau, G.and Denzau, H. (1999) Wildesel. Jan Thorbecke Verlag, Stuttgart. Ducos, P. (1986) The equid of Tell Muraibit, Syria. In: Meadow, R.H. and Uerpmann, H.-P. (eds), Equids in the Ancient World. Dr Ludwig Reichert Verlag, Wiesbaden, pp. 237–245. Eisenmann, V. (1986) Comparative osteology of modern and fossil horses, half-asses, and asses. In: Meadow, R.H. and Uerpmann, H.-P. (eds), Equids in the Ancient World. Dr Ludwig Reichert Verlag, Wiesbaden, pp. 67–116. Eisenmann, V. (1995) L’origine des ânes: questions et réponses paléontologiques. Ethnozootechnie 56, 5–26. Eisenmann, V. (1996) Quaternary horses: possible candidates to domestication. Proceedings of the XIII Congress, International Union of Prehistoric and Protohistoric Sciences 6, 27–36.

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Eisenmann, V. and de Giuli, C. (1974) Caractères distinctifs entre vrais zèbres (Equus zebra) et zèbres de Chapman (Equus burchelli antiquorum) d’après l’étude de 60 têtes osseuses. Mammalia 38, 509–543. Eisenmann, V. and Shah, N. (1996) Some craniological observations on the Iranian, Transcaspian, Mongolian and Indian hemiones. EEP Yearbook, 1995/6, 396–399. Evander, R.L. (1989) Phylogeny of the family Equidae. In: Prothero, D.R. and Schoch, R.M. (eds), The Evolution of Periisodactyls. Oxford University Press, Oxford, pp. 109–127. Forsten, A. (1988) The small caballoid horse of the upper Pleistocene and Holocene. Journal of Animal Breeding and Genetics 105, 161–176. George, M. and Ryder, O.A. (1986) Mitochondrial DNA evolution in the genus Equus. Molecular Biology and Evolution 3, 535–546. Groves, C.P. (1986) The taxonomy, distribution, and adaptations of recent equids. In: Meadow, R.H. and Uerpmann, H.-P. (eds), Equids in the Ancient World. Dr Ludwig Reichert Verlag, Wiesbaden, pp. 11–65. Groves, C.P. (1993) Morphology, habitat and taxonomy. In: Boyd, L. and Houpt, K.A. (eds), Przewalski’s Horse: the History and Biology of an Endangered Species. State University of New York Press, Albany, pp. 39–59. Groves, C.P. (1995) On the nomenclature of domestic animals. Bulletin of Zoological Nomenclature 52, 137–141. Groves, C.P. and Mazák, V. (1967) On some taxonomic problems of Asiatic wild asses with a description of a new subspecies (Perissodactyla, Equidae). Zeitschrift für Säugetierkunde 32, 321–355. Groves, C.P. and Willoughby, D.P. (1981) Studies on the taxonomy and phylogeny of the genus Equus. 1. Subgeneric clasification of the recent species. Mammalia 45, 321–354. Higuchi, R., Bowman, B., Freiberger, M., Ryder, O.A. and Wilson, A.C. (1984) DNA sequences from the quagga, an extinct member of the horse family. Nature 312, 282–284. Hooker, J.J. (1984) A primitive ceratomorph (Perissodactyla, Mammalia) from the early Tertiary of Europe. Zoological Journal of the Linnaean Society 82, 229–244. Houck, M.H., Kumamoto, A.T., Cabrera, R.M. and Benirschke, K. (1998) Chromosomal rearrangements in a Somali wild ass pedigree, Equus africanus somaliensis (Perissodactyla, Equidae). Cytogenetics and Cell Genetics 80, 117–122. Hufnagel, E. (1965) Libyan Mammals. Oleander, Harrow. Hulbert, R.C. (1989) Phylogenetic interrelationships and evolution of North American late Neogene Equinae. In: Prothero, D.R. and Schoch, R.M. (eds), The Evolution of Periisodactyls. Oxford University Press, Oxford, pp. 176–196. Ishida, N., Oyunseren, T., Mashima, S., Mukoyama, H. and Saitou, N. (1995) Mitochondrial DNA sequences of various species of the genus Equus with special reference to the phylogenetic relationship between Przewalski’s wild horse and domestic horse. Journal of Molecular Evolution 41, 180–188. Lydekker, R. (1905) Notes on the specimens of wild asses in English collections. Novitates Zoologicae 11, 583–596. Mayr, E. (1942) Systematics and the Origin of Species. Columbia University Press, New York. Oakenfull, E.A. and Ryder, O.A. (1998) Control region and 12S rRNA variation in all the extant mitochondrial lineages of Przewalski’s horse (Equus przewalskii). Animal Genetics 29, 456–459.

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C.P. Groves and O.A. Ryder Prothero, D.R. and Schoch, R.M. (1989) Classification of the Perissodactyla: summary and synthesis. In: Prothero, D.R. and Schoch, R.M. (eds), The Evolution of Periisodactyls. Oxford University Press, Oxford, pp. 530–537. Quinn, J. (1957) Pleistocene Equidae of Texas. Bureau of Economic Geology, University of Texas, Austin, Report no. 33, pp. 1–51. Rau, R.E. (1974) Revised list of the preserved material of the extinct Cape Colony Quagga, Equus quagga quagga (Gmelin). Annals of the South African Museum 65, 41–86. Rau, R.E. (1978) Additions to the revised list of preserved material of the extinct Cape Colony Quagga and notes on the relationship and distribution of southern Plains Zebras. Annals of the South African Museum 77, 27–45. Ryder, O.A. (1986) Genetic investigations: tools for supporting breeding programme goals. International Zoo Yearbook 24/25, 157–162. Ryder, O.A. and Chemnick, L.G. (1990) Chromosomal and molecular evolution in Asiatic wild Asses. Genetica 83, 67–72. Ryder, O.A., Epel, N.C. and Benirschke, K. (1978) Chromosome banding studies of the Equidae. Cytogenetics and Cell Genetics 20, 323–350. Sakagami, M., Hiromura, K., Chemnick, L.G. and Ryder, O.A. (1999) Distribution of the ERE-1 family in Perissodactyla. Mammalian Genome 10, 930–933. Skead, C.J. (1980) Historical Mammal Incidence in the Cape Province. 1. The Western and Northern Cape. Department of Nature and Environment Conservation, Provincial Administration of Cape of Good Hope, Cape Town. Smuts, M.M.S. and Penzhorn, B.L. (1988) Descriptions of anatomical differences between skulls and mandibles of Equus zebra and Equus burchelli from Southern Africa. South African Journal of Zoology 23, 328–336. Trumler, E. (1961) Entwurf einer Systematik der rezenten Equiden und ihrer fossilen Verwandten. Säugetierkundliche Mitteilungen 24, 206–218. Turnbull, P.F. 1986. Measurements of Equus hemionus from Palegawra Cave (Zarzian, Iraq). In: Meadow, R.H. and Uerpmann, H.-P. (eds), Equids in the Ancient World. Dr Ludwig Reichert Verlag, Wiesbaden, pp. 319–365. Xu, X. and Arnason, U. (1994) The complete mitochondrial DNA sequence of the horse, Equus caballus: extensive heteroplasmy of the control region. Gene 148, 357–362.

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Linkage and Comparative Maps for the Horse (Equus caballus) 1,2 and Ann T. Bowling2,3 J.D. Murray Linkage 10 James and and Comparative D. A.T.Murray Bowling Maps 1Department of Animal Science, College of Agricultural and Environmental Sciences; 2Department of Population Health and Reproduction, School of Veterinary Medicine; 3Veterinary Genetics Laboratory, University of California, Davis, CA 95616, USA

Introduction Linkage Maps Polymorphic genetic markers DNA polymorphisms Pedigreed families Synteny Comparative Maps The Horse Genetic Map Background The current status of the linkage map The current status of the synteny map The current status of the comparative gene map Conclusions Acknowledgements References

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Introduction Over the past two decades, the rapid and continual development of new tools based on recombinant DNA technology has greatly expanded opportunities to undertake genetic studies across a wide range of species. Prior to the development of highly polymorphic, rapidly scored DNA-based markers, the construction of genetics maps for most mammals was not feasible. Thus, construction of a genetic map for an animal such as the horse, with its long gestational period, late maturity and the production of single young, was severely constrained. The advent of polymerase chain reaction (PCR) technology and ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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automated DNA sequencers has provided a great technological boost to genetic mapping studies. For the first time it has become practical to screen horse DNA sequences directly so that polymorphisms in DNA can be assayed rapidly without reference to phenotype or the need for variation in protein sequences. Several different types of genetic maps now can be constructed for a species, with each yielding different, but overlapping, levels of resolution of the target genome. The principal mapping strategies being used are linkage, somatic cell hybrid (SCH) panels, in situ hybridization (physical mapping), DNA clone libraries and, ultimately, the DNA sequence. In horses, the current efforts are focusing on the first three of these strategies. Physical maps are discussed in Chapter 9 and will not be covered here. Construction of gene sequence by overlapping clones from DNA libraries will be likely to occur for selected genes of particular interest for horses, but has not yet been applied to horse gene mapping. In reality, complete DNA sequences will only be obtained for human and a few model species such as the mouse, and it is unlikely that the horse genome will be sequenced completely. To capitalize on the wealth of genomic information being developed for humans and the mouse, it will be necessary to construct a comparative gene map using the strategies listed above, and this effort is well underway for the horse.

Linkage Maps DNA provides the genetic blueprint for an organism in the form of genes that are contained within pairs of homologous chromosomes. Genes on the same chromosome are transmitted together into the next generation (linked) unless separated by a crossover event during gamete formation. During meiosis, homologous chromosomes undergo pairing and exchange material. Linkage maps, i.e. the order and relative distance apart of linked markers along a chromosome, are defined by the frequency of meiotic crossing over events that occur between markers, a concept first delineated by T.H. Morgan in the fruit fly Drosophila almost 90 years ago (Morgan, 1911). A linkage map shows the relative order and distance apart of markers along a chromosome in units called centiMorgans (cM). However, as recombination frequency along a chromosome, between chromosomes or between the sexes is not constant, centiMorgans do not translate into a set physical distance between markers. Generally, an assumption of approximately 1 ×106 bp of DNA cM−1, with an error of plus or minus a factor of two, is accepted. A species linkage map is a genetic map rather than a physical map of the order of genetic information along the length of each chromosome pair. Linkage maps are important as they establish marker (gene) order along a chromosome and predict the frequency with which alleles along a chromosome will be transmitted together to the next generation. At its simplest, the calculation of the recombination fraction between two or more markers is determined by dividing the number of recombinant

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gametes by the total number of gametes scored. However, in such a case, the linkage phase of the alleles of the markers along both homologous chromosomes in the parents must be known and the assumption must be made that no double crossovers occur between any two markers being analysed. In the horse, where half-sib mapping families have been used, the situation is more complex and the linkage phase usually is not known. Thus, complex mapping functions have been used based on maximum likelihood estimations of the fraction of gametes that are recombinant. Archibald and Haley (1998) have prepared an excellent review of linkage mapping considerations, including maximum likelihood methodology and the effects of typing errors, to which the reader is referred for an in-depth discussion. However, two points need to be mentioned here that are requirements for constructing a linkage map – genetic variation and pedigreed families.

Polymorphic genetic markers Study of genetic characteristics is dependent on the existence of genetic variation, or polymorphisms, between individuals. Polymorphisms can be observed in physical traits such as hair colour or markings, as electrophoretic mobility differences in proteins or as DNA sequence variants. Different forms of a single genetic marker are termed alleles and, while any individual can have only two alleles for a marker, one on each member of a chromosome pair, multiple alleles may exist in a population. Individuals are termed heterozygous for a genetic marker when they carry two different alleles at a genetic locus. The term heterozygosity is used to indicate the proportion of individuals within a population that may be expected to be heterozygous at a given locus. Linkage mapping is based upon the ability to follow through successive generations the segregation of, and crossing-over between, two or more polymorphic genes located on a pair of homologous chromosomes. Thus, linkage mapping is completely dependent on the identification of highly polymorphic markers in the parental individuals in a mapping pedigree so that there is a strong likelihood that they will be heterozygous at multiple loci along each chromosome. While genetic polymorphisms often exist in horse populations in the genes underlying physical traits such as coat colour, their use in mapping studies depends upon both the extent of heterozygosity and the ability to score each allele unambiguously. Alleles for coat colour genes are potentially useful as mapping markers, assuming appropriate families can be identified or created. However, for the most part, either the frequency of alleles for most visible traits is too low to be useful or the different genotypes arising from the different allele combinations are not distinctly different (e.g. heterozygous versus homozygous), and thus cannot be scored unambiguously. The importance of mapping of traits such as coat colour is that they are examples of coding genes, also called type I markers, and provide critical points for comparative mapping across species.

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Biochemical polymorphisms are a second class of potential markers for use in mapping studies. They are also type I markers. These protein-based polymorphisms are generally of two types, based either on electrophoretic mobility differences or antigenic determinant differences. Differences in the amino acid sequences of a protein between individuals reflect changes in the underlying genetic code of those individuals and thus represent allelic variation. Because biochemical polymorphisms are based on expressed gene sequences that are under natural selection to maintain function, the number of alleles and average heterozygosity are usually too low for these markers to be very useful in linkage mapping studies. However, when allelic variation is high, such markers do make good mapping markers and blood protein markers contributed substantially to the early linkage mapping efforts in the horse (reviewed in Bowling, 1996).

DNA polymorphisms With the advent of recombinant DNA technology, a number of types of DNA-based markers were developed. These include restriction fragment length polymorphisms (RFLPs) (Botstein et al., 1980), short tandem sequence repeat polymorphisms (microsatellites) (Weber and May, 1989), single-strand conformational polymorphisms (SSCPs) (Orita et al., 1989), single nucleotide sequence polymorphisms (SNPs) (Argüello et al., 1998), amplified fragment length polymorphisms (AFLPs) (Vos et al., 1995) and random amplified polymorphic DNA (RAPDs) (Williams et al., 1990). With the exception of RFLPs, these markers usually are based on anonymous DNA sequences and have been termed type II or non-coding markers. Type II markers may be highly polymorphic and thus can make excellent markers for linkage mapping within a species. While all of these types of polymorphism can be analysed using PCR-based methods, microsatellite markers have been the most commonly used markers for genome mapping because they are easily typed, very abundant and randomly dispersed throughout the genome. RAPDs are the least useful of the type II markers for linkage mapping, as they rely on the use of a single, short primer and are scored as a dominant marker. The principal limitation of type II markers is that they do not transfer across species well, particularly across the higher taxonomic divisions such as family or order level, and thus cannot be used to construct comparative maps. The reader is referred to Archibald and Haley (1998) for a more in-depth discussion of these markers, particularly RFLPs, microsatellites, SSCPs and AFLPs.

Pedigreed families Linkage mapping is based on the observed segregation of heterozygous genetic markers from an individual to his or her offspring. As noted above, the order and relative distance apart of linked markers along a chromosome are

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defined by the frequency of meiotic crossover, or recombination, events that occur between markers. Meiotic crossover events normally occur between one and four times in each pair of chromosomes, depending in part on the size of the chromosome pair. Large chromosome pairs undergo more crossovers per meiosis than do smaller chromosome pairs. Thus, the probability of a crossover occurring between two pairs of heterozygous markers on a chromosome pair increases as the physical distance between the markers increases. To view sufficient meiotic events to validate estimated recombination frequencies of the order of a few per cent requires the screening of large numbers of offspring. Thus, linkage mapping is completely dependent on being able to score recombination events between markers in the offspring of an individual heterozygous at several loci along the length of a chromosome. In the absence of special breeding programmes, the most easily obtained mapping populations for a large domestic animal such as the horse are half-sib families where many offspring are sampled from a single stallion. Such mapping families do not allow mapping of X chromosome-linked traits. They increase in statistical power if the linkage phase and maternal genetic component is known. An ideal mapping pedigree is one in which samples are available from three generations. Four widely genetically divergent individuals (the grandparents) would be mated to produce two individuals that are highly polymorphic at many loci, with in many cases known linkage phases, that would in turn be mated to produce a large number of offspring. In the case of the horse, both of the linkage maps currently available are based on stallion half-sib families (Lindgren et al., 1998; Guérin et al., 1999).

Synteny When markers are known to be located on the same chromosome, they are referred to as syntenic markers. While both linkage mapping and in situ hybridization mapping allow the identification of synteny groups, the most common method of identifying synteny groups is by using an SCH (somatic cell hybrid) clone panel produced by the fusion of primary cells from the species under study (the donor cell) with cells from a transformed cell line. In SCH cell lines, chromosomes from the donor cell line are lost randomly over time. In the case of the UC Davis horse ×mouse SCH panel, horse chromosomes were lost through 40–45 passages (Shiue et al., 1999). Synteny is determined by following the loss or retention together of associated markers across multiple, clonally derived SCH cell lines. Synteny does not rely on meiosis to provide information, so it is not necessary to construct and maintain an animal breeding programme. Unlike linkage mapping, synteny is not based on the incidence of genetic crossing over and does not normally yield any information concerning the order of markers linked on the same chromosome. However, in situations where fragments of chromosomes are retained in an SCH panel and heterozygous marker alleles can be scored, limited information on marker order can be deduced (Shiue et al., 2000). The extreme example of

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chromosome fragment retention is in the creation of a radiation hybrid SCH panel (for a review, see Stewart and Cox, 1997) where chromosomes are fragmented intentionally by radiation treatment prior to fusion of the donor and transformed cells. Chowdhary and Raudsepp (Chapter 9) provide more information on radiation hybrid and SCH panel mapping. Three horse–rodent SCH panels have been produced for use in identifying synteny groups in the horse genome (Williams et al., 1993; Bailey et al., 1995; Shiue et al., 1999). These panels, particularly as described by Shiue et al. (1999, 2000) and Caetano et al. (1999a, b, c), have been used to provide synteny relationships among horse markers prior to the availability of linkage mapping information. Synteny information is extremely valuable in the early stages of developing a genome map as both type I and type II markers can be typed easily, as markers are scored only as present or absent and thus do not depend on polymorphisms.

Comparative Maps Linkage mapping in mammalian species is dominated currently by the use of microsatellite markers. These markers are relatively abundant in any species, widely distributed throughout the genome, and a sufficient proportion of microsatellites has the high level of polymorphism required for linkage mapping. In addition, microsatellite markers are PCR based, thus lending themselves to automated, high-throughput screening. In the near future, a low to medium density microsatellite-based linkage map will be available for the horse, with markers on every chromosome. Such a linkage map will be useful for identifying markers for use in genome scans. However, the density of markers will be too low in most cases to allow a positional cloning approach to be effective for isolating a gene of interest. The alternative is to use a comparative or a modified comparative positional approach. Both of these latter two approaches for cloning a gene of interest require that a comparative gene map be developed for the horse. The value of a comparative map is based on the observation, first made by Ohno (1967) concerning the X chromosomes, that syntenic blocks of genes have been conserved throughout mammalian evolution (Wakefield and Graves, 1996). The degree of synteny conservation varies widely between species. For example, synteny blocks of genes equal to whole chromosome or chromosome arms in humans appear to have been retained in the cat, while in the mouse the same genes have been broken up into smaller groups and dispersed to several different chromosomes (O’Brien et al., 1997). Comparative gene maps are based on typing genes, as opposed to markers occurring in non-coding DNA, as they are under evolutionary constraints to be conserved in order to preserve gene function. However, the same constraints that conserve gene function also limit the amount of polymorphism found in such loci, thus making it more difficult to use coding loci as markers in the construction of linkage maps. The initial comparative maps for a species

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are often based on synteny maps that do not give gene order. When linkage maps incorporating several type I markers on a chromosome, or a radiation hybrid map become available for a species, it turns out that even though large blocks of genes have been conserved at the level of synteny, the order of those genes within the conserved segment has been changed by inversions (Schlapfer et al., 1997; Yang et al., 1998).

The Horse Genetic Map Background The domestic horse has 64 chromosomes, 31 pairs of autosomes and the X and Y chromosomes, giving 33 linkage groups. Nearly 75 traits have been identified as simple Mendelian genes documented with published segregation data. These traits include genes that control coat colour and pattern variation; erythrocyte and lymphocyte alloantigens; electrophoretically assayed serum, erythrocyte and lymphocyte protein variants; protein loci assayed with DNA probes; and diseases. Prior to 1990, only 25 genes were assigned in the horse to five separate autosomal linkage groups and the X chromosome. Allelic variation used to map these loci consisted of antigen epitopes for red blood cell alloantigens, electrophoretic variation of serum proteins and coat colour segregation. Linkage group II was the most extensive horse linkage group, containing eight genes coding for serum proteins, a mitochondrial enzyme and coat colour genes (RN, ALB, GC, TO, E, HP, ES and GOT2) (Sandberg and Juneja, 1978; Trommershausen-Smith, 1978; Andersson and Sandberg, 1982; Andersson et al., 1983b; Sponenberg et al., 1984; Weitkamp et al., 1985). Linkage group III included the major histocompatibility complex (MHC) which shares a conserved association of homologous genes among mammals (F13A, EAA, ELA-A, ELA-B, C4, 21-OH and ESCI) (Bailey et al., 1979; Lew et al., 1986; Bernoco et al., 1987; Kay et al., 1987, Weitkamp et al., 1989). Linkage group IV had three assigned genes (GPI, A1B and MEI) (Weitkamp et al., 1982; Andersson et al., 1983a) and linkage group V (EAU and PI) was composed of the blood group locus U and the serum protease inhibitor complex (Bowling, 1986). Two linkage groups had been localized to specific autosomes using in situ hybridization: linkage group III and IV were assigned to ECA20 (Ansari et al., 1988) and ECA10 (Harbitz et al., 1990), respectively. Comparative mapping in the horse began when coding genes first began to be mapped and linkage groups identified. For example, Sandberg and Juneja (1978) determined that ALB and GC were linked in the horse just as they are in humans, while Trommershausen-Smith (1978) demonstrated that ALB and a white spotting pattern (Tobiano, TO) were linked in the horse just as they are in the mouse. Andersson et al. (1983b) and Weitkamp et al. (1985) identified two additional genes in this horse linkage group (GOT2 and HP, respectively) that are also linked in the mouse but are not linked in humans.

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Williams et al. (1993) demonstrated two synteny groups in the horse, one (LDHB, PEPB and IGF-I) that contained three genes that are linked in humans, and a second group (NP, MPI and IDH2) that contained two genes that are linked in humans (MPI and IDH2) with a third gene (NP) that is not linked in humans. X chromosome linkage assignments (e.g. G6PD, HEMA) also appeared to be highly conserved in horses as well as in other mammals. Thus, the beginning of a comparative horse gene map demonstrated that, as in other mammals, there is considerable conservation of groups of linked genes in the horse. However, the number of mapped genes was too small to determine the degree of conservation. The current status of the linkage map Recently, two preliminary linkage maps have been published for the horse (Lindgren et al., 1998; Guérin et al., 1999) (Fig. 10.1, Tables 10.1 and 10.2). Both of these maps are male linkage maps based on stallion half-sib families and, thus, do not provide information for X-linked markers. The total number of mapped markers is less than 200, with fewer than 30 type I markers currently integrated into the horse linkage map. The few discrepancies in marker order and orientation of linkage groups between the two maps (e.g. ECA4, ECA10 and ECA21) will probably be resolved in subsequent studies. Lindgren et al. (1998) tested 140 markers, including 121 microsatellites. One hundred markers were placed into 25 linkage groups. Twenty two of the linkage groups could be assigned to 18 chromosomes, while three linkage groups could not be assigned to a chromosome. The average distance between markers in the linkage groups was 12.6 cM, with this map covering 679 cM. The international collaborative efforts of the international Equine Gene Mapping Workshop (EGMW) are described in Guérin et al. (1999). In Phase I, 161 markers were tested, of which 144 were microsatellites. In this effort, 124 markers were placed into 29 linkage groups that were assigned to 26 of the autosomes. One linkage group was not assigned to a chromosome. In this linkage map, the average distance between markers was 14.2 cM, with the total map distance covered being 936 cM. The linkage map of the horse is still in the preliminary development phase. The average total map distance in mammals is approximately 3000 cM, Fig. 10.1 (pp. 251–254). Linkage groups assigned to chromosomes according to the efforts of Lindgren et al. (1998) and the EGMW (Guérin et al., 1999). Markers in common to both maps are connected by lines. Markers in boldface have been FISHed to the location indicated. Numbers next to the vertical lines represent distances in cM between markers linked at odds greater than 1000 : 1. On the EGMW map, those markers to the left of the major vertical line could not be given a linear position at odds greater than 30 : 1. The thin vertical line indicates the range of their most likely position. Provided underneath each chromosome linkage sketch is additional information of human painting probes on horse (Zoo-FISH) (Raudsepp et al., 1996), other linkage and microsatellite and type I assignments from synteny, as detailed in Tables 10.1 and 10.2.

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Fig. 10.1a.

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Fig. 10.1b.

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Fig. 10.1c.

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Fig. 10.1d.

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although total map distance differs between the sexes, with males usually having a shorter total genetic map distance than females. Thus, these initial low-density linkage maps for the horse cover less than half of the map distance of the male linkage map and their principal use is to provide a framework for the addition of more markers.

The current status of the synteny map Three groups have contributed synteny information for the horse using horse × mouse somatic cell hybrid clones. In 1993, Williams et al. published three synteny groups based on eight type I loci mapped using isozymes and Southern blotting techniques. Bailey et al. (1995) provided data establishing six synteny groups based on 15 microsatellite and two type I markers. In both cases, linkage group or chromosomal assignments were not made. The UC Davis team has assigned more than 500 markers to 33 synteny groups using our horse ×mouse SCH panel, which includes RAPD, microsatellite and type I markers (Caetano et al., 1999a, b, c; Hopman et al., 1999; Murphie et al., 1999; Ruth et al., 1999; Shiue et al., 1999, 2000; Tallmadge et al., 1999; Bowling et al., 2000). Based on in situ hybridization studies of individual markers and Zoo-FISH (see Chapter 9), it has been possible to assign all synteny groups to chromosomes (Fig. 10.1, Tables 10.1 and 10.2). We assigned multiple microsatellite markers to each synteny group with the exception of the Y chromosome (Fig. 10.1) (Hopman et al., 1999; Murphie et al., 1999; Ruth et al., 1999; Shiue et al., 1999). Type I markers have been assigned to 31 of the 33 synteny groups, including those corresponding to the X and Y chromosomes, but not to the synteny groups corresponding to ECA27 and ECA30 (Fig. 10.1) (Caetano et al., 1999a, b, c; Bowling et al., 2000; Shiue et al., 2000). Early in the process of assembling genetic maps for a species, synteny mapping is a valuable approach in that neither extensive families nor high levels of polymorphism are required, thus allowing the assignment of coding genes. Basic information about which markers are physically linked on a chromosome is obtained, but not marker order. However, the use of labelled PCR microsatellite primers can allow alleles to be identified in SCH clones and thus haplotypes constructed based on the fragmentation and differential retention of segments of homologous chromosomes. Furthermore, if a male is used in SCH panel construction, fragmentation of the single X chromosome allows regional marker order to be determined. Using this principle, we have been able to determine marker order for four microsatellite markers and nine coding genes on the horse X chromosome (Shiue et al., 2000; Fig. 10.2). Thus, synteny mapping provides valuable information that can be used to verify and corroborate initial linkage assignments and aid in the development of radiation hybrid mapping panels. Finally, as it is possible to map large numbers of coding genes, or type I markers, synteny maps provide a mechanism for the development of comparative gene maps.

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Marti et al. (1998)

Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997)

A-14, ECA-3

HBA ASB19 HMS06, HTG09 ASB05 ASB04 ASB09 ASB12 HMS02, A1BG, ASB06

nt nt nt nt nt UCD13 UCD15 nt UCD09 nt UCD10 UCD19 UCD01 UCD10 nt nt WS16 WS12 WS20 WS13 WS15 nt nt nt nt nt nt WS10 04 nt nt nt nt 13 15 04 09 09 10 19 01 10

Binns et al. (1995) Swinburne et al. (1997) Swinburne et al. (1997) Swinburne et al. (1997) Swinburne et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997)

4 2 16 12 20 13 15 4 9 9 10 19 1 10

AHT06 (4) AHT12 (4) AHT14 (2) AHT17 (6) AHT18 (5) ASB01 (4) ASB02 (10) ASB03 (5) ASB04 (6) ASB05 (5) ASB06 (8) ASB07 (4) ASB08 (7) ASB09 (9)

EAQ

UCD06

U

[C]

Binns et al. (1995)

8

AHT05 (6)

Bowling et al. (1996); Shiue et al. (1999) Bowling et al. (1996); Shiue et al. (1999) PI, EAU

nt UCD24

nt WS24

06 U

Bowling et al. (1996); Shiue et al. (1999)

HTG06

Binns et al. (1995) Binns et al. (1995)

8 24

AHT03 (5) AHT04 (4)

Marti et al. (1998)

Other synteny and linkage references

AHT12, ECA-3

Other linkage or synteny

UCD01# UCD01# UCD01# UCD01# UCD01# UCD01# UCD01# UCD02* UCD26* UCD15

nt nt nt nt nt nt nt nt nt nt

nt nt nt nt nt nt nt 02 U 15

Millon et al. (unpublished observations) Millon et al. (unpublished observations) Millon et al. (unpublished observations) Millon et al. (unpublished observations) Millon et al. (unpublished observations) Millon et al. (unpublished observations) Millon et al. (unpublished observations) Marti et al. (1998) Marti et al. (1998) Binns et al. (1995)

1 1 1 1 1 1 1 2 26 15

1CA01 (5) 1CA12 (5) 1CA20 (3) 1CA24 (6) 1CA25 (4) 1CA30 (4) 1CA32 (4) A-14 (8) A-17 (8) AHT02 (5)

UCD synteny groupa

ECA (Lindgren et al., 1998)

EGMW (Guérin, et al., 1999)

Marker reference

ECA

Three hundred and thirty two microsatellites assigned to a chromosome by linkage or synteny (or both).

Locus (no. of alleles)

Table 10.1.

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ASB10 (6) ASB11 (6) ASB12 (5) ASB13 (8) ASB14 (8) ASB15 (5) ASB17 (10) ASB19 (9) ASB22 (9) ASB23 (7) ASB35 ASB36 ASB37 (6) ASB38 (5) B-8 (7) COR001 (4) COR002 (5) COR003 (8) COR004 (6) COR005 (2) COR006 (4) COR007 (9) COR008 (12) COR009 (2) COR010 (4) COR011 (6) COR012 (6) COR013 (4) COR014 (12) COR015 (9)

5 19 1 2 8 15 2 15 4 3 11 1 13 27 15 22 14 8 7 3 1 17 9 12 6 16 8 9 15 10

Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Irwin et al. (1998) Breen et al. (unpublished) Breen et al. (unpublished) Irwin et al. (1998) Irwin et al. (1998) Marti et al. (1998) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999)

05 19 01 02 06 15 02 15 04 03 11 01 U [B] U nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt

nt nt nt nt nt nt WS02 nt WS04 nt nt nt nt nt WS15 nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt

nt UCD19* nt UCD02* UCD06 UCD15 UCD02 nt nt nt nt nt UCD13* UCD-A* UCD15* UCD22+ UCD14+ UCD06+ UCD07+ UCD03+ UCD01+ UCD17+ UCD09+ UCD12+ UCD-D+ UCD16+ UCD06+ UCD09+ UCD15+ UCD10+

Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997) Breen et al. (1997)

Marti et al. (1998)

HMS05, HTG15, PGM ASB08 ASB02, ASB19 HTG06, ASB02, ASB15 HTG09, HTG07

ASB02

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Continued.

Marker reference

Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Hopman et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Murphie et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999)

ECA

22 27 25 6 10 29 22 5 24 24 2 29 3 20 12 27 17 3 4 2 1 2 31 16 27 2 15 2 19 10

Table 10.1.

Locus (no. of alleles)

COR016 (7) COR017 (9) COR018 (7) COR019 (2) COR020 (8) COR021 (3) COR022 (5) COR023 (4) COR024 (7) COR025 (3) COR026 (3) COR027 (8) COR028 (7) COR029 (4) COR030 (4) COR031 (6) COR032 (4) COR033 (11) COR034 (1) COR035 (1) COR036 (1) COR037 (5) COR038 (4) COR039 (1) COR040 (8) COR041 (9) COR042 (2) COR043 (2) COR044 (4) COR045 (8)

UCD synteny groupa UCD22+ UCD-A+ UCD25+ UCD-D+ UCD10+ UCD29+ UCD22+ UCD05+ UCD24+ UCD24+ UCD02+ UCD29+ UCD03+ UCD20+ UCD12+ UCD-A+ UCD17+ UCD03+ UCD04+ UCD02+ UCD01+ UCD02+ UCD31+ UCD16+ UCD-A+ UCD02+ UCD15+ UCD02+ UCD19+ UCD10+

EGMW (Guérin, et al., 1999) nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt

Other linkage or synteny

Other synteny and linkage references

258

nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt

ECA (Lindgren et al., 1998)

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COR046 (6) COR047 (3) COR048 (10) COR049 (2) COR050 (6) COR051 (2) COR052 (2) COR053 (7) COR054 (5) COR055 (9) COR056 (11) COR057 (6) COR058 (12) COR059 (4) COR060 (9) COR061 (9) COR062 (8) COR063 (1) COR064 (4) COR065 (8) COR066 (2) COR067 (1) COR068 (7) COR069 (6) COR070 (9) COR071 (7) COR072 (6) COR073 (8) COR074 (2) COR075 (11)

1 4 10 2 20 2 16 1 1 23 8 19 12 1 23 24 19 1 16 2 1 17 21 13 6 26 17 21 X 15

Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Ruth et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999)

nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt

nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt

UCD01+ UCD04+ UCD10+ UCD02+ UCD20+ UCD02+ UCD16+ UCD01+ UCD01+ UCD23+ UCD06+ UCD19+ UCD12+ UCD01+ UCD23+ UCD24+ UCD19+ UCD01+ UCD16+ UCD02+ UCD01+ UCD17+ UCD21+ UCD13+ UCD-D+ UCD26+ UCD17+ UCD21+ UCD-X+ UCD15+

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Bailey et al. (1995) Bailey et al. (1995)

260

HTG07, HTG09 ECA2, HMS07

UCD05 UCD04 UCD01 UCD19 UCD04 UCD01 UCD30 UCD04 WS05 U WS01 WS19 WS04 WS01 WS30 WS04 05 04 U nt nt nt 30 04

Guérin et al. (1994) Guérin et al. (1994) Guérin et al. (1994) Guérin et al. (1994) Godard et al. (1997) Guérin and Bertaud (1996) Godard et al. (1997) Godard et al. (1997)

5 4 1 19 4 1 30 4

HMS05 (3) HMS06 (5) HMS07 (6) HMS08 (5) HMS09 (3) HMS15 (8) HMS18 (4) HMS19 (3)

HTG04, HTG08 UCD09

WS09

09

Guérin et al. (1994)

9

HMS03 (7)

SG30, A1B

UCD10

WS10

10

Bailey et al. (1995); Bowling et al. (1996); Guérin et al. (1996); Godard et al. (1997) Guérin et al. (1996); Godard et al. (1997) Bailey et al. (1995); Bowling et al. (1996)

HTG06, SG06

Guérin et al. (1994)

10

HMS02 (8)

Bailey et al. (1995)

Other synteny and linkage references

HTG12, HMS07

Other linkage or synteny

UCD15+ UCD15+ UCD02+ UCD01+ UCD25+ UCD11* UCD26 UCD01 UCD02 UCD23* nt UCD14* UCD11* UCD18 UCD01* UCD15

nt nt nt nt nt nt nt nt nt WS23 WS01 WS14 nt nt nt WS15

nt nt nt nt nt 11 nt 01 02 nt nt nt nt U nt 15

Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Tallmadge et al. (1999) Marti et al. (1998) Gralak et al. (1994) Sakagami et al. (1995) Tozaki et al. (1995) Mathiason et al. (1999) Mathiason et al. (1999) Mathiason et al. (1999) Vega-Pla et al. (1996) Vega-Pla et al. (1996) Vega-Pla et al. (1996) Guérin et al. (1994)

15 15 2 1 25 11 26 1 2 23 1 14 11 18 1 15

COR076 (1) COR077 (2) COR078 (1) COR079 (4) COR080 (5) D-8 (5) EB2E8 (5) ECA-2 (3) ECA-3 (4) H13B2 H27A1 H4B2–2 HLM2 (4) HLM9 (3) HLM5 (3) HMS01 (4)

ECA (Lindgren et al., 1998)

UCD synteny groupa

Marker reference

ECA

Locus (no. of alleles)

EGMW (Guérin, et al., 1999)

Continued.

Table 10.1.

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Bailey et al. (1995)

HTG09, HMS06 HMS03, HTG04 HTG07, HMS06 ECA2, HMS07

UCD04 UCD09 UCD04 UCD21 UCD14 UCD01 UCD16 UCD22 UCD15 UCD18*

WS04 nt nt WS21 nt nt nt nt nt WS18

04 09 04 21 U 01 16 [A] 05 U

Marklund et al. (1994) Marklund et al. (1994) Marklund et al. (1994) Marklund et al. (1994) Marklund et al. (1994) Marklund et al. (1994) Marklund et al. (1994) Marklund et al. (1994) Marklund et al. (1994) Lindgren et al. (1999)

4 9 4 21 14 1 16 22 5 18

HTG07 (4) HTG08 (7) HTG09 (5) HTG10 (7) HTG11 (5) HTG12 (4) HTG13 (7) HTG14 (5) HTG15 (5) HTG17 (5)

TNF, DRA AHT02, HMS01

UCD20 UCD15

nt WS15

U 15

Ellegren et al. (1992) Ellegren et al. (1992)

20 15

HTG05 (7) HTG06 (4)

HMS03, HTG08

UCD09

WS09

09

Ellegren et al. (1992)

9

Marklund et al. (1994) Bailey et al. (1995); Guérin et al. (1996); Godard et al. (1997) Bailey et al. (1995); Bowling et al. (1996) Bailey et al. (1995) Bailey et al. (1995); Bowling et al. (1996); Guérin et al. (1996) Bailey et al. (1995) Bailey et al. (1995) Bailey et al. (1995) ALB, GC HTG13; HMS20

HTG04 (6)

Godard et al. (1997)

SG07

UCD04 UCD10 UCD17 UCD17 UCD20 UCD-A UCD18 UCD22 nt UCD16

WS04 U U WS17 nt WS-A WS18 WS22 nt nt

nt 10 nt nt nt [B] 18 22 03 U

Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1998) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Ellegren et al. (1992) Ellegren et al. (1992)

4 10 17 17 20 27 18 22 3 16

HMS22 (3) HMS23 (4) HMS25 (3) HMS41 (4) HMS42 HMS45 (3) HMS46 (4) HMS47 (5) HTG02 (3) HTG03 (5)

Guérin et al. (1996); Godard et al. (1997)

HTG03

UCD16

WS16

16

Guérin and Bertaud (1996)

16

HMS20 (8)

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Continued.

Marker reference

Lindgren et al. (1999) Lindgren et al. (1999) Lindgren et al. (1999) Marti et al. (1998) Marti et al. (1998) Caetano and Bowling (1998) Mathiason et al. (unpublished) Mathiason et al. (unpublished) Coogle et al. (1996a) Coogle et al. (1996a) Coogle et al. (1996a) Coogle et al. (1996a) Coogle et al. (1996a) Coogle et al. (1996a) Coogle et al. (1996a) Coogle et al. (1996a) Coogle et al. (1996a) Coogle et al. (1996a) Coogle et al. (1996b) Coogle et al. (1996b) Coogle et al. (1996b) Coogle et al. (1996b) Coogle et al. (1996b) Coogle et al. (1996b) Coogle et al. (1996b) Coogle et al. (1996b) Coogle et al. (1996c) Coogle et al. (1996c) Coogle et al. (1996c) Coogle et al. (1996c)

ECA

7 18 6 19 16 28 29 16 22 X 5 27 3 10 10 X 20 5 7 18 10 9 1 X 8 X 30 X X X

Table 10.1.

Locus (no. of alleles)

HTG20 (7) HTG28 (3) HTG31 (5) I-12 (10) I-18 (8) IGF1 (5) L12.2 L15.2 LEX02 (3) LEX03 (8) LEX04 (6) LEX05 (7) LEX07 (5) LEX08 (6) LEX09 (5) LEX10 (4) LEX11 (2) LEX14 (5) LEX15 (3) LEX16 (3) LEX17 (5) LEX19 (3) LEX20 (7) LEX22 (7) LEX23 (6) LEX24 (7) LEX25 (6) LEX26 (6) LEX27 (7) LEX28 (3)

UCD synteny groupa UCD07* UCD18* UCD-D* UCD19* UCD16* UCD-C UCD29* nt UCD22 UCD-X UCD05 UCD-A UCD03 UCD10 UCD10 UCD-X UCD20 UCD05 UCD07 UCD18 UCD10 UCD09 UCD01 UCD-X UCD06 UCD-X UCD30 UCD-X UCD-X UCD-X

EGMW (Guérin, et al., 1999) U U U nt nt nt U WS16 nt nt nt WS-A WS03 WS10 nt nt WS20 WS05 nt nt WS10 nt WS01 nt WS08 nt WS30 nt nt nt

Other linkage or synteny

Other synteny and linkage references

262

nt nt nt 19 16 nt nt nt U nt U [B] 03 10 10 nt U 05 07 18 10 09 01 U 06 nt U nt U nt

ECA (Lindgren et al., 1998)

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LEX29 (4) LEX30 (7) LEX31 (3) LEX32 (7) LEX33 (8) LEX34 (6) LEX35 (8) LEX36 (8) LEX37 (5) LEX39 (2) LEX40 (4) LEX41 (6) LEX42 (6) LEX43 (4) LEX44 (6) LEX45 (5) LEX46 (6) LEX47 (4) LEX48 (3) LEX50 (4) LEX51 (4) LEX52 (7) LEX53 (5) LEX54 (7) LEX55 (7) LEX56 (7) LEX57 (5) LEX58 (6) LEX59 (4) LEX60 (5)

8 1 21 24 4 5 19 19 21 1 19 13 24 14 26 7 15 14 16 4 15 20 23 18 17 16 3 1 16 21

Coogle et al. (1996c) Coogle et al. (1996c) Coogle et al. (1996c) Coogle et al. (1996c) Coogle et al. (1996c) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle et al. (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997)

06 01 21 U 04 05 19 nt U 01 19 nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt

WS08 nt nt WS24 WS04 WS05 WS19 WS19 U nt nt WS13 WS24 U U nt WS15 WS14 nt WS04 WS15 WS20 WS23 WS18 U WS16 WS03 WS01 U WS21

UCD06 UCD01 nt UCD24 UCD04 UCD05 UCD19 UCD19 UCD21 UCD01 UCD19 UCD13 UCD24 UCD14 UCD26 UCD07 UCD15 UCD14 UCD16 UCD04 UCD15 UCD20 UCD23 UCD18 UCD17 UCD16 UCD03 UCD01 UCD16 UCD21

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Continued.

Marker reference

Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1997) Coogle and Bailey (1999) Coogle and Bailey (1999) Coogle and Bailey (1999) Coogle and Bailey (1999) Coogle and Bailey (1999) Breen et al. (1994b) Breen et al. (1994b) Røed et al. (1997) Røed et al. (1997) Røed et al. (1997) Røed et al. (1997) Mathiason et al. (unpublished) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997)

ECA

4 10 23 20 6 10 11 9 16 19 20 10 19 10 9 22 1 23 15 18 12 12 11 21 21 10 3 22 15 11

Table 10.1.

Locus (no. of alleles)

LEX61 (6) LEX62 (4) LEX63 (4) LEX64 (5) LEX65 (5) LEX66 (4) LEX68 (7) LEX70 (6) MPZ002 (7) MPZ003 (4) NVHEQ05 (5) NVHEQ07 (7) NVHEQ11 (5) NVHEQ18 (11) PK9TET SGCV01 (2) SGCV02 (1) SGCV04 (4) SGCV06 (2) SGCV07 (5) SGCV08 (8) SGCV10 (6) SGCV13 (5) SGCV14 (3) SGCV16 (6) SGCV17 (2) SGCV18 (6) SGCV19 (4) SGCV21 (1) SGCV22 (1)

UCD synteny groupa UCD04 UCD10 UCD23 nt UCD-D UCD10 UCD11 UCD09 UCD16* UCD19 nt nt nt nt nt UCD22 nt UCD23 UCD15 UCD18 nt UCD12 UCD11 UCD21 UCD21 UCD10 UCD03 nt UCD15 UCD11

EGMW (Guérin, et al., 1999) nt WS10 WS23 WS20 nt nt nt nt nt nt WS20 WS10 WS19 WS10 WS09 U nt WS23 WS15 WS18 WS12 WS12 WS11 WS21 WS21 WS10 WS03 WS22 nt nt

Godard et al. (1997) Godard et al. (1997) Godard et al. (1997)

SG24

Other synteny and linkage references

HTG06, HMS01 HMS46

Other linkage or synteny

264

nt nt nt nt nt nt nt nt 16 nt nt nt nt nt nt [A] nt U 15 18 U U 11 21 21 10 03 22 nt nt

ECA (Lindgren et al., 1998)

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SGCV23 (8) SGCV24 (10) SGCV25 (5) SGCV28 (5) SGCV30 (5) SGCV31 (1) SGCV32 (3) SGCV35 UCDEQ005 (8) UCDEQ014 (4) UCDEQ046 (5) UCDEQ062 (5) UCDEQ136 (6) UCDEQ304 (6) UCDEQ380 (3) UCDEQ387 (5) UCDEQ405 (8) UCDEQ411 (6) UCDEQ412 (12) UCDEQ425 (10) UCDEQ428 UCDEQ437 (7) UCDEQ439 (5) UCDEQ440 (4) UCDEQ455 (4) UCDEQ457 (9) UCDEQ464 (7) UCDEQ465 (4) UCDEQ467 (8) UCDEQ482 (3)

4 11 1 7 10 X 8 19 27 17 8 11 18 5 2 18 25 12 10 28 X 3 11 1 30 11 25 6 24 10

Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Godard et al. (1997) Unpublished Eggleston-Stott et al. (1996) Eggleston-Stott et al. (1996) Eggleston-Stott et al. (1996) Eggleston-Stott et al. (1996) Eggleston-Stott et al. (1997) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1997) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1997) Eggleston-Stott et al. (1997) U67407 Eggleston-Stott et al. (1997) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1997) Eggleston-Stott et al. (1999)

04 11 nt 07 10 nt 06 nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt

U WS11 nt U WS10 nt WS08 nt WS-A WS17 WS08 WS11 WS18 WS05 WS02 WS18 WS25 WS12 WS10 U nt WS03 WS11 WS01 WS30 U WS25 U WS24 WS10

UCD04 nt UCD01 UCD07 UCD10 UCD-X UCD06 UCD19 UCD-A UCD17 UCD06 UCD11 UCD18 UCD05 UCD02 UCD18 UCD25 UCD12 UCD10 UCD-C UCD-X UCD03 UCD11 UCD01 UCD30 UCD11 UCD25 UCD-D UCD24 UCD10 Godard et al. (1997) Godard et al. (1997)

SG13 HMS02

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Continued.

Marker reference

Eggleston-Stott et al. (1997) U67418 Eggleston-Stott et al. (1999) Eggleston-Stott et al. (1997) Eggleston-Stott et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) Meyer et al. (1997) George et al. (1998) George et al. (1998) George et al. (1998) George et al. (1998) George et al. (1998) George et al. (1998) George et al. (1998) George et al. (1998) George et al. (1998) George et al. (1998) van Haeringen et al. (1994) van Haeringen et al. (1998) van Haeringen et al. (1998)

ECA

1 1 12 X 16 X 1 28 1 26 2 14 20 24 6 23 23 1 23 10 2 26 8 9 X 16 1 30 13 2

Table 10.1.

Locus (no. of alleles)

UCDEQ487 (7) UCDEQ493 UCDEQ497 (3) UCDEQ502 (10) UCDEQ505 (10) UM001 (6) UM002 (5) UM003 (4) UM004 (5) UM005 (6) UM007 (7) UM010 (5) UM011 (8) UM012 (5) UM015 (6) UM019 (3) UM022 (5) UM026 (4) UM027 (3) UM028 (2) UM029 (3) UM031 (5) UM033 (4) UM037 (5) UM038 (7) UM042 (4) UM043 (3) VHL020 (10) VHL047 (8) VHL078 (5)

UCD synteny groupa UCD01 UCD01 UCD12 UCD-X UCD16 UCD-X UCD01 UCD-C UCD01 UCD26 UCD02 UCD14 UCD20 UCD24 UCD-D UCD23 UCD23 UCD01* UCD23* UCD10* UCD02* UCD26* UCD06* UCD09* UCD-X* UCD16* UCD01* UCD30 nt nt

EGMW (Guérin, et al., 1999) WS01 nt WS12 nt WS16 nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt WS30 WS13 WS02

Other linkage or synteny

Other synteny and linkage references

266

nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt 30 U U

ECA (Lindgren et al., 1998)

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2 9 1 16 13 14 14 9 7 2 31 1 29 20

van Haeringen et al. (1998) van Haeringen et al. (1998) van Haeringen et al. (1998) Unpublished van Haeringen et al. (1998) van Haeringen et al. (1998) van Haeringen et al. (1998) van Haeringen et al. (1998) Ewen and Matthews (1994b) Ewen and Matthews (1994a) L10926 Ewen and Matthews (1994a) Ewen and Matthews (1994c) Ewen and Matthews (1995)

nt nt nt nt nt nt nt nt nt nt nt U U nt

U WS09 WS01 WS16 U U WS14 U nt nt nt WS01 U nt

UCD02* UCD09* UCD01* nt UCD13* UCD14* UCD14* UCD09* UCD07 UCD02 UCD-B UCD01 UCD29 UCD20

a UCD synteny group reference is Shiue et al. (1999), except for # (Millon et al., unpublished observations), +(in marker reference) or *(unpublished information). nt = not tested.

VHL123 (5) VHL126 (2) VHL134 (4) VHL145 VHL161 (3) VHL204 (2) VHL209 (7) VHL219 VIAS-H7 (12) VIAS-H17 (4) VIAS-H21 VIAS-H34 (7) VIAS-H39 (2) VIAS-H64 (8)

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nt nt UCD22 UCD03 UCD03 UCD14 UCD12 UCD-X UCD03 UCD-C UCD22 UCD05 UCD-X UCD07 nt UCD21 UCD14 UCD12 nt UCD10 UCD18 UCD-D UCD01 UCD10 UCD-X UCD20 UCD23

nt U nt nt nt nt nt nt WS03 nt nt nt nt nt nt nt nt nt U nt nt nt nt nt nt nt nt

nt 10 nt nt nt nt nt nt 03 nt nt nt nt nt nt nt nt nt U nt nt nt nt nt nt nt nt

DR PE DS DS DS DS DS DS PE DS DS DS DS DS DR DS DS DS PE DS DS DS DS DS DS DS DS

ECA 20 10 22 3 3 14 12 X 3 28 22 5 X 7 20 21 14 12 3 10 18 6 1 10 X 20 23

Gene

21-hydroxylase A1 B glycoprotein

Adenosine deaminase Alcohol dehydrogenase 2 Alcohol dehydrogenase 3 Adrenergic receptor, B2 Adrenergic receptor B kinase 1 Delta-aminolevulinate synthetase 2 Albumin

Arylsulphatase A Agouti signalling protein Antithrombin 3 Biglycan Complement component 3 Complement component 4 Complement component 9 Calmodulin-dependent protein kinase 4 CD20 antigen Carboxylesterase

Glycoprotein hormone A Cholinergic receptor Cholinergic receptor Chymase, mast cell Creatine kinase, muscle Chloride channel 4, voltage-gated Colipase Ciliary neurotrophic factor receptor

21-OH (CYP21) A1BG (XK)

ADA ADH2 ADH3 ADRB2 ADRBK1 ALAS2 ALB

ARSA ASP AT3 BGN C3 C4 C9 CAMK4 CD20 CES1 (ES)

CGA CHRNA CHRNG CHY CKM CLCN4 CLPS CNFTR

Type of ECA EGMW UCD synteny markera linkageb linkagec groupd

Kay et al. (1987) Weitkamp et al. (1982); Andersson et al. (1983); Guérin et al. (1996) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999b) Caetano et al. (1999c) Caetano et al. (1999c) Trommershausen-Smith (1978); Sandberg and Juneja (1978); Andersson and Sandberg (1982); Marklund et al. (1999) Caetano et al. (1999c) Bowling et al. (2000) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999a) Kay et al. (1987) Caetano et al. (1999b) Caetano et al. (1999b) Caetano et al. (1999c) Andersson and Sandberg (1982); Andersson et al. (1983) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Shiue et al. (2000) Caetano et al. (1999a) Caetano et al. (1999c)

Linkage or synteny references

268

ALB, GC, E, RN; GOT2

21-OH

GC; TO; ES, RN, E; KIT; PDGFRA

C4 ME1; GPI; HMS02

Other linkage reports

One hundred and thirty six genes assigned to horse chromosomes by linkage or synteny (or both).

Marker

Table 10.2.

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F13A F9 F18 FES FGFR3

CYP1A2 CYP2D DNAPK DRD2 EAA EAK EAQ EAU EDN1 EDN3 EDNRB ELA-DRA ELN EN2 ESR1 ETS2 EVI2A

COL9A1 COL10A CP CSF1R CSF2 CTLA3

Collagen 9 Collagen 10 Ceruloplasmin Colony-stimulating factor 1 receptor Colony-stimulating factor 2 Cytotoxic T-1-associated serine esterase 3 Cytochrome P450 Cytochrome P450 DNA protein kinase, catalytic subunit Dopamine receptor D2 Erythrocyte antigen A Erythrocyte antigen K Erythrocyte antigen Q Erythrocyte antigen U Endothelin 1 Endothelin 3 Endothelin receptor B Equine lymphocyte antigen Elastin Engrailed Oestrogen receptor Avian erythroblastosis virus homologue Murine myeloid leukaemia, human homologue Coagulation factor 13, A Coagulation factor IX Factor 18 Feline sarcoma Fibroblast growth factor receptor ELA, EAA

Weitkamp et al. (1989) Caetano et al. (1999c) Shiue et al. (2000) Caetano et al. (1999c) Caetano et al. (1999a)

nt UCD-X UCD-X UCD01 UCD03

nt nt nt nt nt

nt nt nt nt nt

PE DS DS DS DS

20 X X 1 3

EAA

ELA PGD AHT5 PI, AHT4

Caetano et al. (1999c) Caetano et al. (1999c) Bailey et al. (1997); Caetano et al. (1999a) Caetano et al. (1999c) Bailey et al. (1979) Sandberg (1974) Bowling et al. (1996) Bowling (1986); Bowling et al. (1996) Caetano et al. (1999c) Bowling et al. (2000) Bowling et al. (2000) Bailey et al. (1979); Bailey et al. (1995) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c)

UCD01 UCD-C UCD09 UCD07 nt nt nt nt UCD20 UCD22 UCD17 UCD20 UCD13 UCD04 UCD31 UCD26 UCD11

nt nt nt nt WS20 U U WS24 nt nt nt nt nt nt nt nt nt

nt nt nt nt U nt [C] nt nt nt nt nt nt nt nt nt nt

DS DS DP, DS DS RA RA RA RA DS DS DS LA, DS DS DS DS DS DS

1 28 9 7 20 2 8 24 20 22 17 20 13 4 31 26 11 HTG04, HTG08

Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999b) Caetano et al. (1999b) Caetano et al. (1999c)

UCD20 UCD10 UCD16 UCD14 UCD14 UCD21

nt nt nt nt nt nt

nt nt nt nt nt nt

DS DS DS DS DS DS

20 10 16 14 4 21

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Fibrinogen, gamma polypeptide Fibronectin Glucose-6-phosphate dehydrogenase Glucosidase B acid Group-specific component (vitamin D-binding protein) Growth hormone Growth hormone receptor Growth hormone-releasing hormone Galactosidase, B, 1 Guanine nucleotide-binding protein Glutamic acid transaminase, mitochondrial Glucose phosphate isomerase Glucose-regulated protein Glucuronidase B Haemoglobin alpha Haemoglobin beta Hexoseaminidase B Horse fatty acid-binding protein-like Haptoglobin Hyperkalaemic periodic paralysis Isocitrate dehydrogenase 2 Interferon alpha 1 Interferon beta 1 Interferon G Insulin-like growth factor Insulin-like growth factor receptor Insulin-like growth factor 2 Interleukin 1B Interleukin 7 receptor

FGG FN1 G6PD GBA GC (DBP)

GPI (PHI) GRP78 GUSB HBA HBB HEXB HFABP-L HP HYPP IDH2 IFNA1 IFNB1 IFNG IGF1 IGF1R IGF2 IL1B IL7R

GH GHR GHRH GLB GNAS1 GOT2

Gene

Continued.

Marker

Table 10.2.

Andersson et al. (1983) Caetano et al. (1999c) Caetano et al. (1999c) Breen et al. (1997) Caetano et al. (1999c) Caetano et al. (1999b) Caetano et al. (1999a) Weitkamp et al. (1985) Caetano et al. (1999a) Williams et al. (1993) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Williams et al. (1993); Caetano et al. (1999a) Caetano et al. (1999a) Caetano et al. (1999a) Caetano et al. (1999c) Caetano et al. (1999c)

UCD11 UCD21 UCD22 UCD16 UCD22 nt nt UCD25 UCD13 UCD13 UCD07 UCD14 UCD02 nt UCD11 nt UCD23 UCD23 UCD-D UCD-C UCD01 UCD12 UCD15 UCD21

nt nt nt nt nt nt U nt nt WS13 nt nt nt nt nt nt nt nt nt nt nt nt nt nt

nt nt nt nt nt nt nt nt nt 13 nt nt nt nt nt nt nt nt nt nt nt U nt nt

DS DS DS DS DS PE PE DS DS PE DS DS DS PE DS PS DS DS DS DR, DS DS DS DS DS

11 21 22 16 22 3 10 25 13 13 7 14 2 3 11 1 23 23 6 28 1 12 15 21 MPI, NP

270

ALB, GC

ASB01

A1BG

Caetano et al. (1999c) Caetano et al. (1999c) Trujillo et al. (1965); Shiue et al. (2000) Caetano et al. (1999c) ALB; ES, E, RN; KIT, Sandberg and Juneja (1978); Andersson and Sandberg (1982); Marklund et al. (1999) PDGFRA Caetano et al. (1999a) Caetano et al. (1999a) Caetano et al. (1999a) Caetano et al. (1999c) Caetano et al. (1999c) Andersson et al. (1983b) CES

UCD02 UCD-D UCD-X UCD05 nt

nt nt nt nt WS03

Linkage or synteny references

nt nt nt nt nt

Other linkage reports

DS DS PE DS PE

Type of ECA EGMW UCD synteny markera linkageb linkagec groupd

2 6 X 5 3

ECA

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UCD05 nt UCD04 UCD03 nt nt UCD18 UCD01 UCD11 UCD11 UCD18 UCD05 nt UCD02 UCD11 UCD-D UCD15 nt nt UCD-D nt nt nt UCD31 UCD-D UCD22 UCD07 UCD16 UCD17

nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt WS02 U WS24 nt nt nt nt nt nt

nt nt nt 03 nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt U 05 nt nt nt nt nt nt nt

DS PS DS DS, DP, PT PE PE, PS DS DS DS DS DS DS PS DS DS DS DS DR PS DS PE PE PE DS DS DS DS DS DS

5 28 4 3

Laminin C1 Lactate dehydrogenase B Leptin Melanocortin 1 receptor (extension) (chestnut) Malic enzyme Mannose phosphate isomerase Myostatin Myosin heavy 6 Myosin light 4 Neurofibromatosis 1 Nebulin Nerve growth factor B Nucleoside phosphorylase Natriuretic peptide precursor B Prolyl-4 hydroxylase B Paired box homeotic gene 3 Human paired domain Platelet-derived growth factor receptor Peptidase B Phosphofructokinase, muscle Phosphogluconate dehydrogense Phosphoglucomutase Protease inhibitor Plasminogen Melanocyte protein precursor Prion protein Parathyroid hormone Retinoic acid receptor B Retinoblastoma

LAMC1 LDHB LEP MC1R (MSHR) (E) ME1 MPI MSTN MYH6 MYL4 NF1 NEB NGFB NP NPPA P4HB PAX3 PAX8 PDGFA PEPB PFKM PGD PGM PI PLG PMEL17 PRNP PTH RARB RB1 10 1 18 1 11 11 18 5 1 2 11 6 15 3 28 6 2 5 24 31 6 22 7 16 17

nt

nt

nt

DP

3

Kit oncogene or stem cell factor receptor

KIT

DBP (GC), PDGFRA, Marklund et al. (1999) ALB Caetano et al. (1999c) Williams et al. (1993) PEPB Caetano et al. (1999a) Andersson and Sandberg (1982); Bowling ES, ALB, GC et al. (2000) Weitkamp et al. (1982) A1BG Williams et al. (1993) IDH2, NP Caetano et al. (1999a) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Williams et al. (1993) IDH2, MPI Caetano et al. (1999c) Caetano et al. (1999c) Bowling et al. (2000) Caetano et al. (1999c) GC (DBP), ALB, KIT Marklund et al. (1999) Williams et al. (1993) LDHB Caetano et al. (1999c) Sandberg (1974) EAK Breen et al. (1997) ASB10 Bowling (1986); Bowling et al. (1996) EAU, AHT4 Caetano et al. (1999a) Bowling et al. (2000) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999c) Caetano et al. (1999a)

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UCD-C UCD14 UCD15 UCD19 UCD-X UCD06 UCD04 UCD16 UCD09 UCD07 nt nt UCD06 UCD07 UCD25 UCD07 UCD23 UCD29 UCD-X UCD-X

nt nt nt nt nt nt nt WS16 nt nt nt nt nt nt nt nt nt nt nt nt

nt nt nt nt nt nt nt 16 nt nt nt nt nt nt nt nt nt nt nt nt

DS DS DS DS DS DS DS PE DS DS DS PT DS DS DS DS DS DS DS DS

28 14 15 19 X 8 4 16 9 7 20 3 8 7 25 7 23 29 X X

Stem cell factor (mast cell growth factor) Secreted protein, acid, cysteine-rich Surfactant-associated protein Somatostatin Steroid sulphatase Transcription factor 1 T-cell receptor G Transferrin Thyroglobulin Thy-1 cell surface antigen Tumour necrosis factor Tobiano Thymidylate synthetase Tubby Thioredoxin Tyrosinase Tyrosinase-related protein 1 Vimentin X inactivation-specific transcript Zinc finger protein-X

SCF (MGF)

DRA (ELA) ALB, GC

ALB, GC, E

Other linkage reports

Caetano et al. (1999b) Caetano et al. (1999c) Caetano et al. (1999c) Shiue et al. (2000) Caetano et al. (1999c) Caetano et al. (1999c) Bailey et al. (1995) Caetano et al. (1999c) Caetano et al. (1999c) Bailey et al. (1995) Trommershausen-Smith (1978) Caetano et al. (1999c) Caetano et al. (1999a) Caetano et al. (1999c) Bowling et al. (2000) Bowling et al. (2000) Caetano et al. (1999c) Shiue et al. (2000) Shiue et al. (2000)

Caetano et al. (1999c) Andersson and Sandberg (1982), Sponenberg et al. (1984) Bowling et al. (2000)

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272

U = unassigned; nt = not tested. a Type of marker and method of detection: DP, DNA polymorphism by sequencing or PCR and electrophoresis; DR, RFLP, by enzymatic digestion of DNA, electrophoresis and Southern blot; DS, synteny, using PCR primers to detect horse DNA sequence across an SCH; LA, lymphocyte antigen by serology; PE, blood protein polymorphism, by electrophoresis; PS, synteny, using protein electrophoresis on SCH; PT, physical trait, visually from phenotype; RA, red cell blood group, by antiserum. b Lindgren et al. (1998). Marker assignments in parentheses show linkage unassigned to chromosomes. c Guérin et al. (1999). d Caetano et al. (1999a, b, c); Shiue et al. (2000); Bowling et al. (2000).

SPARC SPTBN1 SST STS TCF1 TCRG TF TG THY1 TNFA TO TS TUB TXN TYR TYRP1 VIM XIST ZFX

UCD16 nt

nt nt

nt nt

DS PT

16 3

Rhodopsin Roan

RHO RN

Type of ECA EGMW UCD synteny markera linkageb linkagec groupd

ECA

Gene

Continued.

Marker

Table 10.2.

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Fig. 10.2. Order of four microsatellite markers (LEX003, LEX010, LEX024 and UCDEQ28), nine coding genes (ALAS2, BGN, CCLN4, F9, F19, G6PD, STS, XIST and ZFX) and one pseudogene (psALDH2) on the horse X chromosome as determined by breakpoints on X chromosome fragments retained in a somatic cell hybrid panel. Markers aligned with a horizontal line indicate the marker defining a breakpoint and are the last marker retained on the fragment when viewed from Xp to Xq. Genes listed between the horizontal lines and offset are located between breakpoint markers. F19, G6PD and BGN are distal to LEX003, but their order relative to each other is unknown. F19 and XIST have been ‘FISHed’ to proximal and distal Xq, respectively (Tozaki et al., 1998; Shiue et al., 2000). The figure is modified from Shiue et al. (2000).

The current status of the comparative gene map Chowdhary and Raudsepp (Chapter 9) present a summary of the results of Zoo-FISH studies, where human chromosome-specific probes were hybridized to horse metaphase chromosomes. This work was very important as the cumulative results determined the broad degree of chromosome conservation between the horse genome and that of humans. Too few coding genes have been placed on the horse linkage map to allow a meaningful comparative gene map to be compiled. However, the combination of genes mapped by linkage mapping and synteny allows for the development of a comparative gene map for the horse that covers all chromosomes except ECA27 and 30 and includes over 130 loci (Fig. 10.1). Caetano et al. (1999c) reported that the majority of gene assignments to horse chromosomes follow the location predicated by Zoo-FISH (Raudsepp et al., 1997). However, synteny mapping has allowed for two refinements of the Zoo-FISH comparative map. While the q arm of ECA13 hybridized with a HSA16 probe, the p arm did not paint with any human chromosome probe. Two genes mapped by synteny to ECA13 (ELN and GUSB) are orthologous to genes on HSA7, thus suggesting that part of ECA13 is orthologous to HSA7 even though HSA7-derived Zoo-FISH probes did not paint ECA13. Similarly, the synteny group assigned to ECA6 contains genes orthologous to genes

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located on HSA2, even though HSA2-derived painting probes did not hybridize to ECA6.

Conclusions The development of genetic maps of the horse genome is now underway, although only preliminary low marker density linkage maps with incomplete coverage currently are available. However, the data now available are encouraging, as they are evidence of a strong international collaborative effort being focused on the horse genome. There are abundant plans for future research directions, but the major limiting factor for horse gene mapping is insufficient funding. The use of SCH mapping panels and the development of radiation hybrid-SCH panels will greatly aid the continued development of the horse linkage map. Three or four evenly spaced type I markers need to be placed on each chromosome in the linkage map in order to orient markers and determine relative gene order for the comparative map. More microsatellite and type I markers must be developed. A high priority collaborative project is the integration of the different linkage maps. The culmination of the development of a medium-density microsatellite-based linkage map, i.e. a map with more than 1000 linked markers, will provide the resources to prepare marker panels for carrying out genome- and chromosome-specific scans in families segregating traits of interest. The availability of a good comparative gene map for the horse will then allow the candidate-positional cloning approach to identify the gene or genes responsible for the trait.

Acknowledgements We would like to thank all of our many collaborators who have generously provided us with PCR primer pairs for markers they have developed. We would also like to thank them for the interesting and stimulating conversations we have had during the course of our work on mapping the horse genome. Finally, special thanks go to our former PhD students, Drs A. Caetano and Y.-L. Shiue, for their hard work, enthusiasm and intellectual contributions to our programme. As university-based scientists, we need always to remember and appreciate the contributions of our students, who make it all worthwhile.

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J.D. Murray and A.T. Bowling Caetano, A. and Bowling, A.T. (1998) Characterization of a microsatellite in the promoter region of the IGF1 gene in domestic horses and other equids. Genome 41, 70–73. Caetano, A.R., Pomp, D., Murray, J.D. and Bowling, A.T. (1999a) Comparative mapping of 18 equine type I genes assigned by somatic cell hybrid analysis. Mammalian Genome 10, 271–276. Caetano, A.R., Lyons, L.A,. Laughlin, T.F., O’Brien, S.J., Murray, J.D. and Bowling A.T. (1999b) Equine synteny mapping of comparative anchor-tagged sequences (CATS) from human chromosome 5. Mammalian Genome 10, 1082–1084. Caetano, A.R., Shiue, Y.-L., Lyons, L.A., Laughlin, T.F., O’Brien, S.J., Murray J.D. and Bowling A.T. (1999c) A comparative gene map of the horse (Equus caballus). Genome Research 9, 1239–1249. Coogle, L. and Bailey, E. (1997) Equine dinucleotide repeat loci LEX049–LEX063. Animal Genetics 28, 378. Coogle, L. and Bailey, E. (1999) Equine dinucleotide repeat loci LEX064–LEX070. Animal Genetics 30, 71–72. Coogle, L., Bailey, E., Reid, R. and Russ, M. (1996a) Equine dinucleotide repeat polymorphisms at loci LEX002, -003, -004, -005, -007, -008, -009, -010, -011, -013 and -014. Animal Genetics 27, 126–127. Coogle, L., Reid, R. and Bailey, E. (1996b) Equine dinucleotide repeat loci LEX015–LEX024. Animal Genetics 27, 217–218. Coogle, L., Reid, R. and Bailey, E. (1996c) Equine dinucleotide repeat loci from LEX025 to LEX033. Animal Genetics 27, 289–290. Coogle, L., Reid, R. and Bailey, E. (1997) Equine dinucleotide repeat loci LEX034–LEX048. Animal Genetics 28, 309. Eggleston-Stott, M.L., DelValle, A., Bowling, A.T., Bautista, M., Zahorchak, R. and Malyj, W. (1996) Four equine dinucleotide repeats at microsatellite loci UCDEQ5, UCDEQ14, UCDEQ46 and UCDEQ62. Animal Genetics 27, 129. Eggleston-Stott, M.L., DelValle, A., Bautista, M., Dileanis, S., Wictum, E. and Bowling, A.T. (1997) Nine equine dinucleotide repeats at microsatellite loci UCDEQ136, UCDEQ405, UCDEQ412, UCDEQ425, UCDEQ437, UCDEQ467, UCDEQ487, UCDEQ502 and UCDEQ505. Animal Genetics 28, 370. Eggleston-Stott, M.L., DelValle, A., Bautista, M., Dileanis, S. and Wictum, E. (1999) Twelve equine dinucleotide repeats at microsatellite loci UCDEQ304, UCDEQ380, UCDEQ387, UCDEQ411, UCDEQ439, UCDEQ440, UCDEQ455, UCDEQ457, UCDEQ464, UCDEQ465, UCDEQ482 and UCDEQ497. Animal Genetics 30, 69–70. Ellegren, H., Johansson, M., Sandberg, K. and Andersson, L. (1992) Cloning of highly polymorphic microsatellites in the horse. Animal Genetics 23, 133–142. Ewen, K.R. and Matthews, M.E. (1994a) VIAS-H17 and VIAS-H34, two new polymorphic equine microsatellite loci. Animal Genetics 25, 63. Ewen, K.R. and Matthews, M.E. (1994b) Equine dinucleotide repeat polymorphism at the VIAS-H7 locus. Animal Genetics 25, 292. Ewen, K.R. and Matthews, M.E. (1994c) VIAS-H39, an equine tetranucleotide microsatellite repeat polymorphism. Animal Genetics 25, 433. Ewen, K.R. and Matthews, M.E. (1995) An equine microsatellite repeat at the VIAS-H64 locus. Animal Genetics 26, 282. George, L.A., Miller, L.M., Valberg, S.J. and Mickelson, J.R. (1998) Fourteen new polymorphic equine microsatellites. Animal Genetics 29, 469–470. Godard, S., Vaiman, D., Oustry, A., Nocart, M., Bertaud, M., Guzylack, S., Meriaux, J.-C., Cribiu, E.P. and Guérin, G. (1997) Characterization, genetic and physical mapping

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analysis of 36 horse plasmid and cosmid derived microsatellites. Mammalian Genome 8, 745–750. Godard, S., Schibler, L., Oustry, A., Cribiu, E.P. and Guérin, G. (1998) Construction of a horse BAC library and cytogenetical assignment of 20 type I and type II markers. Mammalian Genome 9, 633–637. Gralak, B., Coppieters, W. and Van de Weghe, A. (1994) Two new equine dinucleotide repeat microsatellites at the EA2C4 and EB2E8 loci. Animal Genetics 25, 285. Guérin, G. and Bertaud, M. (1996) Characterization of two polymorphic horse microsatellites, HMS15 and HMS20. Animal Genetics 27, 123. Guérin, G., Bertaud, M. and Amigues, Y. (1994) Characterization of seven new horse microsatellites, HMS1, HMS2, HMS3, HMS5, HMS6, HMS7 and HMS8. Animal Genetics 25, 62. Guérin, G., Bertaud, M., Nocart, M. and Meriaux, J.C. (1996) Linkage analysis between genetic markers in the horse. Animal Genetics 27 (Suppl. 2), 73. Guérin, G., Bailey, E., Bernoco, D., Anderson, I., Antczak, D.F., Bell, K., Binns, M.M., Bowling, A.T., Brandon, R., Cholewinski, G., Cothran, E.G., Ellegren, H., Förster, M., Godard, S., Horin, P., Ketchum, M., Lindgren, G., McPartlan, H., Mériaux, J.-C., Mickelson, J.R., Millon, L.V., Murray, J.D., Neau, A., Røed, K., Sandberg, K., Shiue, Y.-L., Skow, L.C., Stott, M., Swinburne, J., Valberg, S.J.,. Van Haeringen, H., Van Haeringen, W.A. and Ziegle, J. (1999) Report of the International Equine Gene Mapping Workshop: male linkage map. Animal Genetics 30, 341–354. Harbitz, I., Chowdhary, B.P., Saether, H., Hauge, J.G. and Gustavsson, I. (1990) A porcine genomic glucosephosphate isomerase probe detects a multiallelic restriction fragment length polymorphism assigned to chromosome 10pter in horse. Hereditas 112, 151–156. Hopman, T.J., Han, E.B., Story, M.R., Schug, M.D., Aquadro, C.F., Bowling, A.T., Murray, J.D., Caetano, A.R. and Antczak, D.F. (1999) Equine dinucleotide repeat loci COR001–COR020. Animal Genetics 30, 225–226. Irwin, Z., Giffard, J., Brandon, R., Breen, M. and Bell, K. (1998) Equine dinucleotide repeat polymorphisms at loci ASB21, 23, 25 and 37–43. Animal Genetics 29, 67. Juneja, R.K., Gahne, B. and Sandberg, K. (1978) Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum. Animal Blood Groups and Biochemical Genetics 9, 29–36. Kay, P.H., Dawkins, R.L., Bowling, A.T. and Bernoco, D. (1987) Heterogeneity and linkage of equine C4 and steroid 21-hydroxylase genes. Journal of Immunogenetics 14, 247–253. Lew, A.M., Bailey, E., Valas, R.B. and Coligan, J. (1986) The gene encoding the equine soluble class I molecule is linked to the horse MHC. Immunogenetics 24, 128–130. Lindgren, G., Sandberg, K., Persson, H., Marklund, S., Breen, M., Sandgren, B., Carlstén, J. and Ellegren, H. (1998) A primary male autosomal linkage map of the horse genome. Genome Research 8, 951–966. Lindgren, G., Persson, H. and Ellegren, H. (1999) Five equine dinucleotide microsatellite loci HTG17, HTG20, HTG21, HTG28 and HTG31. Animal Genetics 30, 70–71. Marklund, L., Johanssonn Moller, M., Sandberg, K. and Andersson, L. (1996) A missense mutation in the gene for melanocyte-stimulating homone receptor (MC1R) is associated with the chestnut coat colour in horses. Mammalian Genome 7, 895–899. Marklund, S., Ellegren, H., Eriksson, S., Sandberg, K. and Andersson, L. (1994) Parentage testing and linkage analysis in the horse using a set of highly polymorphic microsatellites. Animal Genetics 25, 19–24.

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J.D. Murray and A.T. Bowling Marklund, S., Moller, M., Sandberg, K. and Andersson, L. (1999) Close association between sequence polymorphism in the KIT gene and the roan coat colour in horses. Mammalian Genome 10, 283–288. Marti, E., Breen, M., Fischer, P., Swinburne, J. and Binns, M.M. (1998) Six new cosmid derived and physically mapped equine dinucleotide repeat microsatellites. Animal Genetics 29, 236–238. Meyer, A.H., Valberg, S.J., Hillers, K.R., Schweitzer, J.K. and Mickelson, J.R. (1997) Sixteen new polymorphic equine microsatellites. Animal Genetics 28, 69–70. Millon, L.V., Bowling, A.T. and Bickel, L.A. (1993) Fluorescence in situ hybridization of C3 and 18S rDNA to horse chromosomes. Proceedings of the 8th North American Colloquium on Domestic Animal Cytogenetics and Gene Mapping, Guelph, p.163. Morgan, T.H. (1911) Random segregation versus coupling in Mendelian inheritance. Science 34, 384. [reprinted in L. Levin, Papers on Genetics. Mosby, St Louis, 1971.] Murphie, A.M., Hopman, T.J., Schug, M.D., Aquadro, C.F., Bowling, A.T., Murray, J.D., Caetano, A.R. and Antczak, D.F. (1999) Equine dinucleotide repeat loci COR021–COR040. Animal Genetics 30, 235–237. O’Brien, S.J., Wienberg, J. and Lyons, LA. (1997) Comparative genomics: lessons from cats. Trends in Genetics 13, 393–399. Oakenfull, E.A., Buckle, V.J. and Clegg, J.B. (1993) Localization of the horse (Equus caballus) alpha-globin gene complex to chromosome 13 by fluorescence in situ hybridization. Cytogenetics and Cell Genetics 62, 136–138. Ohno, S. (1967) Sex Chromosomes and Sex-Linked Genes. Springer-Verlag, Berlin. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K. and Sekiya, T. (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proceeding of the National Academy of Sciences of the United States of America 86, 2766–2770. Raudsepp, T., Fronicke, L., Scherthan, H., Gustavsson, I. and Chowdhary, B.P. (1996) Zoo-FISH delineates conserved chromosomal segments in horse and man. Chromosome Research 4, 1–8. Raudsepp, T., Otte, K. and Chowdhary, B. (1997) FISH mapping of the IGF2 gene in horse and donkey – detection of homoeology with HSA11. Mammalian Genome 8, 569–572. Røed, K.H., Midthjell, L., Bjørnstad, G. and Olsaker, I. (1997) Equine dinucleotide repeat microsatellites at the NVHEQ5, NVHEQ7, NVHEQ11, NVHEQ18 and NVHEQ24 loci. Animal Genetics 28, 380. Ruth, L., Hopman, T.J., Schug, M.D., Aquadro, C.F., Bowling, A.T., Murray, J.D., Caetano, A.R. and Antczak, D.F. (1999) Equine dinucleotide repeat loci COR041–COR060. Animal Genetics 30, 320–321. Sakagami, M., Tozaki, T., Mashima, S., Hirota, K. and Mukoyama, H. (1995) Equine parentage testing by microsatellite locus at chromosome 1q2.1. Animal Genetics 26, 123–124. Sandberg, K. (1974) Linkage between the K blood group locus and the 6-PGD locus in horses. Animal Blood Groups and Biochemical Genetics 5, 137–141. Sandberg, K. and Juneja, R.K. (1978) Close linkage between the albumin and Gc loci in the horse. Animal Blood Groups and Biochemical Genetics 9, 169–173. Schlapfer, J., Yang, Y., Rexroad, C. and Womack, J.E. (1997) A radiation hybrid framework map of bovine chromosome 13. Chromosome Research 5, 511–519. Shiue, Y.-L., Bickel, L.A., Caetano, A.R., Millon, L.V., Clark, R.S., Eggleston, M.L., Michelmore, R., Bailey, E., Guérin, G., Godard, S., Mickelson, J.R., Valberg, S.J.,

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Murray, J.D. and Bowling, A.T. (1999) A synteny map of the horse genome comprised of 240 microsatellite and RAPD markers. Animal Genetics 30, 1–9. Shiue, Y.-L., Millon, L.V., Skow, L.C., Honeycutt, D., Murray, J.D. and Bowling, A.T. (2000) Synteny and regional marker order assignment of 26 type I and microsatellite markers to the horse X- and Y-chromosomes. Chromosome Research, in press. Sponenberg, D.P., Harper, H.T. and Harper, A.L. (1984) Direct evidence for linkage of roan and extension loci in Belgian horses. Journal of Heredity 75, 413–414. Stewart, E.A. and Cox, D.R. (1997) Radiation hybrid mapping. In: Dear, P.H. (ed.), Genome Mapping. IRL Press, New York, pp. 73–93. Swinburne, J.E., Marti, E., Breen, M. and Binns, M.M. (1997) Characterization of twelve new horse microsatellite loci, AHT12–AHT23. Animal Genetics 28, 453. Tallmadge, R.L., Hopman, T.J., Schug, M.D., Aquadro, C.F., Bowling, A.T., Murray, J.D., Caetano, A.R. and Antczak, D.F. (1999) Equine dinucleotide repeat loci COR061–COR080. Animal Genetics 30, 462–463. Tozaki, T., Sakagami, M., Mashima, S., Hirota, K. and Mukoyama, H. (1995) ECA-3: equine (CA) repeat polymorphism at chromosome 2p1.3–4. Animal Genetics 26, 283. Tozaki, T., Hirota, K., Mashima, S., Tomita, M. and Mukoyama, H. (1998) Cloning and characterization of the equine F18 gene, which has a novel exon. Animal Genetics 29, 381–384. Trommershausen-Smith, A. (1978) Linkage of tobiano coat spotting and albumin markers in a pony family. Journal of Heredity 69, 214–216. Trujillo, J.M., Walden, B., O’Neil, P. and Anstall, H.B. (1965) Sex-linkage of glucose-6phosphate dehydrogenase in the horse and donkey. Science 148, 1603–1604. van Haeringen, H., Bowling, A.T., Stott, M.L., Lenstra, J.A. and Zwaagstra, K.A. (1994) A highly polymorphic horse microsatellite locus, VHL20. Animal Genetics 25, 207. van Haeringen, W.A., van de Goor, L.H.P., van der Hout, N. and Lenstra, J.A. (1998) Characterization of twenty four equine microsatellite loci. Animal Genetics 29, 153–156. Vega-Pla, J.L., Garrido, J.J., Dorado, F. and de Andres-Cara, D.R. (1996) Three new polymorphic equine microsatellites: HLM2, HLM3, HLM5. Animal Genetics 27, 215. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23, 4407–4414. Wakefield, M.J. and Graves, J.A.M. (1996) Comparative maps of vertebrates. Mammalian Genome 7, 715–726. Weber, J.L. and May. P.E. (1989) Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. American Journal of Human Genetics 44, 388–396. Weitkamp, L.R., Guttormsen, S.A. and Costello-Leary, P. (1982) Equine gene mapping: close linkage between the loci for soluble malic enzyme and Xk (Pa). Animal Blood Groups and Biochemical Genetics 13, 279–284. Weitkamp, L.R., Costello-Leary, P. and Guttormsen, S.A. (1985) Equine marker genes: polymorphism for haptoglobin and assignment of the locus for haptoglobin to equine linkage group II. Animal Blood Groups and Biochemical Genetics 16 (Suppl. 1), 78–79. Weitkamp, L.R., Bailey, E., MacCluer, J.W. and Guttormsen, S.A. (1989) Polymorphism for equine F13A: linkage of F13A with ELA and A. Animal Genetics 20 (Suppl. 1), 10–11.

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J.D. Murray and A.T. Bowling Williams, H., Richards, C.M., Konfortov, B.A., Miller, J.R. and Tucker, E.M. (1993) Synteny mapping the horse using horse–mouse heterohybridomas. Animal Genetics 24, 257–260. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531–6535. Yang, Y.P., Rexroad, C.E., III, Schläpfer, J. and Womack, J.E. (1998) An integrated radiation hybrid map of bovine chromosome 19 and ordered comparative mapping with human chromosome 17. Genomics 48, 93–99.

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Genetics of Behaviour

K.A. Houpt Genetics 11 Katherine of and Behaviour R. Kusunose Albro Houpt1 and Ryo Kusunose2 1Animal Behaviour Clinic, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA; 2Japan Racing Association, JRA Equine Research Institute, 321–4, Togami-cho, Ustunomiya, Japan Introduction Learning Activity Patterns Feeding behaviour Sexual Behaviour Maternal and Foal Behaviour Maternal Abnormal maternal behaviour Foal Behaviour Temperament Aggression Breed differences in temperament Temperament tests Sire effects Abnormal Behaviours Breed and genetic differences in stereotypies Evidence for a hereditary basis of cribbing Other abnormal behaviour Conclusions References

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Introduction Equine behavioural genetics is in its infancy. At a time when a single gene can be ‘knocked-out’ of a mouse’s genome and the resulting behavioural aberration identified, we are still relying on pedigree information and breed differences to determine the heritable behaviours of horses. Nevertheless, what has been found so far gives us a tantalizing glimpse into the genetic and biochemical bases of behaviour. ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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Some years ago, Van Vleck (1990) hypothesized the probable ranges for heritability of several factors in horses. Weight and height may have heritabilities of 0.5–0.9, but gestation length is lower (0.25–0.40). The probable ranges for temperament scores are 0.20–0.45. These estimates are not based on data, but reflect what has been found in quantitative studies of other species. Behaviour depends on genetic and environmental factors, of course. The role of the genetic factors can be at any step in the animal’s perception and reaction to a stimulus. If the horse has a genetic problem with its rods as some Appaloosa horses do (Rebhun et al., 1984), it would not see the piece of paper blowing across the paddock at night. If his vision were normal, but he was suffering from one of the genetically determined myopathies, he could see the paper and be startled by it but might not be able to move (Valberg et al., 1996). In this chapter, we are most interested in what happens between perception and motor movement, but even here genes can be acting at many levels. A mutation in a gene controlling hippocampal development would affect learning ability (Zhao et al., 1999). An animal with an anatomically normal hippocampus, but with a mutation in the CREB gene would also have a learning disability (Kogan et al., 1996). Behaviour depends on the nervous system, and within the nervous system the communication between neurons depends on neurochemicals. There are genetic determinants of every step from formation of the neuron and its myelin through production of neurotransmitters and the enzymes that inactivate them to the receptors for the neurotransmitters, and the transcription factors necessary for production of the gene product. The neurochemicals or neurotransmitters which are most important are acetylcholine, dopamine, and serotonin. Serotonin has a generally calming action and is associated with satiety and non-aggressiveness. Serotonin is the neurotransmitter affected by anti-depressants, such as clomipramine (Clomicalm®) and the specific serotonin re-uptake blockers such as fluoxetine (Prozac®). Dopamine is associated with rewarding behaviour and with coordinated activity. The clinical application is that acepromazine is a dopamine receptor antagonist and amphetamine is a dopamine agonist. Acetylcholine has been implicated in learning in the central nervous system (CNS), in addition to its autonomic and peripheral nervous system activities. Although most behaviour is a product of the CNS, hormones and other non-neural factors affect the nervous system and, therefore, affect behaviour. For this reason, there is unlikely to be a gene for maternal aggression or sexual behaviour, but rather a series of genes for these quantitative traits. Our approach is to present genetic or breed differences in behaviour of other domestic and laboratory species to indicate what might apply to horses. When available, examples of breed and genetic differences in horses will be given. Emphasis will be given to temperament tests and genetic influences on equine temperament. The best examples of the genetic basis of behaviour are found in mice because this species has high-resolution genetic maps, highly polymorphic markers and availability of inbred strains of genetically identical animals. The

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behaviour of transgenic and knockout mice can be assessed in valid and reliable behavioural tests. As a baseline, food and water intake, sexual cycles and activity are measured. An even more exciting approach is a transgenic animal with a receptor gene driven by the receptor gene promoter of another species with a different social system (Young et al., 1997). Even in inbred strains of mice, subtle environmental differences between laboratories influence the results of behavioural tests (Crabbe et al., 1999).

Learning There are marked breed differences in learning ability and in learning particular tasks (Hart and Hart, 1985). In contrast with dogs which have been selected for trainability, pigs have not been selected for learning ability. Nevertheless, there are breed differences in learning ability. Durocs learn more quickly than Hampshires, and Yorkshires learn more quickly than Poland– China (Wieckert and Barr, 1966; Kratzer, 1969). Breed differences in learning ability of horses have been found by several authors. Mader and Price (1980) taught Quarter Horses (n = 10) and Thoroughbreds (n = 11) to make visual discriminations for a food reward by pushing one of three doors behind which was sweet feed (molasses, corn and barley). Only the correct door was unlocked. The first task was to distinguish the door with a checkerboard card on it from the two doors with plain white cards on them. If the horses chose correctly 80% of the time they moved on to the next test in which they were rewarded for choosing the door with the checkerboard rather than a door with either a heart or a horse silhouette. Finally, they had to choose the door with the original checkerboard pattern from two doors with a different checkerboard pattern (larger blocks). In that study, Quarter Horses learned faster than the Thoroughbreds. All (nine of nine) Quarter Horses reached criteria on the first and second task, but only seven of ten Thoroughbreds. The Quarter Horses took 5.4 ± 1.2 days to reach criteria on the first test; the Thoroughbreds took 8.4 ± 2.7 days. The authors hypothesized that the Thoroughbreds were more reactive to extraneous stimuli such as noises or the confinement of the stall in which the testing was done and were, therefore, distracted from the learning task. Another interpretation could be that the Thoroughbreds were less hungry. Lindberg et al. (1999) found that warmblooded horses learned an operant less quickly than coldblooded horses. Neither type of horse seemed able to learn by observation of other horses. Wolff and Hausberger (1996) performed the only test of paternal effects on learning ability of horses. Offspring of stallion A and of stallion B were compared on two learning tasks. When tested for their ability to learn to open a wooden chest, the offspring of stallion A were slower to learn than those of other stallions. More offspring of stallion B were successful in a spatial learning test in which the horse had to solve a detour problem in which food was located inside an electric fence with a gate on the side opposite to the horse’s initial position. On tests of their reaction to novel objects, the offspring of

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sire A were more emotional, which supports the hypothesis that emotional responses interfere with learning and may be responsible for the breed differences noted above.

Activity Patterns Activity patterns or time budgets of the horse appear to vary more with the environment than with the breed of horse. There have been a large number of studies of activity patterns or time budgets of horses. What is most impressive is that horses spend a large percentage of their time grazing – more time than is spent by any other domestic ungulate. The percentages vary from 92% of the evening hours by free-ranging Equus caballus in Alberta, Canada (Salter and Hudson, 1979) to 25% of the daylight hours by free-ranging horses in Colorado, USA (Berger, 1977). In general, horses spend 50–60% of their time feeding. This is true of stabled horses when hay is available ad libitum (Sweeting et al., 1985). Studies of the Przewalski’s horse (Equus ferus przewalskii) have revealed very similar feeding patterns (Boyd et al., 1988; Berger et al., 1999). Horses, as a species, show similar grazing behaviour, but there are subtle differences in selectivity among horses (Marinier and Alexander, 1992). These individual differences have survival value because more selective horses avoid poisonous plants and, therefore, are more likely to reproduce. A genetic basis should be sought.

Feeding behaviour There are breed differences in susceptibility to obesity – the Thelwellian Shetland pony or the Belgian can be compared with the Thoroughbred, but the genetic basis of appetite and controls of intake have been studied most thoroughly in rodents. The Zucker fatty rat (formerly fa/fa, currently the gene renamed as Lepr, leptin receptor) and the obese mouse (formerly ob/ob, currently the gene renamed as Lep) are good examples (Truett et al., 1995). The obese mouse does not produce the normal Lep gene product, leptin, which is a feedback signal from the fat to the brain which normally inhibits the neurotransmitter neuropeptide Y (NPY) (a stimulant of food intake) and increases energy loss though sympathetic activation (Baskin et al., 1999).

Sexual Behaviour There are genetic differences in sexual behaviour. For example, strains of chickens have been developed which have high and low breeding capacities (Cook and Siegel, 1974). Identical twin bulls had nearly identical scores on a sexual capacity test on mounting, mounts with intromission and complete services (Bane, 1954). There are marked differences between Bos taurus and

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Bos indicus; the latter exhibit much lower libido (Chenoweth and LandaetaHernandez, 1998). Breed or other genetic differences in libido have not been described for horses, but are probably there. Owners of a breeding stallion are reluctant, understandably, to admit that he might have low libido or has difficulty intromitting or ejaculating. Nitric oxidase synthetase knock-out mice are not only aggressive, but also have abnormal sexual behaviour in that they will copulate with a female whether or not she is receptive (Nelson et al., 1995). A good example of genetic effects on sexual behaviour are the two oestrogen receptors – alpha and beta. If the gene for female oestrogen receptor β is knocked-out, sexual behaviour is normal in females although their ovarian efficiency is diminished, but knock-out of the oestrogen receptor α results in females who do not show normal receptive behaviour. They are also deficient in maternal behaviour – failing to retrieve pups and biting the pups (Ogawa et al., 1996). There may be breed and heritable differences in equine female sexual behaviour. For example, we have noted that Standardbreds are more likely to exhibit ‘yawing’ behaviour when in oestrus than Arabians. Some oestrus mares respond to stallion vocalization by squatting and urinating as they would in response to a stallion (Veeckman and Ödberg, 1978), other mares do not (McCall, 1991). This could be due to a genetic difference.

Maternal and Foal Behaviour Maternal A null mutation of the prolactin receptor gene produces a defect in maternal behaviour in mice as well as deficits in learning. Prolactin appears to stimulate nest building in sows (Castrén et al., 1993; Lucas et al., 1998) and is necessary for normal lactation. Its role in maternal behaviour of follower (non-nest builders) species such as horses is unknown. Recently, the Mest gene (LeFebvre et al., 1998), has been identified as important in maternal behaviour because deletion of the gene in mice resulted in lack of maternal behaviour and lack of placentophagia, a normal component of maternal behaviour in the mouse. Examination of the equine genome and comparison of the genes of rejecting and non-rejecting mares might be worthwhile. The physiology of maternal behaviour involves a variety of hormones, experience as a mother, hereditary tendencies and the stimulus of the neonate (Keverne et al., 1983; Kendrick and Keverne, 1991; Kendrick et al., 1992; Houpt, 1998). Sheep have been investigated in more depth than any other domestic species; therefore, the physiological basis of maternal behaviour in ewes will be described. Maternal behaviour can be induced in multiparous, non-pregnant ewes by a regime of decreasing progesterone, increasing oestrogen and vaginal–cervical stimulation (Kendrick and Keverne, 1991). Vaginal– cervical stimulation, which normally would be caused by the passage of the

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lamb through the birth canal, causes oxytocin release via spinal afferents with neural connections to the hypothalamus. The cells that produce oxytocin are located in the hypothalamus. These cells have axons that carry the hormone either to the posterior pituitary where it is released into the peripheral blood stream or to other parts of the brain, including the olfactory bulb. In the olfactory bulb, oxytocin stimulates the release of monoamines and opiates which in turn initiate a sensitive period during which the ewe will identify the smell of the lamb as her own. The period during which a ewe will form a bond with a specific lamb is about 24 h. Exposure to the lamb for only a few minutes is necessary (Kendrick and Keverene, 1991) for permanent recognition of the lamb. There are breed differences in bovine maternal behaviour. Angus are more defensive of their calves than Hereford or Charolais. Sheep also show breed differences in maternal behaviour; Merinos are less maternal than Dorsets or Romneys (Alexander et al., 1983). The behaviour of mares toward their foals seems to be independent of breed and is exhibited to horse foals, to mule foals and even to foals born to mules by embryo transfer (Shaw and Houpt, 1985). Nevertheless, there are individual differences in maternal behaviour. These differences persist from foal to foal and indicate that there may be genetic differences (Crowell-Davis, 1986). There are variations in equine maternal behaviour within the normal range. Individual differences in mare–foal distance have been noted by Kusunose and Sawazaki (1984a). Crowell-Davis (1985) found that some mares maintained much closer proximity to their foals than others. This was true over several foaling seasons, so it was a maternal not an offspring attribute.

Abnormal maternal behaviour Not all mares exhibit normal maternal behaviour. Some reject their foals. Foal rejection can take several forms. In the mildest form, the mare accepts and licks the foal, but will not allow it to nurse. In another form, the mare will have nothing to do with the foal and may kick it if it approaches too closely. The most severe form is the mare who attacks the foal, attempts to bite its neck and throw it. The behaviour is similar to that of a stallion repulsing a male challenger or driving a male offspring out of his band. All of these forms of rejection are most likely to occur in primiparous mares, an indication that maternal experience is important. Apparently, there is a learning component even to this innate behaviour. Nevertheless, some mares will reject one foal after another. Two findings led us to investigate the genetic aspects of this behaviour. Matoock (1992) noted that nine mares of 107 Arabian horses at the Egyptian Agricultural Organization (EAO) rejected or partially rejected their foals. The mares that rejected were all from one of several lines kept at the EAO.

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The other finding was the result of a request for information from owners of foal-rejecting mares. The request and a sample history form were published in a variety of equine magazines (Equus, Compendium of Continuing Education, Horse and Rider, The Bloodhorse, Equine Practice, etc.). Of the 135 cases of foal rejection reported, 70 (52%) were Arabian, 24 were Quarter Horses, and 14 were Thoroughbreds. Most (101) of the mares were primiparous. Rejection occurred immediately after parturition by 95 mares, 12 h after parturition by 11, and 24 h after parturition by 14. Ten foals were killed or died as a result of rejection (Houpt and Lieb, 1994). No firm conclusion could be drawn from non-randomly collected data because Arabian owners might have been more willing to fill in a form or less able to cope with a behaviour problem than owners of other breeds. To address the question of breed predisposition more accurately, the cooperation of the Arabian Horse Registry of America and The American Paint Horse Association was obtained. Letters were sent to 800 owners of foals of each breed born in 1993. The owners were asked to indicate on a stamped, self-addressed postcard whether the mother of the foal they registered had rejected it. If a response was not received within 3 weeks, a reminder postcard was sent. If a response was not received within another 3 weeks, a second letter containing another stamped, self-addressed postcard was sent. Responses were obtained from 720 Arabian owners (90%) and 657 Paint horse owners (82%). Five per cent of Arabian foals had been rejected, but only 2% of Paint foals. That is a significant breed difference (χ2, P < 0.001). The pedigree of each rejecting Arabian mare and of two non-rejecting mares whose foals had the closest registration number to the rejected foals were examined. Two sires (father and son) were found to be over-represented in the pedigrees of rejecting mares in comparison with those of non-rejecting mares (Juarbe-Díaz et al., 1998). The sires were Egyptian Arabians of the line noted to reject by Matoock (1992). Twenty-one Thoroughbred farms were surveyed in 1992 and 1993 in Japan. The incidence of foal rejection was 0 of 371 foaling in 1993 and 0.5 (one of 411) in 1994 (Juarbe-Díaz et al., 1998). Studies in sheep indicate that there should be a physiological basis for abnormal maternal behaviour. In order to determine if either oestrogen or progesterone were involved, blood samples were taken from Arabian mares at the EAO 30 min before foaling and 15, 30, 60 and 90 min after foaling. The levels of both hormones were lower in the four mares who rejected. The pre-foaling progesterone levels were significantly lower.

Foal behaviour There are breed differences in development of foals. These are most pronounced in the first few hours of life. Pony foals stand and suckle in their first hour of life. Thoroughbreds need another 30 min. Saddlebred foals are intermediate (Rossdale, 1967; Waring, 1982). These differences may be due to the size of the foal or to the relationship between leg length and total height

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which may handicap Thoroughbreds. In addition to these genetic differences, the earlier birth dates of most Thoroughbreds also puts them at a disadvantage. We have observed Thoroughbred foals in Ithaca, New York, where winter temperatures are lower than in the UK and where foals do not stand or suckle for 3 h. Foal behaviour does not differ markedly between breeds of horses or even between species of equids. New Forest ponies (Tyler, 1972), English Thoroughbreds (Carson and Wood-Gush, 1983), Japanese Thoroughbreds (Kusunose and Sawazaki, 1984b), Welsh ponies (Crowell-Davis, 1985), Belgians (Barber and Crowell-Davis, 1994), feral horses (Berger, 1986), Przewalski’s horses (Boyd, 1988), donkeys (Moehlman, 1998) and mules (SmithFunk and Crowell-Davis, 1992) all suckle two to five times an hour during the first few weeks of life. The duration is approximately 1 min. The mule foals suckled most frequently, possibly a sign of hybrid vigour. The donkey foals suckled least in the arid environment of Death Valley, but at a rate comparable with E. caballus in the more hospitable climate of Ossabaw Island. Foals, for example Przewalski foals, are recumbent 35% of the day, domestic Welsh pony foals for 37%. Przewalski’s stand rest for 6% of the time, Welsh pony foals for 4% (Boyd et al., 1988). Grazing behaviour is also similar, slowly rising to adult levels by 6 months of age (Crowell-Davis et al., 1985; Boy and Duncan, 1979). Drinking water is more dependent on the environment. Foals on lush pasture do not drink, but those in the desert of Wyoming do (Boyd, 1980). Paternal effects on foal behaviour Evidence for a paternal effect on foal behaviour was found by Wolff and Hausberger (1994), who studied 13 suckling French Saddlebred foals sired by five stallions. They found that the foals of one sire rested more, both stand resting and lying laterally, than those of other sires. The foals of another sire suckled more, approached their dams more often and spent more time within 10 m of their dams. The foals of another sire played more. The small numbers make it difficult to draw definite conclusions, but indicate that there are genetically determined differences in temperament. The use of suckling foals removes many of the environmental (training and use) influences on behaviour.

Temperament Aggression In the case of aggression, there are many candidate genes: monoamine oxidase A in humans (Brunner et al., 1993) and mice (Cases et al., 1995), serotonin 5-HT1B receptor in mice (Saudou et al., 1994) and α-calmodulin-dependent protein kinase II (CaMKII) in mice. This enzyme is required for activation of tryptophan hydroxylase which is the rate-limiting step in serotonin synthesis.

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In humans, a genetic mutation in the gene for tryptophan hydroxylase is associated with aggression (Manuck et al., 1999). Transgenic mice overexpressing transforming growth factor-α (TGF-α) are also aggressive, but the relationship of this to neurotransmitter or brain lesions is unknown (Hen, 1996). Finally, nitric oxide is involved in neural events throughout the body and, as a result, affects aggression. Knock-out mice, lacking the neural form of nitric oxide synthetase, are aggressive (Nelson et al., 1995). There are differences in aggressiveness among breeds of cattle. Angus are more aggressive than Hereford, which is reflected in dominance relations. Ayrshires are dominant over Holsteins. The heritability of dominance in cattle is calculated to be 0.4–0.5. Unfortunately, no one has examined dominance in enough horses to be able to calculate heritability. Several studies have indicated that foals of dominant mares are dominant (Houpt and Wolski, 1980). This could be learned or inherited, but the dominance rank of pony foals weaned shortly after birth is not related to that of their mothers (Houpt et al., 1982).

Breed differences in temperament We are all aware that horse breeds differ in temperament and, although some of this depends on the training and use to which the horses are put, other differences are genetic. Table 11.1 lists the character of ten horse breeds according to one author (Hermsen, 1997). There are no breed standards for temperament, although some breeds are scored for temperament in the show ring. The temperament a Table 11.1.

Breed temperament (from Hermsen, 1997).

Breed

Temperament

Appaloosa

They have tremendous stamina and are docile and very tough. They are willing and good-natured They are exceptionally intelligent with tremendous stamina. They are also renowned for their spirited character Miniatures enjoy human companionship. They are mischievous and playful, and learn quickly Spirited, intelligent and easily managed; courageous and willing They are generally intelligent with a first class temperament Generally have an excellent temperament and are very intelligent. They are quite calm and reliable A superior riding horse with an exceptionally friendly nature They have an excellent sportive temperament and considerable stamina They are very intelligent horses with a fine character

Arabian Miniature Horse Morgan Paint horse Quarter Horse American Saddlebred Standardbred Tennessee Walking Horse Thoroughbred

Thoroughbred are renowned for their brave, almost fearless, and lively nature

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breed should have and the temperament perceived by those who interact with the horses may be different. A survey of 50 trainers and veterinarians was carried out by Karen Hayes (1998). She asked for comments concerning ten breeds on the following six characteristics (see Table 11.2). 1. Training (ability to learn new tasks, such as leading, backing, giving to the bit, presenting a foot for farriery, submitting to non-painful but invasive procedures such as nasogastric intubation or palpation, trailer loading, etc.) 2. Work ethic (willingness to perform repetitive work or tasks already welllearned, stamina in the face of mild fatigue) 3. Temperament (in general, as when approached in pasture or in a stall, behaviour with other horses in a group, response to non-equine animals such as dogs and cats, behaviour with children, etc.) 4. When asked to do something he does not want to do (e.g. walk through water, cross a footbridge, enter stocks, submit to non-painful but invasive treatment such as nasogastric intubation or palpation, hold foot up for farriery, etc.) 5. Fearfulness and flight impulse (in response to benign stimuli such as unexpected noise, a cat dropping off a nearby ledge, a bird flushed from a nearby bush, etc.) 6. Self-protection impulse (chiefly in response to painful stimuli, such as vet ministrations or intramuscular injections). This survey is based on subjective opinions of equine professionals, but is probably a fairly accurate predictor of behaviour of the various breeds.

Temperament tests There are a variety of equine temperament tests. Most have been performed on only one breed or on a small numbers of horses, but both Wolff and Hausberger (1996) using two sires and the authors using 25 sires have found paternal effects in their tests. Application of these tests to a variety of horses of known lineage would enhance our understanding of equine genetics. McCann et al. (1988) developed a test for emotionality for horses. The horses (32 yearling Quarter Horses, in this case) were restrained in a chute. Their reaction to the chute and to the approach of people was noted. After a 3 min period when humans were at least 5 m away, the horse was released into a 6 ×30 m pen. A handler attempted to catch the horse. Four people rated the yearling as highly nervous, nervous, normal or quiet based on their response to approach of an unfamiliar person. Highly nervous horses tried to escape from the chute when approached and galloped away from the chute and the handler. Nervous horses kept moving during the evaluation, but did not attempt to escape. Normal horses settled in the chute and moved out of it with less intensity. They would move away from the handler in the pen, but would stand rather than move. Quiet horses could be caught in the pen.

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Good learner but needs lessons repeated Fastest learner

Appaloosa

Temperament

Fast, receptive

Fast learner

Good

American Saddlebred

Standardbred

Tennessee Walking Horse Thoroughbred

Reasonably fast Hard worker but lacks ability to concentrate

Not enthusiastic

Obedient

Willing, hard worker

Inconsistent; can be extremely self-protective or extremely tolerant High Low; tolerates pain Average

High; self-protection

Response to pain

High

Low

Low

Average (Tobiano low, Overo high) Low Average or below average Low Slightly above average

Average Low

High strung, personable, Compliant, eager to Easily affectionate, plays with please frightened stall toys Easy going, levelheaded, Gives in Low, sensible Low kind Good, calm, adaptable, Does it anyway Unflappable Low easy going High strung, emotional, May react with fearfulness, High High reactive refusal, does it anyway

Does what it is asked

Not reactive

Easy going

Fast, reasonable Patient, compliant

Low

Flight

Over-reactive High

Low

Quarter Horse

Eager to please but evades

Acts dumb; passive aggressive

Fearfulness

Average Not easily frightened Average

Needs incentive; Easy going, inquisitive does not like change in routine Energetic Easily bored, playful, impressionable

Work ethic

When asked to do something the horse does not want to do

Miniature Horse Fast learner, smart Hardworker, not lazy High strung Stubborn Morgan Good, fast learner Willing, diligent Easy going, spirited but Adaptable and compliant easily managed Paint horse Fast learner Hard worker Reasonable, easy going Does it anyway

Arabian

Trainability

Behavioural characteristics of breeds of horses (from Hayer, 1998).

Breed

Table 11.2.

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An equine temperament test has been developed by Mackenzie and Thiboutot (1997). This test requires that the horse be led, but the handler is not allowed to coax, reward or threaten the horse. The handler should not have had previous experience with the horse. After measuring the time taken for the horse to walk between two lines 10 m apart, the horse is removed from the test area while a 3 ×3 m sheet of clear plastic is placed at the end of the 10 m. The time taken for the horse to walk 10 m ending at the edge of the plastic is measured. The horse must approach an umbrella at a 45° angle. The umbrella is opened as the horse reaches it and the time for the horse to pass the umbrella is noted. Reactivity to loud sounds is measured by dropping seven pots and pans from 3.3 m on to a metal folding chair behind a visual barrier. The time required to lead the horse to the centre of the visual barrier after the pans are dropped is measured. The response of the horse to tactile stimulation is determined by measuring the amount of pressure it is necessary to apply before the horse moves away. A penetrometer, a fruit testing gauge, is used to determine the pressure. The Mackenzie and Thiboutot test has been used by Holland et al. (1996) who were testing the effect of adding oil to the diet on the horses’ temperaments. Although there were no significant differences on temperament scores, there were strong tendencies. Horses fed a diet containing soy lecithin, corn oil (10% fat) did not hesitate to approach the umbrella, in contrast to horses fed a control diet (0% fat). Horses fed corn oil tended to require more pressure before they moved than horses fed the control diet (Holland et al., 1996). A suggestion of not only breed differences in temperament, but also underlying physiological differences between breeds is found in the study of Bagshaw et al. (1994). They measured heart rate, activity and defaecation rate in response to isolation, as well as serum levels of serotonin, dopamine and tryptophan (the precursor of serotonin) in response to isolation. The two Arabian mares had a significantly lower level of serum serotonin in comparison with the eight Standardbred mares. Lower serotonin may predict a more anxious, fearful or aggressive horse, which is certainly the reputation of Arabians. Nevertheless, in this small study, Bagshaw et al. (1994) found no breed differences in behaviour in response to isolation. All the mares defaecated during the tests and the incidence increased significantly during isolation to 25 from 11 when in visual contact with horses. The heart rate for Arabians ranges from 59 to 96 beats min−1 and for Standardbreds from 39 to 68 beats min−1.

Sire effects Wolff et al. (1997) have developed a temperament test for horses and have found paternal effects. The test consists of three parts: (i) isolation in a familiar arena; (ii) reaction to being led across a wooden surface; and (iii) reaction to a collection of novel objects in a 1 m circle: red and white striped bands, grey tubes and green poles. Behaviours were recorded at 1 s intervals and included

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standing, exploratory walk (head lowered, sniffing), sustained rapid walking, trotting or galloping. The horse was ranked 0 to 3 for these locomotor behaviours. Vigilance (the horse stands with head erect and ears pricked), whinnying, ‘passage’, snort and raised tail were ranked 5–6 for these postural components of an index of emotions. Forty-two French Saddle Horses were tested; 18 were sired by stallion A and 24 by sire B. When isolated, the offspring of sire A had lower indices of emotionality than those of sire B when specific behaviours were compared. They performed significantly more sustained walking than those of sire B. On this test, full sisters showed less variability than half-sisters. When confronted with a novel object, more offspring of stallion A showed excited behaviours (passage and raised tail) than those of stallion B who exhibited more exploratory behaviour. The offspring of stallion A had a higher index of emotionality than those of stallion B. Half-siblings showed similar proportions of the various behaviours in contrast to unrelated horses. When led over a wooden surface, the offspring of stallion A required more time to cross the bridge and showed a more similar reaction in comparison with unrelated horses. It is interesting that the offspring of stallion A were calmer when isolated, but reacted more emotionally to novel objects. In their study of foals (see maternal and foal behaviour; Wolff and Hausberger, 1994), the same authors found that foals of stallion B remained closer to their mother. Motivation for proximity may be the characteristic inherited. There are two major training centres in Japan: Ritto in the West and Miho in the East. All horses that race at Japan Racing Association-sponsored races must be trained at one of these tracks. The best evidence for a genetic effect on temperament is based on the behaviour of 9644 young Thoroughbred horses at these tracks. The horses were sired by 62 stallions. Each stallion’s offspring were represented at each training centre. The number of offspring per stallion ranged from eight to 34 at Miho Training Center and from seven to 39 at Ritto Training Center. Because the horses were coming from many locations, the chance of epidemics of infectious disease is high. Therefore, every horse was examined when it came to a training centre. A veterinarian checked the mucous membrane of the eye, auscultated the chest and took a blood sample. The veterinarians gave each horse a grade for ease of handling for each procedure. If the horse was calm, it received an A. If it moved, but could be restrained without a twitch, it received a B. If a twitch had to be applied, it received a C. Each horse received three grades AAA, AAB, BCA, etc. for its behaviour during the three procedures, manipulation of the eyelids, auscultation and venipuncture. The majority of the horses were tractable; 75% received AAA. When the ratio of offspring graded AAA to the total number of offspring of that sire at each training track was calculated, the ratios varied from 1 (all offspring received AAA) to 0.26. When the ratios at Miho are plotted against that stallion’s ratios at Ritto, the correlation coefficient is 0.523 (P < 0.001). In other words, stallions whose offspring were well behaved at one training centre were well behaved at another (Fig. 11.1). This is strong evidence for a heritable factor in reactivity.

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Fig. 11.1. The relationship between the tractability scores of the offspring of a sire at Miho Training Center and the same sire’s offspring at Ritto Training Center. There is a significant correlation (r = 0.523, P < 0.001) between the scores.

Abnormal Behaviours Breed and genetic differences in stereotypies Stereotypies are repetitive functionless behaviour patterns. They are often called stable ‘vices’ in the lay literature. Tongue rolling by cattle and pacing in front of cage bars by lions are examples of stereotypic behaviours in other species. The stereotypic behaviours of horses can be divided into locomotor stereotypies such as stall walking or weaving and oral ones such as cribbing (Luescher et al., 1991). In most of the studies (McGreevy et al., 1995b; Luescher et al., 1998; Redbo et al., l998), the horses were adults, but Prince (1987) and McGreevey et al. (1995a) studied training stables where the average age would be lower because 2- and 3-year-old horses were present. Surveys can yield inaccurate perspectives between breeds if they fail to control for postnatal experience, learning and current management. The latter is probably the most important variable. Nevertheless, the consistency of breed differences in stereotypic behaviour strongly indicates a heritable basis. Cribbing or crib biting is the behaviour for which there is most evidence of a genetic predisposition. Cribbing is the term used for arching the neck while grasping a horizontal surface with the incisors and aspirating air into the pharynx and upper oesophagus with a characteristic sound (McGreevy et al., 1995c) (Fig. 11.2). Weaving is walking in place as the horse shifts from side to side. Stall walking is repetitive locomotion in the stall, usually in a circular pattern but occasionally along one or two sides. The clearest indication of breed differences in behaviour is in susceptibilities to abnormal behaviour. Table 11.2 lists the breed incidence of the various stereotypies. In a survey of 769 horses from 32 Thoroughbred, Standardbred

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Fig. 11.2.

A horse cribbing.

and pleasure horse stables in southwestern Ontario, Thoroughbred and pleasure horse stables had an incidence of stereotypic behaviour of 12%. Warmbloods (Hannoverian, Trakehners, Thoroughbred–draught crosses, Hunters, Morgans, Anglo-Arabs and Saddlebreds) were more likely to stall kick (Luescher et al., 1998). All the professional trainers in Sweden were surveyed by Redbo et al. (1998). Sixty-one per cent of trotting horse trainers and 72% of Thoroughbred trainers responded. The incidence of stereotypies in Thoroughbreds (n = 644) and trotters (Standardbreds and North Swedish trotters, n = 4597) was compared. They found a much higher incidence of stereotypies in Thoroughbreds (see Table 11.2). The authors concluded that differences in management might be the most important cause of the breed difference because the Thoroughbreds had less social contact with other horses, had less free time outside the stall, were fed more concentrates and trained more than trotters. There were no breed differences in wood chewing, but the incidence of wood chewing was higher in horses fed less roughage. The management hypothesis is strengthened by the findings of McGreevy et al. (1995a) in a large survey of Thoroughbred training stables in which the number of meals, type of bedding and visual contact with other horses were correlated with the incidence of stereotypic behaviour. McGreevy et al. (1995a) found that factors which decreased the risk of stereotypic behaviour were: feeding roughage more than three times a day, bedding with straw, providing more than 7 kg roughage and more than one type of roughage, stabling a large number of horses and providing visual access among the horses. The use to which the horse is put also influences the prevalence of stereotypic behaviour. Letters were sent to members of the British Horse Society dressage, eventing and endurance groups requesting information on stereotypic behaviour. Information was received concerning 1750 horses. Information was also obtained on wood chewing, but wood chewing is not a

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stereotypic behaviour. Wood is actually ingested. A lack of roughage and the accompanying low caecal pH (Williard et al., 1977) and possibly lack of exercise (Krzak et al., 1991) are important. Twenty per cent of dressage horses, 15% of eventing horses and 8% of endurance horses wood chewed in the McGreevy survey (1995b). The rate of all abnormal behaviour was highest in dressage and eventing horses and lowest in endurance horses (McGreevy et al., 1995b). That may reflect the time spent out of the stall by endurance horses or reflect a breed difference because Arabians are used for endurance and Thoroughbreds or warmbloods for dressage and eventing.

Evidence for a hereditary basis of cribbing As mentioned above, cribbing must be distinguished from wood chewing in which the wood is actually ingested and splintered wood will be found on the ground. Horses that crib make semicircular erosions on a fence rail but do not eat wood. Any damage is caused by the pulling force as the horse flexes his neck. It is regarded as an unsoundness and is the stereotypy of most concern to professional horsemen. It is widely believed that horses learn cribbing by observing other horses crib, but there is no evidence for that. In fact, numerous attempts to make horses learn other activities by observing demonstrator horses have failed. The activities, all for food rewards, were: choosing one of two buckets (Baer et al., 1983; Baker and Crawford, 1986; Clarke et al., 1996) or pushing a foot pedal (Lindberg et al., 1999). Cribbing has been linked to endogenous opiates because naloxone, an opiate blocker, inhibits cribbing temporarily (Dodman et al., 1987). The hypothetical pathogenesis is that when a horse eats grain or another sweet feed, endogenous opiates are released and these, in turn, stimulate cribbing. Figure 11.3 illustrates the temporal relationship of cribbing to feeding of grain but not to feeding of hay. Cribbing occurs in 7.5% of dressage horses, 8.3% of eventing horses, but in only 3.1% of endurance horses (McGreevy et al., 1995b). Breed differences in cribbing incidence are given in Table 11.3. The first evidence for a genetic predisposition to crib was presented by Hosoda (1950) who found that fewer than 1% of the 1503 Thoroughbreds and Japanese native horses cribbed, but in some families the percentage was 7% (see Fig. 11.4). For example, 8.3% of the offspring and 6.4% of the grandsons of the cribbing stallion, Ryopan, also cribbed. The horses were distributed over several farms so the environment differed. The mode of inheritance was not clear, but because 25% of offspring of one cribbing parent and 50% of offspring of two cribbing parents cribbed, it may be a dominant gene. Vecchioti and Galanti (1986) found an incidence of 7.4% for all stereotypic behaviours in Italian Thoroughbreds. The incidence of cribbing was 2.4% among 1035 Thoroughbred horses in Italy, but in 14 families the incidence was 44%. They proposed inheritance of predisposition to perform stereotypic behaviours rather than inheritance of the tendency to crib (Fig. 11.5).

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Fig. 11.3. Mean frequency of cribbing before and after a roughage meal (upper graph) and a concentrate meal. The dotted line indicates the means level: *P < 0.05, **P < 0.01 (From Kusunose, 1992. Reprinted with permission of the Japanese Journal of Equine Science.)

Marsden and Henderson (1994) surveyed zoos containing a total of 275 Przewalski’s horses. They found an incidence of stereotypic behaviour of 8% similar to that reported by Luescher et al. (1998), but higher than that found by McGreevy et al. (1995a). They concluded that if a horse had grandparents or a sibling, but not parents, with stereotypic behaviour, their chance of developing a stereotypy was 25%, but if either parent had a stereotypy, the chance rose to 60%. If both of the parents exhibited stereotypic behaviour, the risk was 89%. The behaviour of a particular Przewalski dam and offspring illustrates the importance of environment in the expression of cribbing. The dam, Colleen (Brookfield no. 3), cribbed even when in a fairly large enclosure with other mares. She cribbed on the backs of other horses, as well as on the edge of the hay rack. None of the other horses cribbed, nor did any of her foals while living with her. When her colt, Rococo (Topeka no. 1), was sent as an adult to

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the Denver Zoo, he was free of stereotypies until he was stalled away from the mares with whom he usually lived. He began to crib on the stall door. This is another indication of the environmental effect on a genetic predisposition to crib. The behaviour of these horses emphasizes that environmental stress such as a poor social environment, confinement or lack of grazing opportunity is permissive for cribbing behaviour if the horse is genetically susceptible. An immediate stimulus is a sweet taste which releases endogenous opiates in human infants for example. Pell and McGreevy (1999) hypothesize that the genetic susceptibility to cribbing is in the response of opiate receptors to endogenous opiates.

Other abnormal behaviour Breed differences in self-mutilation Self-mutilation is a more abnormal behaviour because it can result in tissue damage. Mildly affected horses turn their necks to their flanks and grasp the hair or skin in their teeth, but severely affected ones actually bite themselves. The biting action is usually accompanied by a squealing vocalization and by kicking. The flank is the usual target, but some horses bite their chests or limbs. It is primarily a stallion behaviour problem, although some geldings and a few mares also exhibit it. In a 1994 survey based on histories obtained from owners who responded to requests in a number of lay and veterinary journals, Arabians were most affected (12 of 52) followed by Quarter Horses (nine) and American Saddlebreds (eight). At that time, Quarter Horses were the most numerous breed. In 15 of 42 cases in which the behaviour of relatives of the horse was known, related horses were also affected. The breed incidence and familial tendency indicate heritability (Dodman et al., 1994). Sexual behaviour in geldings The causes of misbehaviour, especially sexual misbehaviour, in geldings begins before their birth. The embryo begins with an undifferentiated gonad and two sets of tubular structures, the Müllerian ducts and the Wolffian ducts, both of which are paired structures. The fetal gonad under the influence of the chromosome (X or Y) differentiates into either an ovary or a testis (see Chapters 12 and 13). Initially, the fetal gonad consists of a medulla and a cortex. If the fetus is male, the cortex atrophies and the medulla becomes the seminiferous tubules, but if the fetus is female, the cortex atrophies and the medulla becomes the ovary. During fetal life, the testes are large – the colt’s testes are as large as his mother’s ovary even when she outweighs him by a factor of 10. The testes are large because placental gonadotrophin stimulates the fetal testis to produce androgen which cause the Wolffian ducts to develop and the Müllerian ducts to atrophy. The Wolffian ducts become the ductus deferens. In filly foals, the Wolffian ducts atrophy and the Müllerian ducts persist as the oviducts, fusing caudally to form the uterus. These gross

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Genetics of Behaviour Table 11.3.

Incidence of stall stereotypies.

Behaviour

Breed

Cribbing

Thoroughbred

Stall walking

Standardbred Quarter Horse Arabian Warmblood Thoroughbred

Standardbred

Weaving

299

Arabian Warmblood Quarter Horse Thoroughbred

Standardbred Warmblood Quarter Horse Arabian

% 4.2 4.0 2.8 2.4 1.1 0. 5.94 3.66 5.56 3.03 2.5 1.7 1.5 1.1 0.59 0.2 7.32 0. 0. 5. 4.17 4.0 2.5 1.1 0.3 0. 1.8 1.98 1.22

Reference Prince (1987) McGreevy et al. (1995a) Luescher et al. (1998); Redbo et al. (1998) Vecchiotti and Galant (1986) Hosoda (1950) Luescher et al. (1998) Luescher et al. (1998) Luescher et al. (1998) Vecchiotti and Galant (1986) McGreevy et al. (1995a) Redbo et al. (1998) Prince (1987) Luescher et al. (1998) Redbo et al. (1998) Luescher et al. (1998) Redbo et al. (1998) Luescher et al. (1998) McGreevy et al. (1995a) Vecchiotti and Galant (1986) Prince (1987) Redbo et al. (1998) Luescher et al. (1998) Luescher et al. (1998)

anatomical changes have been known for some time, but there are more recently discovered sex differences that may account for our misbehaving geldings. In addition to the obvious differences in external genitalia between the sexes, there are also differences in the brain. Males have a much larger sexually dimorphic nucleus in the preoptic area of the hypothalamus. This is also due to the organization effect of androgen (Nelson, 1995). The sex differences should disappear when a male horse is castrated because the source of testosterone has been removed. This does not always happen. In fact, almost 30% of geldings still demonstrate some aspect of male sexual behaviour (Line et al., 1985). They may flehmen, they may mount and even copulate, they may herd mares away from other geldings, they may fight with other geldings. Owners are especially upset when their gelding protects ‘his’ mares from them, threatening the hand that feeds him. Many owners think that the veterinarian has not done the surgery properly and has left

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Ryupan

1

2

I Kaen

II

Hasyu

1

4 Ryuten

3

Shusen 2 Seien

III 1

IV

3 Harunami

Hoshitani

2

3

1

Kasuga

2

Dyoright

4 Hokyo

Primero

Fryarsmedon

1

2

1 No. 4 Fryarsmedon

2 No. 19 Primero

3 Azumarai

3 Sentright 4 Yamarai

4 Toshitani

5 Iwarai

Suigun Normal stallion Normal mare dead foal

Stallion acquired the habit Mare acquired the habit

Fig. 11.4. Pedigrees of families of Japanese Thoroughbreds that contained a high percentage of cribbers (Hosoda, 1950. Reprinted with permission of the Japanese Journal of Zootechnical Science.)

different stallions

1

3

2

4

5

6 N

N

N

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9

8 N

different stallions

10

N

13 11

12

14

Proband male and female, normal male and female, vicious female flapping its lips

N

N

male and female, nervous no information reformed male

Fig. 11.5. Pedigrees of families of Italian Thoroughbreds that contained a high percentage of cribbers. Vicious, in this case, indicates that the horse has vices not that it is aggressive. (Vecchiotti and Galanti, 1986. Reprinted with permission of Livestock Production Science.)

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some testicular tissue behind. This is rarely the case. Occasionally, when a cryptorchid testicle is removed, some testicular tissue remains behind. Fortunately, there is a blood test that can be done to measure testosterone which should be ≤0.2 ng ml−1 in a gelding. The most accurate test is to measure baseline oestrone sulphate, then administer human chorionic gonadotrophin 10,000 IU intravenously and take a second sample 2 h later. If the horse is a gelding or a stallion, the level will be more than 500 pg ml−1. If he is cryptorchid, the levels will be > 2000 pg ml−1. In response to a request published in Equus, a magazine for those interested in horses, 85 owners of older geldings exhibiting sexual behaviour completed history forms. The mean age of geldings was 16.5 years. Forty per cent of the horses were Quarter Horses, the most numerous breed in the US, the next most frequent breeds were Thoroughbred, Arabian, Appaloosa and Morgan; 78% of the horses were purebreds. The objectionable behaviours were herding, mounting or attempting to mount mares, aggression to other geldings, aggression to people and masturbation. Masturbation is exhibited by all geldings, but at a lower rate than in stallions (McDonnell et al., 1991). Trailer problems A short article was placed in the lay journal, Equus, requesting that owners of horses with trailer problems get in contact with us. Those owners who wrote or reached us by facsimile were sent a 2-page questionnaire. The questions included the reactions of the horse, the type of problem, the type of trailer, the flooring of the trailer and the treatments attempted. Responses were obtained from owners of 25 Quarter Horses, 21 Thoroughbreds, 14 Arabians and eight Appaloosas. Problem behaviour occurred during loading, travelling, unloading and when the trailer was stationary. Of 103 trailer problems, 55 (53%) were loading problems and 53 (51%) were problems during travel. Some horses had both problems. There was no apparent breed predisposition.

Conclusions So far, there have been no other quantitative studies of heritability of equine behaviour, but the marked breed differences in foal rejection, stereotypies and reactivity (emotionality) indicate that temperament is heritable.

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McGreevy, P.D., French, N.P. and Nicol, C.J. (1995b) The prevalence of abnormal behaviours in dressage, eventing and endurance horses in relation to stabling. Veterinary Record 137, 36–37. McGreevy, P.D., Richardson, J.D., Nicol, C.J. and Lane, J.G. (1995c) Radiographic and endoscopic study of horses performing an oral based stereotypy. Equine Veterinary Journal 27, 92–95. Moehlman, P.D. (1998) Feral asses (Equus africanus): intraspecific variation in social organization in arid and mesic habitats. Applied Animal Behaviour Science 60, 171–195. Nelson, R.J. (1995) An Introduction To Behavioural Endocrinology. Sinauer Associates, Sunderland, Massachusetts. Nelson, R.J., Demas, G.E., Huang, P.L., Fishman, M.C., Dawson, V.L., Dawson, T.M. and Snyder, S.H. (1995) Behavioural abnormalities in male mice lacking neuronal nitric oxide synthase. Nature 378, 383–386. Ogawa, S., Taylor, J.A., Lubahn, D.B., Korach, K.S. and Pfaff, D.W. (1996) Reversal of sex roles in genetic female mice by disruption of oestrogen receptor gene. Neuroendocrinology 64, 467–470. Pell, S.M. and McGreevy, P.D. (1999) A study of cortisol and beta-endorphin levels in stereotypic and normal thoroughbreds. Applied Animal Behaviour Science 64, 81–90. Prince, D. (1987) Stable vices. In: McBane, S. (ed.) Behaviour Problems in Horses. David & Charles, North Pomfret, Vermont. Rebhun, W.C., Loew, E.R., Riis, R.C. and Laratta, L.J. (1984) Clinical manifestations of night blindness in the Appaloosa horse. Compendium of Continuing Education for the Practicing Veterinarian 6, S103-S106. Redbo, I., Redbo-Torstensson, P., Ödberg, F.O., Hedendahl, A. and Holm, J. (1998) Factors affecting behavioural disturbances in race-horses. Animal Science 66, 475–481. Rossdale, P.D. (1967) Clinical studies on the newborn thoroughbred foal I: perinatal behaviour. British Veterinary Journal 123, 470–481. Salter, R.E. and Hudson, R.J. (1979) Feeding ecology of feral horses in western Alberta. Journal of Range Management 32, 221–225. Saudou, F., Amara, D.A., Dierich, A., LeMeur, M., Ramboz, S., Segu, L., Buhot, M.-C. and Hen, R. (1994) Enhanced aggressive behaviour in mice lacking 5-HT1B receptor. Science 265, 1875–1878. Shaw, E. and Houpt, K.A. (1985) Pre- and post-partum behaviour in mules impregnated by embryo transfer. Equine Veterinary Journal 17 (Suppl. 3), 73. Smith-Funk, E.D. and Crowell-Davis, S.L. (1992) Maternal behaviour of draught mares (Equus caballus) and mule foals (Equus asinus ×Equus caballus). Applied Animal Behaviour Science 33, 93–119. Sweeting, M.P., Houpt, C.E. and Houpt, K.A. (1985) Social facilitation of feeding and time budgets in stabled ponies. Journal of Animal Science 160, 369–374. Truett, G.E., Jacob, H.J., Miller, J., Drouin, G., Bahary, N., Smoller, J.W., Lander, E.S. and Leibel, R.L. (1995) Genetic map of rat chromosome 5 including the fatty (fa) locus. Mammalian Genome 6, 25–30. Tyler, S.J. (1972) The behaviour and social organization of the New Forest ponies. Animal Behaviour Monographs 5, 85–196. Valberg, S.J., Geyer, C., Sorum, S.A. and Cardinet, G.H. III. (1996) Familial basis of exertional rhabdomyolysis in Quarter Horse-related breeds. American Journal of Veterinary Research 57, 286–290.

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Reproduction and Modern Breeding Technologies in the Mare 1 and 2 W.R. Allen Reproduction 12 W.R. andand Allen D.F. Modern Antczak Breeding D.F. Antczak Technologies 1University of Cambridge Department of Clinical Veterinary Medicine, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket CB8 9BH, UK; 2James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA

Introduction Seasonality, Cyclicity, and Ovarian and Oviducal Function Ovarian function Oviducal function Uterine Structure and Function Embryogenesis Membrane Differentiation Placentation The Oestrogens of Pregnancy Modern Breeding Technologies Embryo Transfer Embryo Technologies Gender Selection of Spermatozoa Conclusions Acknowledgements References

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Introduction The horse provides several advantages in genetic studies. These include extensive and well-maintained pedigrees and quantitative methods for assessing performance through the use of race records and race times. Furthermore, interest in inheritance patterns is high among virtually all horse breeders. Coupled with these advantages, however, come a number of disadvantages, many of which originate from the unique aspects of reproductive physiology in the genus Equus. For example, mares produce only a single foal per year (or very rarely twins), and that after a long, 11 month gestation. This means ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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that very few full sibling families are available for genetic investigations. A similar situation exists in cattle, but superovulation, followed by artificial insemination, and then subsequent recovery and transfer of multiple embryos, has dramatically changed dairy cattle breeding. To date, however, reliable techniques for superovulation of mares have not yet been achieved, despite considerable effort. Thus, some of the evolutionary adaptations of the reproductive system of the horse appear designed to frustrate those interested in ‘improving the breed’ using classical genetic techniques. Other difficulties arise from horse management practices. For example, in aged Thoroughbred broodmares from racing stock, which may have had ten or more foals, the combination of the stretching of the cervix during successive foalings and the parity-related stretching of the myometrium and broad ligament creates the situation in which the uterus is unable to expel the excess seminal fluid and debris deposited in it at natural mating before ovulation occurs and constricts the cervix to seal it off from the outside world. Thus, instead of a nice sterile ‘feather bed’ in which the young embryo may be nurtured, the uterus becomes a cauldron of trapped fluid and bacteria in which even the most stalwart of embryos has no chance of survival. This situation often occurs when the mare achieves her highest value as a potential producer of racing stock, as a result of the record of her previous offspring. On the other hand, in non-racehorse competition types of horses, such as eventers or show-jumpers, competing mares are not normally retired to stud until they are 14 or more years of age when they are still maidens. The same practical problems of trapped intrauterine fluid and associated infection occurs after natural mating in these animals, but this time it is an excessively muscled and tortuous cervix which has never been stretched previously by the birth of a foal and which fails to relax adequately at any time during oestrus and prevents expulsion of the accumulated seminal and uterine fluids and other debris. Is there any hope for the horse breeder and amateur geneticist? We believe there is. In this chapter, we attempt to highlight the various ways in which the wealth of modern breeding technologies that have been developed in recent years can be applied to combat some of the natural and artificial problems that confront horse breeders.

Seasonality, Cyclicity, and Ovarian and Oviducal Function Mares are long-day seasonal breeders which, in the temperate zones of the world, show regular oestrous cycles of around 21–23 days in length during the spring, summer and autumn months, followed by a period of sexual quiescence or anoestrus during winter (Fig. 12.1). The cycle is composed of 4–6 days of oestrus or sexual receptivity when one, or occasionally two, dominant ovarian follicles are developing towards ovulation, followed by 15–17 days of dioestrus when a functional corpus luteum (CL) in the ovaries is secreting the

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Fig. 12.1. Diagrammatic summary of ovarian activity throughout the year in the long-day seasonally polyoestrous mare. Note the transitional phases of shallow anoestrus between ovarian cyclicity and winter anoestrus.

high concentrations of progesterone into the bloodstream which make her reject the advances of the stallion (Hughes et al., 1975). The gonadotrophic control of ovarian cyclicity in the mare is unusual and interesting. During the transition phase of shallow anoestrus between true winter anoestrus and the resumption of ovulatory oestrous cycles in spring, the steady reduction in the prolonged periods of elevated melatonin secretion from the pineal gland caused by the shortening of the long nights increasingly removes the brake from the underlying hypothalamus which responds by increasing both the frequency and amplitude of its spike-like releases of gonadotrophin-releasing hormone (GnRH) into the portal circulation connecting it directly to the pituitary gland beneath (Alexander and Irvine, 1987). The latter responds, in turn, by increasing its secretion rate of the two principal gonadotrophic hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which together act upon the ovaries to, between them, stimulate the processes of follicle recruitment, growth, maturation and eventual ovulation (Fig. 12.2). As the follicles grow, they secrete increasing amounts of inhibin into the bloodstream which exerts a negative feedback action on the hypothalamic–pituitary axis, thereby causing blood FSH concentrations to decline. Coincidentally, the increasing amounts of oestradiol and oestrone secreted by the growing follicles provide positive feedback stimulation to the pituitary to begin synthesizing and releasing increasing amounts of LH, which then takes over the role of stimulating the continued growth and maturation of the dominant follicle, inducing the production of steroids within the follicle and the necessary maturational changes of the oocyte, and the eventual rupture of the follicle and the process of luteinization of the granulosa cells to form the progesterone-secreting corpus luteum.

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Fig. 12.2. mare.

Summary of the principal exteroreceptive factors controlling ovarian cyclicity in the

The importance of LH in the whole process of follicular development and ovulation in the mare is well illustrated by two unusual phenomena. First, by the common occurrence of the condition known as prolonged spring oestrus in barren and maiden mares during the transition phase between anoestrus and cyclicity whereby, as a result of continuously elevated levels of pituitary FSH secretion, but inadequate production of pituitary LH (Fitzgerald et al., 1987), as many as 30–40 small follicles of around 5–15 mm diameter develop in the ovaries, giving them the feel of a ‘bunch of grapes’ when palpated per rectum. Such mares frequently will show strong but erratic oestrous behaviour, often for many weeks, which is presumed to stem from the cumulative effects

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of the small amounts of oestrogen secreted by each partly stimulated follicle. Due to the lack of sufficient exteroreceptive stimulation of the pineal– hypothalamic–pituitary axis at this very early stage of the breeding season, the inadequate release of LH means that none of these small follicles can be driven towards full maturation and ovulation, and the mare becomes ‘stuck’ in a half-stimulated twilight world until sufficient exteroreceptive stimulation releases sufficient LH from the pituitary to progress the situation further. Prolonged spring oestrus was formerly a significant management problem in breeds such as the Thoroughbred in which commercial pressure to produce early foals demands an unnaturally early start to the mating season (Osborne, 1966). Repeated covering of mares showing persistent oestrous behaviour increased their chances of succumbing to post-mating infective endometritis and it caused overuse of the stallion at a time when his rate of spermatogenesis and libido were similarly reduced by a lack of exteroreceptive stimulation. Nowadays, however, the condition is easily diagnosed by ultrasound examination of the mare’s ovaries (Simpson et al., 1982) and it can be treated effectively (Evans et al., 1982; Allen et al., 1987b). A second feature of pituitary LH in the mare is the unusual biological activities of the molecule and its prolonged period of secretion during oestrus. In marked contrast with LH in humans and the other common domestic animal species, equine LH possesses an abnormally high content of sialic acid which gives it a much longer half-life in mares’ blood of some 3–5 h compared with a half-life of only 20 min for sheep LH (Irvine, 1979). Furthermore, like the equine chorionic gonadotrophin (eCG) molecule secreted by the endometrial cups in early pregnancy (see later), equine pituitary LH expresses a large component of FSH-like biological activity in addition to its conventional LH-like properties in non-equine species (Combarnous, 1982). Thus, stimulated by the rising oestrogen concentrations in early oestrus, the secretion rate of pituitary LH begins to increase so that plasma LH levels rise steadily over the next 4 or 5 days to reach an ill-defined peak some 1–3 days after, rather than before, ovulation (Fig. 12.3). This is in marked contrast with the short, sharp spike-like ovulatory release of LH which occurs some 24–28 h before ovulation in other animal species. High concentrations of the mixture of LH isomers of varying biological activities persist in the mare’s blood for some days after ovulation of the dominant follicle and this may lead to the gonadotrophic ‘rescue’ of a partly atretic follicle from the original cohort which can then go on to mature and ovulate ‘silently’ some days after ovulation of the primary follicle that dominated the previous oestrus (Hughes et al., 1975). Since stallion spermatozoa can live for at least 3, and sometimes up to as many as 5 or 6, days in the mare’s reproductive tract (Clément et al., 1999), the situation is created whereby fertilization of the second ‘silently’ ovulated oocyte can give rise to asynchronous twin conceptuses that may differ by as much as 5 days in developmental age. This, in turn, can cause diagnostic problems when scanning mares for pregnancy as early as 12–14 days after ovulation; failure to observe a day 8 or 9 embryo which is an asynchronous twin to a day 13 or 14 conceptus may have disastrous consequences if the mare is not rescanned and the

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Fig. 12.3. Plasma profiles of gonadotrophic and steroid hormones in relation to follicular growth, ovulation, corpus luteum development and luteolysis in the cycling mare. PGFM = 13,14 dihydro-15-keto prostaglandin F2a.

presence of the twins realized in time for manual rupture of one conceptus to be undertaken to convert the twin pregnancy to an ongoing singleton before development of the endometrial cups at day 36–38 (Allen et al., 1973).

Ovarian function The ovaries of the mare are unusual on two counts. First, they are bigger than the ovaries of other large mammals and the sizes of both the mature preovulatory follicle at 3.5–6.0 cm and the resulting corpus luteum are much greater than those found in all other mammals, including elephants. This makes both the rectal palpation and the transrectal ultrasound scanning of follicular development, ovulation and corpus luteum development very simple and straightforward in the mare which, in turn, has enabled mating and ovulation to be much better synchronized, resulting in improved pregnancy rates on well-managed farms. The second feature of the mare’s ovary is the very tough and fibrous tunica albuginea which completely envelops it, apart from a small, welldefined dent or fossa in the medial surface through which ovulation occurs.

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This has two consequences. First, the dilated fimbria of the equine oviduct is relatively small compared with that in other species since it only has to envelop the ovulation fossa to pick up the ovulated oocyte. Second, despite the potential to induce the growth and partial maturation of multiple follicles in the ovaries of mares by treatment with exogenous gonadotrophins (Palmer et al., 1993) by active or passive immunization against the β-subunit of inhibin (McCue et al., 1993; Nambo et al., 1999), competition between these multiple follicles in their attempts to track through the ovarian stroma to reach the ovulation fossa results in degeneration or luteinization without ovulation in many of them, with a consequential and disappointing fall-off in the recovery of multiple embryos (Palmer et al., 1993).

Oviducal function The oviduct in the mare is long and very convoluted and its ampullary section opens to the fimbria through a small aperture in the folds to one edge of the structure, rather than in the centre. The uterotubal connection, on the other hand, takes the form of a pronounced papillum which protrudes into the uterine lumen of the tip of each horn. Oguri and Tsutsumi (1972) first reported the very protracted oviducal transport time of 6 days in the mare which was subsequently confirmed by Webel et al. (1977), Freeman et al. (1991) and others. Battut et al. (1997) flushed the uteri of fertile inseminated mares at fixed intervals after accurately recording the time of ovulation and noted that the embryo enters the uterus between 144 and 169 h post-ovulation when it is at the late morula stage of development. This is considerably longer than the 48 h oviducal sojourn of the four-cell pig embryo, or the 72 h required for ruminant embryos to reach the uterus (Moor, 1968; Allen, 1984). Experiments undertaken by Woods and his group revealed some of the explanation for the slow transit of embryos in the equine oviduct (Weber et al., 1991a, b, 1992a, b, 1995). These authors noted appreciable secretion of prostaglandin E2 (PGE2) into the medium by day 5 and 6 embryos cultured in vitro, but not by day 3 and 4 embryos cultivated in a similar fashion (Weber et al., 1991a), and when they implanted mini-pumps on to the mesosalpinx of mated mares on the third or fourth day after ovulation which released a constant low dose of PGE2 into the surrounding tissues, the embryo reached the uterus 24–36 h earlier than those in untreated or saline-infused control animals (Weber et al., 1991b). In a more recent study, the same hastening of oviducal transport of the embryo was achieved by laparoscopic deposition of a small quantity of PGE2-impregnated gel material on to the surface of the oviduct on day 4 after ovulation (Robinson et al., 1999). This more practical method of applying the exogenous PGE2 to achieve the desired effect of more rapid entry of the embryo into the uterus has considerable practical implications for the development of modern breeding technologies in the horse, including the deep-freezing of embryos and their bisection to produce monozygotic twins.

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Thus, it appears that the equine embryo, uniquely among the species studied to date, controls its own passage through the oviduct by releasing PGE2 to induce relaxation of the smooth muscle which surrounds and governs the motility of this tube. It is indeed an unusual evolutionary adaptation and whether its underlying purpose is to hold the dividing embryo in a preferential nutritional environment of the oviduct before releasing it into the wider uterine world, or to hold an otherwise unsophisticated embryo in check in the oviduct simply to allow the uterus time to resolve the inflammation stimulated by natural mating or insemination, remains to be determined.

Uterine Structure and Function The equine uterus is a simple T-shaped structure composed of a straight body and two straight horns which is suspended in the abdomen and pelvis by the broad uterovarian ligament through which the supplying arteries and draining veins run. Unlike the pig and ruminants, in which a complicated plexus between the uterine vein and ovarian artery enables a direct local countercurrent transfer of endometrial PGF2α at the end of dioestrus into the ovarian artery to affect luteolysis in the ipsilateral ovary (McCracken, 1971), no such plexus exists in the mare (Ginther et al., 1972), and the PGF2α released in spike-like bursts at the end of dioestrus reaches the ovary and effects luteolysis via the peripheral circulation. The endometrium which provides the mucosal lining of the whole of the interior of the uterus is of special importance in the mare for a number of physiological reasons. It is arranged in a series of longitudinal folds and circular rugae which, in the healthy young mare, give a soft, velvety appearance throughout the uterine lumen. Histologically, the surface region of the endometrial stroma, the stratum compactum, is lined by a continuous layer of cuboidal-to-columnar lumenal epithelial cells which invaginate frequently into the stroma to form the tightly coiled, branching, endometrial secretory glands which penetrate through the stratum compactum into the underlying stratum spongiosum where they are distributed densely and evenly among the blood and lymph vessels (Fig. 12.4a). They serve two vital functions in the mare. First, when she is in oestrus and the uterus is dominated by the oestrogens secreted by the enlarging follicles, the glands respond with an outpouring of thin, watery secretory material which performs an important lavaging function to assist with the expulsion from the uterus of the seminal fluid and other foreign debris that accrued from mating or artificial insemination. Second, under the dominance of progesterone secreted by the corpus luteum in dioestrus or early pregnancy, the endometrial glands secrete smaller quantities of protein-rich histotrophe which forms the sole source of nutrients for the unimplanted but rapidly growing conceptus during its first 40 days of life (van Niekerk and Allen, 1975) and which continues to play an important role in fetal nutrition throughout gestation, even after development of the haemotrophic placental exchange mechanism which accompanies attachment and

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Fig. 12.4. Diagrammatic representation of the histological structure of the endometrium of the mare. (a) A healthy young mare showing the frequent invagination of an intact lumenal epithelium to form myriads of multibranched secretory glands in the stroma. (b) An aged, subfertile mare suffering endometrosis. Note the erosions of the lumenal epithelium, accumulations of mononuclear cells in the stratum compactum, fibrous blockage of lymph sinuses to form endometrial cysts which protrude into the uterine lumen and concentric deposition of fibrous tissue around the fundic portions of the endometrial glands to form ‘gland nests’.

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interdigitation of the allantochorion to the endometrium from day 40 (Samuel et al., 1975). Like other non-primate mammals, mares show no form of menopause and most mares continue to cycle and ovulate regularly throughout their lives. However, from around 15 years of age onwards, their endometrium begins to show increasingly a range of age-related degenerative changes that formerly were termed chronic degenerative endometritis (Kenney, 1975; Ricketts, 1975) but are now known collectively as endometrosis (Allen, 1993). These include: (i) accumulations of mononuclear cells and generalized fibrosis in the stratum compactum; (ii) fibrous constrictions of the lymph drainage vessels causing dilatations (lacunae) in the stroma and the formation of lymph-filled endometrial cysts which protrude into the uterine lumen; and (iii) concentric depositions of fibrous tissue around the basal portions of the endometrial glands in the stratum spongiosum to form so-called ‘gland nests’ (Kenney, 1975), which increasingly block the gland outlets and defunctionalize them (Fig. 12.4b; Bracher et al., 1992). Thus, as the mare ages, fewer and fewer functional glands persist in the increasingly degenerate endometrium, with the result that both the oestrogen-induced watery secretions of oestrus which play their important roles in sperm transport and uterine cleansing, and the protein-rich histotrophe upon which the growing young embryo depends so heavily, are reduced correspondingly. A diminished ability of such ageing uteri to withstand and overcome the considerable bacterial challenge of natural mating, thereby leading to an increased incidence of acute infective endometritis, and an increased incidence of pregnancy failure or the birth of small and weak foals due to placental insufficiency causing intrauterine growth retardation (IUGR), are inevitable consequences of these age-related degenerative changes. They affect Thoroughbred racehorse mares and other high-class sport horse mares especially, which tend to be left at stud long after the optimum ‘uterine sell-by date’ due to their superior pedigree and/or the competitive success of their existing progeny. Endometrial degeneration is clearly Nature’s evolutionary mechanism to limit the fecundity of ageing mares, and the use of embryo transfer and related modern breeding technologies to enable gestation of the foal with the proven superior genotype in a uterus with a young and healthy endometrium is the obvious way forward to overcome the problem.

Embryogenesis The young equine embryo differs strikingly from those of other large domestic species, both in the manner in which it informs its mother biochemically of its presence in the uterus (the ‘maternal recognition of pregnancy signal’; Short, 1969) and in the growth and differentiation of its constituent membranes and their attachment to the endometrium to form the definitive allantochorionic placenta that will effect fetomaternal exchange throughout pregnancy.

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In equids, the trophectoderm does not elongate during the critical period between days 10 and 14 after ovulation due to the presence of an elastic, glycoprotein capsule which forms initially between the trophectoderm and the covering zona pellucida on day 6 after ovulation (Betteridge, 1989) and completely envelops the embryo from this time until around day 23, when it simply ‘melts away’, presumably due to action of enzymes liberated by the trophoblast cells and/or the endometrium (Oriol et al., 1993a). Thus, wrapped in its tough, resilient capsule, the conceptus remains spherical and moves continuously throughout the entire uterine lumen between days 6 and 17 after ovulation, driven by peristaltic contractions of the myometrium (Ginther, 1985) that are stimulated by releases of PGF2α and PGE2 from the conceptus itself (Stout, 1997). It is reasonable to presume that this constant movement of the conceptus within the uterine lumen may be necessary to achieve distribution of the embryonic signal to a sufficient portion of the surface of the endometrium to induce suppression of PGF2α release and, indeed, surgical restriction of conceptus mobility has been shown to result in luteolysis at the expected time (McDowell et al., 1985). Movement of the conceptus ceases abruptly around day 17 after ovulation due to a dramatic increase in myometrial tone which holds the still spherical conceptus stationary at the base of one or other of the uterine horns (Fig. 12.5; van Niekerk, 1965). In addition to providing structural support and physical strength to enable the embryo to remain spherical and withstand the pressures of the myometrial contractions that bring about its movement, the capsule is also clearly involved in supplying nutrients. Its negative electrostatic charge and its unusual gycocalyx structure (Oriol et al., 1993b) make it likely that it can accumulate and hold endometrial gland secretions (histotrophe or ‘uterine milk’; Amoroso,

Fig. 12.5.

A summary of some significant events during early pregnancy in the mare.

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1952) on its outer surface as the conceptus rolls around the interior of the uterus. Prominent among these maternal histotrophic nutrients layered on to the capsule is a 19 kDa protein (P19; Stewart et al., 1995) which is a member of the lipocalin family of proteins and is therefore likely to function as a transport molecule of essential vitamins or heavy metals, such as iron, to the young embryo (Crossett et al., 1998). The horse conceptus does not secrete any type of interferon or related molecule during the critical period for maternal recognition of pregnancy (Sharp et al., 1989; Baker et al., 1991) but, like pig and camel embryos (Bazer and Thatcher, 1977; Skidmore et al., 1994), it does secrete appreciable quantities of oestrogens from as early as day 10 after ovulation (Heap et al., 1982). Recent experiments have indicated that these embryonic oestrogens may be the embryonic signal to the mare that brings about luteostasis in early pregnancy (Stout, 1997), by suppressing endometrial oxytocin receptors (Stout et al., 1999) along similar lines to the luteal oxytocin–endometrial PGF2α link that exists in ruminants (Lamming et al., 1995).

Membrane Differentiation From day 17 after ovulation, when movement of the conceptus ceases due to sudden increase in uterine tone (van Niekerk, 1965), until day 40–42 when a close and stable microvillous attachment between the trophoblast and endometrial epithelium is established and simple villi of the allantochorion begin to interdigitate with accommodating crypts in the endometrial surface (Samuel et al., 1975), considerable differentiation takes place in the constituent membranes of the conceptus. At day 21, the embryo, with its primitive beating heart, is situated at one pole of the conceptus. Vascularized mesoderm has developed between the chorion and yolk sac to give trilaminar omphalopleure over half the conceptus, and the allantois is just beginning to form as an outpouching of the hind gut (van Niekerk and Allen, 1975). Over the next 12–15 days, the allantois, also covered in vascularized mesoderm, expands steadily and fuses with the overlying chorion to form the definitive allantochorion. At the point where the enlarging allantois and regressing yolk sac abut each other, a discrete band-like portion of the chorion is thrown up into a series of simple folds. The trophoblast cells that cover the surface of these ridges undergo a remarkable hyperplasia and pile up on each other to give, by day 34–35, a pale, thickened, annulate band of tissue, the chorionic girdle (Fig. 12.6), which is held closely apposed to the overlying endometrium by the uterine tone and by an alcian blue-positive sticky exocrine secretion produced by simple gland-like structures that form between the adjacent folds of now largely binucleate specialized trophoblast cells of the girdle (Allen et al., 1973). Between days 35 and 38, the entire chorionic girdle peels off the fetal membranes and begins to vigorously invade the maternal tissue, initially by dislodging and destroying the lumenal and glandular epithelial cells, and then

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Fig. 12.6. Horse conceptus at 35 days after ovulation showing the development of the thickened annulate chorionic girdle (cg) between the enlarging allantochorion (ac) and the regressing yolk sac (ys). The persisting area of bilaminar omphalopleure (bo) at what was formerly the abembryonic pole of the conceptus is encircled by the sinus terminalis, immediately above which is the embryo (e).

passing through the respective basement membranes and streaming off into the endometrial stroma. Here, they suddenly cease to migrate, round up, enlarge greatly, become tightly packed together and begin secreting the unique equine chorionic gonadotrophin (eCG; Allen et al., 1973). This reaches the maternal circulation via a network of large lymph sinuses which develop beneath each bunch of invaded trophoblast cells, now called an endometrial cup (Clegg et al., 1954), and the LH-like component of the hormone stimulates ovulation and/or luteinization of the most dominant of the cohort of follicles that develop every 10–12 days in the mare’s ovaries as a result of the wave-like releases of pituitary FSH that continue in the early pregnant mare, just as in the cycling mare, throughout the breeding season (Evans and Irvine, 1975; Urwin and Allen, 1982). The endometrial cups can range in size from small, isolated structures of only 0.5–1.0 cm in diameter, to long ribbons of tissue that may be as large as 20 cm in length and 1.5–3.0 cm in width (Fig. 12.7a). They are first seen macroscopically between days 38 and 40 after ovulation as pale, raised plaques on the endometrial surface, and during the next 20–30 days they assume a concave, saucer-like appearance due to overgrowth at the edges and commencing degeneration and desquamation of cells in the central region (Fig. 12.7b). Histologically, each cup consists of a densely packed mass of large, epithelioid fetal endometrial cup cells, interspersed with occasional blood vessels, lymph sinuses and the dilated fundic portions of endometrial glands, the outlets and apical portions of which were destroyed during the initial invasion of the chorionic girdle cells at day 36 (Fig. 12.7c). Increasing numbers of CD4+ and CD8+ lymphocytes, plasma cells, eosinophils and other mononuclear cells accumulate in the endometrial stroma at the edge of the cup tissue and appear to ‘wall off’ the mass of fetal cells from the surrounding maternal tissue (Cole and Goss, 1943; Clegg et al., 1954; Allen, 1975; Grünig et al., 1995).

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B

C

Fig. 12.7. Equine endometrial cups. (A) An unbroken ribbon of newly developed cup tissue in the gravid horn of the uterus at day 47 of gestation. (B) Individual endometrial cups revealed by retracting the allantochorion at day 85 of gestation. The cups have become typically saucershaped due to overgrowth at the periphery and commencing degeneration in the central region. (C) Histological section of an endometrial cup at day 53 of gestation. The densely packed mass of large, binucleate fetal endometrial cup cells interspersed with dilated endometrial glands and occasional blood vessels is becoming surrounded by an accumulation of maternal lycophocytes and other immune cells. Large lymph sinuses have formed in the stroma beneath the cup (×25).

After day 70–80 of gestation, the large cells in the well of the crater-like surface of the cup begin to degenerate and slough off, thereby unblocking outlets of the endometrial glands to release their accumulated exocrine secretion. Coincidentally, the aggregated mononuclear cells begin to invade into the periphery of the cup. Eventually, between days 100 and 120, the whole necrotic cup and its admixed insipated coagulum of dead cup cells and exocrine secretion, rich in eCG activity (Rowlands, 1963), is sloughed off the surface of the endometrium where it may invaginate into the surface of the underlying allantochorion to form a pendulous allantochorionic pouch that hangs into the allantoic cavity (Clegg et al., 1954: Allen, 1975). This curious endometrial cup reaction, which is an unusual developmental strategy that occupies only the first trimester of pregnancy, is found only in

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equids and, to date, defies a logical evolutionary raison d’être. Initially, it was generally believed that the cups had primarily an endocrine role, with eCG stimulating the development of the secondary corpora lutea in the mare’s ovaries during the first half of gestation (Amoroso et al., 1948) and the striking enlargement of the fetal glands (Cole et al., 1931) and their secretion of large quantities of C19 oestrogen precursors during the second half of gestation. However, as mentioned previously, it has now been established that only the LH-like component of eCG acts to cause ovulation and/or luteinization of mature follicles, and it is the continuation of the 10–12 day releases of pituitary FSH, not the FSH-like component of eCG (Stewart and Allen, 1979), which stimulates these waves of follicular development (Fig. 12.8; Evans and Irvine, 1975; Urwin and Allen, 1982). Furthermore, despite the failure of endometrial cup development and complete absence of eCG in maternal blood, and the consequential failure of development of any secondary corpora lutea in the ovaries of mares carrying extraspecific donkey-in-horse pregnancies created by embryo transfer (Allen, 1982), around 30% of such pregnancies make it safely to term without any exogenous hormonal therapy (Allen et al., 1987a). This rate of fetal survival is not improved by administering either exogenous eCG or progesterone (Allen, 1982). Another potential role for the endometrial cups in equine pregnancy is in the fetal–maternal immunological relationship. The specialized invasive trophoblast cells of the chorionic girdle, but not the non-invasive trophoblast cells of the allantochorion, express very high levels of paternally derived major

Fig. 12.8. Endocrine roles of the eCG secreted by the specialized invading trophoblast cells of the chorionic girdle which transform in the endometrial stroma to become the endometrial cups. The LH-like component of eCG ovulates and/or luteinizes secondary follicles that are stimulated to grow in the maternal ovaries by continuing 10–12 day waves of pituitary FSH. eCG also stimulates the primary and secondary corpora lutea to secrete progesterone and oestrogens.

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histocompatibility complex (MHC) class I antigens when they invade the maternal endometrium to form the endometrial cups (Donaldson et al., 1990). Although this expression is greatly down-regulated within a few days after the cells transform into mature, eCG-secreting endometrial cup cells, it is nonetheless sufficient to induce a strong humoral response in the mare. Thus, anti-paternal lymphocytotoxic antibody appears in the serum of all mares carrying fetuses which differ at the MHC class I barrier within 2 weeks after initial development of the endometrial cups at days 36–40, and the titres often remain elevated to beyond term (Antczak et al., 1984). Hence, in a normal intraspecies equine pregnancy, the mare mounts strong immunological responses, both humoral and cell-mediated, to the paternally derived transplantation antigens (Antczak and Allen, 1989) and possibly also other species-related (Allen, 1975) or tissue-specific (Allen, 1982) foreign antigens of her fetus. Despite these potentially harmful reactions, the majority of pregnancies survive unscathed and proceed normally to term (Kydd et al., 1982), and in both interspecific (hybrid) equine pregnancies (e.g. female horse ×male donkey = mule; female donkey ×male horse = hinny) and extraspecific pregnancies created by embryo transfer (e.g. donkey-in-horse, horse-in-donkey and even zebra-in-horse), the cell-mediated reactions against the fetal endometrial cups are greatly increased so that the cups are destroyed prematurely, yet the very foreign fetuses can nevertheless develop normally and they are carried safely to term (Fig. 12.9; Allen, 1975; Kydd et al., 1985). It is only in the donkey-in-horse model of extraspecific pregnancy, and in which endometrial cups fail to develop due to abnormal growth of the donkey chorionic girdle in the horse uterus, that the majority (±70%) of fetuses die and are aborted between days 80 and 100 in conjunction with delayed and abnormal placentation and with an intense, generalized lymphocytic infiltration

Fig. 12.9. ‘Genetic drift’ in the nursery. Three surrogate pony mares (Equus caballus, 2n = 64) with their transferred extraspecific Przewalski’s horse (E. ferus przewalskii, 2n = 66), donkey (E. asinus, 2n = 62) and Grant’s zebra (E. burchelli, 2n = 46) foals. The results of these extraordinary pregnancies highlight the tolerance of the horse uterus and maternal immune system to the successful development of conceptuses from other member species of the genus Equus.

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throughout the area of endometrium in contact with the xenogeneic donkey allantochorion. Thus, it becomes appealing to speculate that some aspect of the expression of foreign fetal antigens by the invading chorionic girdle cells of the endometrial cups is involved importantly, but not exclusively, in both stimulating and controlling true placentation and in stimulating a protective, rather than a destructive, type of immune response from the mare towards its allogeneic or xenogeneic fetus (Kydd et al., 1982; Baker et al., 1999).

Placentation Commencing at day 40–42 after ovulation, a microvillous junction is established between the non-invasive trophoblast cells of the allantochorion and the lumenal epithelial cells of the endometrium, and simple, blunt villi of allantochorion begin to interdigitate with accommodating crypts or sulci in the surface of the endometrium (Samuel et al., 1975; van Niekerk and Allen, 1975). This very late, but none the less precise, ‘commencement of placentation’ in the mare is likely to be driven fundamentally by the tremendous up-regulation in the production of the mitogen, epidermal growth factor (EGF), which occurs between days 35 and 40 in the epithelium lining just the apical portions of the endometrial glands (Stewart et al., 1994). EGF is also likely to be responsible for stimulating the considerable growth and remodelling of the endometrium that must occur between days 40 and 150 to create the elongated and, eventually, very branched and complex endometrial sulci into which the equally complex and branched allantochorionic villi will interdigitate during development of the microcotyledons to supply the haemotrophic portion of placental exchange for the fetus during the second half of pregnancy (Fig. 12.10; Steven, 1982). Between adjacent microcotyledons, the endometrial glands continue to liberate their uterine milk which is taken up by specialized groups of pseudostratified trophoblast cells overlying the gland outlets known as aerolae (Amoroso, 1952; Steven 1982). Thus, just as in oestrus for the supply of watery, lavaging secretions (Le Blanc et al., 1989), early pregnancy for the supply of vital embryonic histotrophe (Stewart et al., 1995), mid-pregnancy for the supply of EGF to induce placentation (Stewart et al., 1994) and mid–late pregnancy for the continued supply of fetal histotrophe, a wealth of healthy and functional endometrial glands are essential for the mare to sustain both her fertility and her fecundity. The early studies of Kenney (1975) and Kenney and Doig (1986) and others described the progression of age-related degenerative changes that occur in the equine endometrium, and the more recent experiments of Bracher et al. (1996) illustrated graphically how fibrous deposition in the stratum compactum and around the basal portions of the endometrial glands may seriously disrupt placental development and thereby cause varying degrees of IUGR in the foal. Furthermore, the classical experiments of Walton and Hammond (1938) involving the reciprocal mating of Shire horses and Shetland ponies using artificial insemination, and more recent experiments in the

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Fig. 12.10. Histological section of the fetomaternal interface in a mare at day 83 of gestation. Branching villi of the allantochorion are closely interdigitated with elongated crypts or sulci in the surface of the endometrium. Note the areolus of pseudostratified trophoblast cells between two endometrial sulci (arrowed) for absorbing histotrophic endometrial gland secretions (×120).

authors’ laboratories involving the transfer of equine embryos to recipients that are appreciably smaller than the true genetic sire and dam of the embryo, have demonstrated convincingly that foals born small and weak due to IUGR occasioned by an inadequate placenta fail to show catch-up growth after birth and they remain runted throughout life (Walton and Hammond, 1938; Davies et al., 1985).

The Oestrogens of Pregnancy The synthesis and secretion of oestrogens figure prominently throughout equine pregnancy. As mentioned above, the young embryo begins to release appreciable quantities of oestrogen from as early as day 10 after ovulation when it is still moving actively throughout the uterine lumen, and both the trophectoderm and the yolk sac endoderm retain a very strong aromatizing capacity until at least day 34 (Heap et al., 1982). The studies of Stout and Allen (1996) and others have indicated that these early embryonic oestrogens are likely to play a pivotal role in bringing about maternal recognition of pregnancy and luteostasis in the mare, but further work is required to prove this point conclusively. A second wave of oestrogen secretion begins between days 35 and 40 after ovulation, coincidentally with the onset of secretion of eCG from the newly formed endometrial cups, but this time the hormone is maternal in

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origin. Peripheral plasma concentrations of both conjugated and unconjugated oestrogens remain basal and steady during the first month of pregnancy, but then show a sudden and sharp rise within 12–24 h after the first appearance of eCG in maternal blood (Terqui and Palmer, 1979). They plateau or decline slowly during the next 20–30 days but then begin to rise steeply again from around day 60–70. Experiments involving bilateral ovariectomy followed by maintenance of pregnancy with exogenous progestagen therapy (Terqui and Palmer, 1979) established the ovarian source of this second wave of oestrogen production, and more recent experiments have indicated that it is the gonadotrophic actions of eCG on the corpora lutea, not the follicles, in the maternal ovaries that give rise to the increases measured (Daels et al., 1991). The third wave of oestrogen production in the pregnant mare is the most dramatic and long lasting, although it is perhaps the most enigmatic in terms of its source and its biological functions. Beginning at around day 70 of gestation, the levels of conjugated oestrogens in both the blood and urine of the mare rise steadily and steeply over the next 150 days to reach microgram concentrations around day 200–240 (Fig. 12.11). Thereafter, they decline again steadily to a nadir just before birth of the foal at around 336 days (Cole et al., 1931; Cox, 1975; Raeside and Liptrap, 1975). These high concentrations are composed of the common phenolic oestrogens, oestrone and oestradiol, and the unusual and less biologically active ring β unsaturated oestrogens, equilin and equilinen, both of which are generated by placental aromatization of appropriate C-19 precursors, synthesized and secreted by the enlarged fetal gonads (Bhavnani et al., 1969, 1971; Bhavnani and Short, 1973a, b; Pashen et al., 1982; Raeside et al., 1982; Tait et al., 1982).

Fig. 12.11. Diagramatic summary of some major endocrinological changes and other events during pregnancy in the mare. Note the parallels in the enlargement and regression of the fetal gonads and the rise and fall of conjugated oestrogen concentrations in blood and urine.

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The rise and subsequent fall in oestrogen production during the second half of pregnancy are paralleled by a dramatic increase in size and subsequent regression of the fetal gonads which stems from a tremendous hyperplasia and hypertrophy of the interstitial cells of both the fetal testes and the fetal ovaries (Hay and Allen, 1975). Around day 220–240 of gestation, the gonads occupy as much as one-third of the abdominal cavity of the fetus (Cole et al., 1931) and they are considerably bigger than the now completely inactive maternal ovaries. The coincidence of the two events in equids, high oestrogen secretion rates and enlargement and regression of the fetal gonads, and their parallel with the production of large quantities of oestriol by the human fetoplacental unit utilizing C-19 precursors secreted by the fetal adrenal glands, threw suspicion upon the fetal gonads as being the source of the C-19 precursors in the pregnant mare (Bhavnani et al., 1969). This was verified subsequently by Pashen and Allen (1979) when they showed an immediate and precipitate fall in maternal plasma-conjugated oestrogen concentrations to baseline values following bilateral gonadectomy of the fetal foal between days 243 and 286 of gestation. Thus, equine pregnancy is heavily involved with oestrogen secretion and synthesis from start to finish. Initially, the free-living 10- to 16-day-old embryo secretes oestrone and oestradiol in remarkable quantities, presumably to preserve it in a progesterone-dominated uterus by suppressing the normal cyclical releases of PGF2α from the endometrium, and also perhaps to stimulate the marked increase in myometrial tone which holds the conceptus stationary from around day 17 and facilitates the uptake of uterine milk via the temporary areolae-like structures on the transitory choriovitelline membrane (Amoroso, 1952). Then, by utilizing its chorionic gonadotrophin to stimulate the maternal ovaries to secrete oestrogens, the fetus may be further preserving its existence by stimulating the onset of the dramatic series of architectural changes which must occur in both the allantochorion and the endometrium in order to achieve stable and functional placentation over the next 60–100 days (Steven, 1982). Finally, the fetus may be furthering its growth and setting the uterus up for its timely expulsion at term by the bucket-loads of oestrogen it produces during the second half of pregnancy.

Modern Breeding Technologies Legend relates that the technique of artificial insemination (AI) was first ever practised in the horse when an Arab chieftain ‘stole’ some semen from the vagina of a neighbouring chieftain’s mare that had been covered by a greatly prized stallion and, after a night time dash across the desert with the semen in a goat skin gourd, successfully impregnated his own mare. However, despite this early start, AI and other modern breeding technologies such as embryo transfer (ET) have been relatively slow to be developed and exploited commercially in horses compared with other farm species such as cattle, sheep and pigs (Pickett, 1995). Part of this apathy towards the use of AI in horses has

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stemmed from the worldwide blanket ban upon its use in Thoroughbreds bred for racing purposes and therefore requiring entry of the foal into the General Stud Book of the country of its birth in order to qualify for entry on to a racecourse in adult life. Similar bans have also been imposed by the Stud Book authorities of many other pure breeds of horses, from Shires to Welsh Ponies. However, despite this slow start, equine AI has shown a dramatic upsurge in use in all types of sport horses in the major horse-producing countries of the world over the past decade, and many hundreds of thousands, possibly millions, of agricultural horses in both the former Soviet Union and the Republic of China are nowadays inseminated artificially each year (Pickett, 1995). Collection of semen from the stallion is a relatively straightforward process, using a water-jacketed artificial vagina and either a mare in oestrus or an ovariectomized mare treated with exogenous oestrogen as the mount animal, or a mechanical ‘dummy mare’ to which most stallions will train readily. An important practical advance was made in Poland in the 1960s when Professor Wadislaw Bielanski and his colleagues in Krakow discovered that the stimulus to ejaculation in the stallion derives from pressure at the base of the penis rather than from sensory perception in the dilated glans penis (Tischner et al., 1974). From this observation, they designed the open-ended Polish artificial vagina (AV) which, apart from its simplicity and cheapness of construction, has two potential advantages. First, when collecting semen destined for deep-freezing, it is possible to collect only the first one or two sperm-rich pulses of the ejaculate and so avoid the need to centrifuge the whole ejaculate to get rid of the potentially harmful accessory sex gland secretions contained in the later ejaculatory squirts. Second, in stallions harbouring a bacterial infection in some part of their reproductive tract causing pus in the ejaculate, it is possible to collect each pulse of the ejaculate into separate sterile tubes. Bacterial culture of each sample can then give a much better idea of which parts of the reproductive tract are infected. Stallion spermatozoa can show remarkable longevity in the reproductive tract of the mare, and the early studies of Day (1942) demonstrated that, for stallions of average fertility, an interval as long as 3 days between a single mating or insemination and subsequent ovulation will maintain full fertility. Indeed, for some especially fertile stallions, an interval as long as 5 or even 6 days between mating and ovulation may still give high conception rates (R.E.S. Greenwood and P.D. Rossdale, personal communication). In a recent very elegant study in France in which normal, fertile mares were inseminated with semen from each of three stallions of known high fertility at various times and combinations of times before ovulation, molecular paternity testing of embryos recovered from the mares on day 15 after ovulation revealed an optimum interval of 2.6 days, not 1.0 or 2.0 days, between the single insemination and fertilization (Clément et al., 1999). This suggests the possibility that, as in sheep, pigs and other animals that have been studied intensively, a reservoir of a small number of spermatozoa at the ampullary isthmus junction of the oviduct is quickly established after insemination in the mare, which, in turn, initiates some sort of mechanism that prevents further spermatozoa derived

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from the same or subsequent inseminates from reaching that favoured fertilization site, thereby reducing the risk of polyspermy (Hunter and Nichol, 1988). Ball and colleagues demonstrated strong binding of stallion spermatozoa to the oviducal epithelium in the mare (Thomas et al., 1994). This causes temporary immobilization of the bound spermatozoa due to Ca2+ reflux between the two types of cell, which no doubt helps to prolong the life span and fertilizing capacity of the sperm. Also, Scott et al. (1999) showed by scanning and transmission electron microscopy that the many deep crypts and crevices which exist in the outer surface and mouth of the prominent uterotubal papilla in the mare act as a type of preliminary sperm reservoir from which the final ampullary isthmus-fertilizing population of sperm are derived. The longevity of stallion spermatozoa, both within and outside the mare, has enabled a worldwide upsurge in the use of cooled, shipped semen in recent years. Spermatozoa from the great majority of stallions of all breeds and types will survive adequately for 48 h, and sometimes up to 72 h, when diluted with a simple skim milk–glucose extender (Kenney et al., 1975) and maintained at 4°C. This has spawned the development of well-designed insulated containers that maintain a remarkably steady temperature of 4°C for up to 3 days (Equitainer, Hamilton Thorn, Massachusetts, USA), which is ample time for road, rail or air transport to any part of the country or continent, or indeed, anywhere in the world. High conception rates are achievable using cooled semen moved in this manner (Hellander, 1992), which has meant that, to date, cooled semen has tended to be used in preference to deep-frozen semen in most parts of the world. Like that of the other large volume ejaculator, the boar, stallion semen does not, on the whole, freeze well and there are clearly some major, and almost certainly genetic, differences between individual stallions in the freezability of their semen. Despite many studies over the past two decades aimed at developing improved methods of extending, deep-freezing and thawing stallion semen, no breakthroughs have occurred and the methods used nowadays have only minor modifications from those developed originally for bull semen. Glycerol remains the preferred cryoprotectant, egg yolk the source of energy for the spermatozoa, 0.5 ml straws the method of packaging, and freezing in liquid nitrogen vapour or in an automatic, computerized freezing machine, the method of preservation (Amman and Pickett, 1987). Most semen samples are centrifuged prior to addition of the freezing extender containing the cryoprotectant, and the recent development of the biological cushions on to which to centrifuge the spermatozoa have significantly reduced physical damage and have therefore improved fertility rates (Harrison, 1997). Similarly, the development of variable density gradients through which to centrifuge the semen sample, and so increase the population of highly motile spermatozoa available for freezing, have added another improvement to the practical freezing of stallion semen (Anderson and Grinsted, 1997). Nevertheless, the two fundamental problems of the genetically based variation in freezability of semen between individual stallions and the reduced life span of frozen–thawed spermatozoa in the mare’s reproductive tract

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remain real hurdles to the full exploitation of frozen semen in horse breeding programmes. The reduced life span has stimulated the very impractical and rather absurd management protocol whereby the ovaries of pre-ovulatory oestrous mares are scanned ultrasonographically as often as three or four times per 24 h in order to be able to inseminate the mare once only within no more than 6 h after ovulation has occurred and while the oocyte is assumed to still retain full fertilizing capabilities. An alternative and perhaps more practical approach is to induce luteolysis followed by oestrus in the mares to be inseminated using one injection of PGF2α or an analogue, given in dioestrus to animals in which the stage of the oestrous cycle can be calculated accurately, or the daily administration of progesterone-in-oil or a progestagen like altrenogest (Hoescht Rousel Vet, Bedfordshire, UK) for 8–10 days, plus an injection of PGF2α to induce luteolysis at the end of progestagen therapy, in mares in which the cycle stage is not known, all followed by the administration of an ovulation-inducing drug such as human chorionic gonadotrophin (hCG) (Chorulon; Intervet, Cambridge, UK) or a slow-release low-dose subcutaneous implant of GnRH analogue (Ovuplant, Peptide Technology, Sydney, Australia), given either when a ≥ 35 mm or larger dominant follicle is scanned in the ovaries, or ‘blind’ at a fixed time (i.e. 6 or 7 days) after the previous PGF2α injection to induce luteolysis, with a single insemination of the frozen–thawed semen given at a fixed time (e.g. 32–36 h) after the ovulation induction treatment (Duchamp et al., 1987). The simultaneous advent of the various hormone preparations required and the ultrasound scanner for monitoring follicular growth and ovulation have together made these fixed-timed treatment protocols feasible and accurate.

Embryo Transfer Due to the straight and readily distendable cervix in the dioestrous mare, coupled to the simple T-shaped equine uterus suspended in the abdominal cavity immediately beneath the rectum, the non-surgical flushing of an embryo from the uterus of a donor mare (Fig. 12.12) and its non-surgical transfer to that of a synchronized recipient mare are relatively simple manipulative procedures compared with the same manoeuvres in the cow. Yet, as for AI, ET and related technologies have been slow to be developed and commercialized in the horse, despite the enormous monetary valued placed on the leading animals in the major equine sporting disciplines. As with AI, the blanket ban on the use of ET and other related technologies in Thoroughbreds and many other pure breeds of horses by the relevant stud book authorities has been a major limiting factor in slowing development and application of these methods. Added to this, a number of equine physiological oddities also act to hinder the practical application of the methodology. First, and perhaps foremost, the mare’s resistance to the biological properties of her own chorionic gonadotrophin (eCG) due to abnormally low levels of specific receptors in her gonads and other tissues (Stewart and Allen, 1979),

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Fig. 12.12. Non-surgical embryo recovery in the mare on day 7 after ovulation. The flexible two-way catheter is passed through the easily distensible cervix and the cuff inflated with air to seal the internal os of the cervix. The whole uterus must be filled with flushing medium in order to pick up the highly mobile embryo.

combined with the unusual evolutionary developments of the ovulation fossa in her ovaries through which ovulation must occur, has, until now, effectively ruled out superovulation in donor mares as a practical option (Palmer et al., 1993). Single or multiple injections of even high doses of eCG are ineffective in stimulating multiple follicles (Allen, 1982), and even serial daily injections of partially purified extracts of equine pituitary gonadotrophin (CEG; Combarnous, 1982; Duchamp et al., 1987) given to cycling mares, (Squires et al., 1987; Palmer et al., 1993), or immunization of mares against the βsubunit of inhibin (McCue et al., 1993), do not effect superovulation. The multiple large follicles that occasionally develop in some treated animals appear to ‘compete’ with each other in their efforts to track through the ovarian stroma in order to reach the ovulation fossa, with the result that many of them become partially luteinized and fail to ovulate (Fig. 12.13). However, some mares are clearly exceptional, and individuals that can develop and ovulate up to eight follicles spontaneously in repeated oestrous cycles without any form of exogenous gonadotrophic stimulation have been reported (Brück et al., 1997). Other mares will respond to twice, instead of once, daily serial treatments with CEG

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Fig. 12.13. A summary of the two major problems that limit the commercial development of embryo transfer in horses: (i) the failure of the ovaries of donor mares to superovulate in response to exogenous gonadotrophic stimulation; and (ii) the long oviducal transport time which means the embryo may be already beginning to blastulate when it enters the uterus and is therefore more susceptible to damage when being deep-frozen or bisected to produce monozygotic twins.

and superovulate to produce as many as seven blastocysts when their uteri are flushed on the seventh or eighth day after ovulation (J. Alvarenga and E. Squires, personal communication). These recent improvements in responsiveness to gonadotrophin therapy are encouraging for the future. A second physiological phenomenon that causes trouble in equids is the exceptionally long oviducal transport time for embryos of 144–168 h (Battut et al., 1997). This is much longer than the 48–72 h oviducal transport times for pig and ruminant embryos (Moor, 1968) and it means that the equine embryo is already at the late (non-compacted) morula stage of development and, in some instances, may already be beginning to blastulate, when it enters the uterus (Fig. 12.13). Two problems stem from this. First, studies to date have

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demonstrated that success rates are closely related to embryo age and stage of development in attempts either to deep-freeze embryos in liquid nitrogen (Czlonkowska et al., 1985; Skidmore et al., 1991) or to bisect them for the production of monozygotic (identical) twins (McKinnon et al., 1989; Skidmore et al., 1989), with optimum results being obtained with non-blastulating morulae in both procedures. The sharp drop-off in success almost certainly relates to the very small size of the inner cell mass (ICM) of the horse embryo and hence its greater susceptibility to either physical (micromanipulation) or freezing damage, and the development of the tough, elastic blastocyst capsule between days 6 and 7 after ovulation which both complicates the cutting process for bisection (Skidmore et al., 1989) and may impede the passage of cryoprotectant into or out of the embryo during the freezing and thawing processes. The previously mentioned discovery of the role of embryonic PGE2 in stimulating oviducal transport in the mare (Freeman et al., 1991, 1992) and the consequential development of the laparoscopic application of PGE2 gel to the oviduct to hasten entry into the uterus (Robinson et al., 1999) holds great promise for the more efficient production of monozygotic twins. Quite apart from the intrinsic interest in, and research potential of, identical twins made in this manner (Fig. 12.14), the mere fact of being able to produce ‘two embryos from one’ and so double embryo production rate is potentially very significant in a species such as the horse which responds so poorly to superovulation procedures.

Fig. 12.14. Romulus and Remus. Monozygotic (genetically identical) twin horse foals produced by bisection of a day 6 morula followed by transfer of the two demi embryos to separate synchronized recipient mares.

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Embryo Technologies Attempts to date to produce horse embryos by conventional in vitro fertilization (IVF) of oocytes recovered from abattoir ovaries (Zhang et al., 1989), or from mature, gonadotrophin-treated pre-ovulatory follicles of living mares by the technique of transvaginal ultrasound-guided follicle puncture, have been remarkably unsuccessful, with only two known living progeny in existence at the time of writing (Palmer et al., 1991). Difficulties in either maturing the oocytes in vitro (IVM), or in inducing capacitation of stallion spermatozoa in vitro (Zhang et al., 1991) no doubt underlie this lack of success in the horse with a technique which nowadays is used routinely for the in vitro production of large numbers of cattle embryos using oocytes recovered from slaughterhouse ovaries and in human-assisted fertilization clinics using oocytes recovered from subfertile patients by transvaginal follicle puncture. The technique of intracytoplasmic sperm injection (ICSI), whereby a single immobilized spermatozoon is injected directly into the cytoplasm of the oocyte using a micromanipulator (Catt and Rhodes, 1995), may be the technological advance needed to break through the present log-jam in the advancement of equine embryo transfer. Live foals have been produced at three laboratories by this method during the past 3 years using oocytes recovered from preovulatory follicles in oestrous mares (Squires et al., 1996; McKinnon et al., 1999), or from large, mature follicles developing in the ovaries of pregnant mares between 40 and 70 days of gestation (Cochran et al., 1999). The method obviates the need for capacitation to be induced in the injected spermatozoon and it opens the way for the production of multiple embryos for transfer to suitable recipient mares using very small numbers of frozen–thawed or epididymal spermatozoa.

Gender Selection of Spermatozoa Another major technological breakthrough in recent years which could have an important practical application in horse breeding programmes is the newfound ability to sort mammalian spermatozoa into X chromosome (female)- or Y chromosome (male)-bearing populations with a high degree of accuracy, by passing them at high speed through coordinated laser beams in a fluorescence-activated cell sorting (FACS) instrument (Johnson, 1995). The ability to pre-determine the sex of the foal at the time of fertilization has been a ‘holy grail’ of horse breeding for many generations past, since horses of one gender tend to out-shine and out-perform those of the other gender in certain competitive horse disciplines. For example, castrated males (geldings) predominate in the sport of 3-day eventing, whereas mares are much preferred by polo players. Thus, considerable sentimental and commercial pressures exist to produce foals of pre-determined gender. One potential limitation to the practical application of gender-sorted spermatozoa in horse breeding is the considerable disparity which exists

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between the maximum rate of sperm sorting that can be achieved with existing FACS machines (e.g. 3–5 ×106 sperm h−1) and the widely accepted minimal dose of motile spermatozoa required to achieve high fertility rates when using conventional intrauterine insemination methods (300–500 ×106 spermatozoa per dose; Pickett and Voss, 1975). However, another recent experiment has yielded hope of a practical solution to the problem, i.e. the use of the flexible strobed light videoscope to achieve simple, visually controlled passage up the uterine horn in lightly sedated mares maintained in stocks (Bracher et al., 1992), where doses of Ficoll-treated stallion spermatozoa as low as 1 ×106, suspended in 50–100 µl of TALP medium containing bovine serum albumin (BSA) and deposited on to the prominent uterine papilla of the uterotubal junction in the uterine horn ipsilateral to the ovary containing a mature, hCG-treated preovulatory follicle, will give conception rates as high as 64% (Morris et al., 2000). Future developments of this practical low-dose insemination method using gender-sorted stallion spermatozoa that has either been ejaculated normally, or may have been recovered from the epididymis of the testes of castrated colts, should produce a powerful and much needed tool for the production of genetically superior offspring from combinations of high performing female and male horses, where the former is still in active athletic competition and the latter may well have been castrated in early life.

Conclusions As can be discerned from the information in this chapter, the horse and the horse industry present many challenges to the implementation of assisted reproductive techniques in horse breeding, and therefore in genetic improvement. However, it is also true that most of the technology that has been applied successfully to domestic livestock and to human reproduction has been demonstrated to work in the horse. The high value assigned to individual horses in many breeds makes the application of reproductive technologies economically feasible in many cases, and expertise in equine reproduction is widespread around the world. Thus, the potential is great for applying the combined information from the emerging genetic maps of the horse with the most modern techniques in reproduction to the propagation and improvement of the horse.

Acknowledgements The authors are grateful to Misses Sally Thake and Sandra Wilsher for their assistance in preparing this manuscript. Mr John Fuller kindly drew the figures.

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immunisation against inhibin may become a new method of control of folliculogenesis and ovulation rate in mares. Journal of Reproduction and Fertility (Suppl. 56), in press. Oguri, N. and Tsutsumi, Y. (1972) Non-surgical recovery of equine eggs and an attempt at non-surgical egg transfer in horses. Journal of Reproduction and Fertility 31, 187–195. Oriol, J.G., Sharom, F.J. and Betteridge, K.J. (1993a) Developmentally regulated changes in the glycoproteins of the equine embryonic capsule. Journal of Reproduction and Fertility 99, 653–664. Oriol, J.G., Betteridge, K.J., Clarke, A.J. and Sharom, F.J. (1993b) Mucin-like glycoproteins in the equine embryonic capsule. Molecular Reproduction and Development 34, 255–265. Osborne, V.E. (1966) An analysis of the pattern of ovulation as it occurs in the annual reproductive cycle of the mare in Australia. Australian Veterinary Journal 42, 149–154. Palmer, E., Bezard, J., Magistrini, M. and Duchamp, G. (1991) In vitro fertilisation in the horse. A retrospective study. Journal of Reproduction and Fertility (Suppl. 44), 375–384. Palmer, E., Hajmeli, G. and Duchamp, G. (1993) Gonadotrophin treatments increase ovulation rate but not embryo production from mares. Equine Veterinary Journal (Suppl. 15), 99–102. Pashen, R.L. and Allen, W.R. (1979) The role of the fetal gonads and placenta in steroid production, maintenance of pregnancy and parturition in the mare. Journal of Reproduction and Fertility (Suppl. 27), 499–509. Pashen, R.L., Sheldrick, E.L., Allen, W.R. and Flint, A.P.F. (1982) Dehydroepiandrosterone synthesis by the fetal foal and its importance as an oestrogen precursor. Journal of Reproduction and Fertility (Suppl. 32), 389–397. Pickett, B.W. (1995) The stallion: retrospective analyses and opinions. Biology of Reproduction, Monograph Series 1, 547–564. Pickett, B.W. and Voss, J.L. (1975) The effect of semen extenders and sperm number on mare fertility. Journal of Reproduction and Fertility (Suppl. 23), 95–98. Raeside, J.L. and Liptrap, R.M. (1975) Patterns of urinary oestrogen excretion in individual pregnant mares. Journal of Reproduction and Fertility (Suppl. 23), 469–475. Raeside, J.L., Gofton, N., Liptrap, R.M. and Milne, F.J. (1982) Isolation and identification of steroids from gonadal vein blood of the fetal horse. Journal of Reproduction and Fertility (Suppl. 32), 383–387. Ricketts, S.W. (1975) Endometrial biopsy as a guide to diagnosis of endometrial pathology in the mare. Journal of Reproduction and Fertility (Suppl. 23), 341–345. Robinson, S.J., Neal, H. and Allen, W.R. (1999) Modulation of oviductal transport in the mare by local application of prostaglandin E2. Journal of Reproduction and Fertility (Suppl. 56), in press. Rowlands, I.W. (1963) Levels of gonadotrophin in tissues and fluids with emphasis on domestic animals. In: Cole, H.H. (ed.), Gonadotrophins: Their Chemical and Biological Properties and Secretory Control. Freeman, San Francisco, pp. 74–107. Samuel, C.A., Allen, W.R. and Steven, D.H. (1975) Ultrastructural development of the equine placenta. Journal of Reproduction and Fertility (Suppl. 23), 575–578. Scott, M.A., Liu, K.M., Overstreet, J.W. and Enders, A.C. (1999) The structural morphology and epithelial association of spermatozoa at the uterotubal junction: a descriptive study of equine spermatozoa in situ using scanning electron microscopy. Journal of Reproduction and Fertility (Suppl. 56), in press.

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Developmental Genetics 1 and F. Stewart2 A. Ruvinsky Developmental 13 A. Ruvinsky andGenetics F. Stewart 1Animal Science, SRSNR, University of New England, Armidale, NSW 2351, Australia; 2TBA Equine Fertility Unit, Woodditton Road, Newmarket CB8 9BH and Developmental Genetics, The Babraham Institute, Babraham, Cambridge CB1 2HN, UK

Introduction Developmental Stages of the Horse Embryo Genetic Control of Pre-implantation Development Maternal regulation of early development Genome activation Embryonic gene expression Trophoblast gene expression Gametic imprinting Maternal Recognition of Pregnancy and Placentation Maternal recognition of pregnancy Development of the placenta Genes Involved in Control of Morphogenesis Gastrulation Notochord formation Hox genes and development of axial identity Organogenesis: T-box and Pax genes Muscle development Developmental effects of coat colour mutations Sex Determination The major steps in gonad differentiation SRY gene and sexual differentiation Cycle of the X chromosome Sex reversals in the horse Development of Interspecies Hybrids Description of interspecies hybrids Contribution of equine hybrids to developmental genetics References ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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A. Ruvinsky and F. Stewart

Introduction Extensive investigation of mammalian development during recent years has contributed significantly to a better understanding of developmental genetics in general. However, the vast majority of information concerning mammalian development has been generated through the use of mouse genetics. Therefore, it may appear premature to write a chapter entirely devoted to developmental genetics of the horse. On the other hand, there are a number of studies on the embryology and genetics of development in the horse which, when placed on a general background of mammalian development, could provide a useful basis for future research in this area. Despite the high level of similarity in mammalian development, there are numerous contrasts between species resulting from their morphological differences, placental structure, longevity and schedule of development. These distinctive features of development based on genetic differences, which have accumulated for tens of millions years of independent evolution, are still awaiting clarification and understanding. Studying the differences often can be very informative and we hope that gathering the available information on the horse now will be a helpful reference and will provide a basis for future comparisons and reviews on developmental genetics in this species.

Developmental Stages of the Horse Embryo Gamete maturation and fertilization, which comprise the first crucial steps in each new developmental cycle in mammals, have been considered in the previous chapter. The embryological events and their genetic determination which follow fertilization are discussed here. Table 13.1 summarizes the essential events and timing of conceptus and fetal development in the horse. Since fertilization occurs within several hours of ovulation (Thibault, 1967; Morris et al., 2000) and ovulation can be timed accurately in the horse, days of gestation are always counted from the day of ovulation. In this chapter, day 0 is taken as the day of ovulation and fertilization is assumed to occur within 24 h of ovulation. Three consecutive stages of development have been defined: vesicular, embryonic and fetal (Table 13.1). The vesicular (ovum) period covers the first 15–16 days after ovulation. It is characterized by several crucial events including cleavage, morula formation and compaction, blastocyst development, gastrulation and the start of extraembryonic mesoderm migration (Van Niekerk and Allen, 1975; Betteridge et al., 1982; Bezard et al., 1989; Ginther, 1992). After compaction of the morula at about day 4.5, the conceptus enters the uterus between days 6 and 7 after ovulation and coincidentally begins to undergo blastocyst formation. Tight intercellular junctions develop and this provides a condition for accumulation of fluid within the central cavity (the blastocoele). The majority of external cells of the blastocyst, called trophoblast or trophoectoderm, develop an epithelial phenotype and are concerned with development of the amnion and

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Developmental Genetics Table 13.1.

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Essential events and timing of prenatal development in the horsea.

Stage of development Vesicular (ovum) period Two-cell Eight-cell Genome activation Morula compaction Capsule formation Entry into the uterus Trophoblast differentiation Blastocyst formation Hatching from zona pellucida Migration of endoderm complete Embryonic disc Loss of polar trophoblast Gastrulation (primitive streak) Mesoderm migration Notochord Maternal recognition of pregnancy First somite pair Embryonic period Neural groove Head fold Closing of neural tube Vascularized yolk sac Beating heart and allantoic bud Visible limb buds Capsule ruptures Optic and otic vesicles visible Chorionic girdle formation Chorionic girdle invades Well-developed tail and elongating limbs Fetal period Chorioallantoic placentation Sex determinable Hooves Further development of limbs Eyelids close Head and neck in normal position First hair on lips Hair coat Birth

Days after ovulation 0–15 1 3 2–3 4.5 6 5–6 6–7 6–7 7–8 8 11–12 12 13–14 13 14 12–14 15 16–40 16 18 18 19 21 23 22–23 26–28 26–35 35–36 38 40–term from 40 40 45–50 60 60 80 100 270 320–340

Cells/stage/crlb 2 8 4–8 ~16 32 16–32 32–64 64+ ~200 200+ 1000+ Thousands Thousands Thousands Thousands Thousands Thousands 14 somites 16 somites 16 somites 18 somites 24 somites 40+ somites 40+ somites Vertebrae developing Vertebrae developing Vertebrae developing crl = 2 cm crl = 2.5 cm crl = 2.5 cm crl = 3–4 cm crl = 6 cm crl = 6 cm crl = 11 cm crl = 19 cm Maturing fetus (Considerable variation)

aCompiled

from: Ewart (1897, 1915); Van Niekerk and Allen (1975); Betteridge et al. (1982); Bezard et al. (1989); Cruz and Pedersen (1991); Ginther (1992); Bazer et al. (1993); Enders et al. (1993); Guillemot et al. (1995); Hafez (1993); Jainudeen and Hafez (1993); Ménézo and Renard (1993); Brinsko et al. (1995); Grondahl and Hytell (1996). bcrl = crown–rump length.

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placenta. The remaining cells, located at one pole beneath the polar trophoblast, form the embryoblast or inner cell mass (ICM). Later during gastrulation, the ICM differentiates into the three primary germ layers of the embryo; ectoderm, mesoderm and endoderm. In addition to having a zona pellucida, the equine conceptus is enveloped by an acellular capsule which starts to develop beneath the zona pellucida at about day 6 after ovulation and disappears between days 22 and 23 (Betteridge et al., 1982). Therefore, when the blastocyst ‘hatches’ from its zona pellucida between days 7 and 8, the capsule persists and expands along with the conceptus so that, by about day 18, it has increased in mass approximately 20-fold, probably due to both fetal and maternal contributions (Oriol et al., 1993). This embryonic capsule not only protects the conceptus physically but is also thought to play a role in communicating with the mother and in promoting the mobility of the conceptus which is important for transmitting the maternal recognition of pregnancy signal (Bazer et al., 1986; Chu et al., 1997; see below). The capsule also ensures that the conceptus remains spherical, and without it the equine conceptus would probably elongate as in the ruminant species. By day 15, the conceptus is about 2 cm in diameter, the embryonic disc and migrating extra-embryonic mesoderm are visible to the naked eye and the first pair of somites have formed. Therefore, despite its vesicular appearance and lack of placental attachment, embryogenesis is well underway. We therefore define the embryonic period as beginning on day 16. During the embryonic period (days 16–40), the yolk sac placenta forms but, although the capsule disappears on about day 23, the conceptus remains more or less spherical and it does not start to elongate significantly until after day 40. Furthermore, although it lodges at the base of one or other of the uterine horns at about day 18 and, when the capsule has disappeared, makes close contact with the maternal endometrium, the true chorioallantoic placenta does not start to form until about day 40. Nevertheless, embryogenesis proceeds at a rate comparable with other large mammals, and essential morphogenetic events, such as development of the head, vertebrae and appendages, occur, as well as development of the nervous system, blood circulation and other major internal organs. The most thorough description of early embryogenesis in the horse can be found in Professor Cosar J. Ewart’s publications (Ewart, 1897, 1915). These papers, which contain detailed descriptions and drawings, have been largely forgotten but they are well worth consulting. To give an example, Fig. 13.1 shows a selection of drawings from his 1915 paper illustrating the external features of a day 21 horse embryo. This paper concentrates on the first 3 weeks and gives an accurate account of the development of the extraembryonic membranes, nervous system, sense organs, alimentary canal, heart and blood vessels. Sex determination also occurs during the late embryonic period, when the crown–rump length of the embryo reaches about 2.5 cm. Furthermore, just prior to transition into the fetal stage at day 40, a discrete portion of the chorion known as the chorionic girdle (Ewart, 1897) invades the maternal endometrium to form the so-called endometrial cups (Allen and Moor, 1972).

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Fig. 13.1. Drawings of a 21-day horse conceptus and embryo (from Ewart, 1915). (A) Section through the left uterine horn of the pregnant mare showing the intact, spherical conceptus in very close contact with the endometrial lining of the uterus (approx. 75% of natural size). (B) Appearance of the 11 mm embryo after removal of the chorion showing the vitelline veins (vv) and left vitelline artey (va). (C) Side view of the embryo showing the right vitelline vein (vv), the amnion (am), the branchial arches (ba), the fonto-nasal process (fp), the depression or stomodaeum (s) which will form the mouth and the heart (ht). (D) Dorsal and ventral views of the embryo showing 20+ somites, the neural canal with its opening to the exterior at the caudal end (np), the amnion (am) and the otic vesicles (ov).

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These structures secrete large amounts of equine chorionic gonadotrophin (eCG) into the maternal bloodstream (Cole and Goss, 1943) to ensure maintenance of progesterone secretion from the maternal ovaries (Amoroso et al., 1948). However, the endometrial cups are completely detached from the fetal membranes and have generally died and been sloughed off the surface of the endometrium by day 120 (Allen and Stewart, 1993). The transition from embryo to fetus is generally considered to be when organogenesis is complete and the taxonomic order of the embryo starts to become identifiable through external features. In most species, this coincides with establishment of the chorioallantoic placenta. Therefore, in agreement with others (Allen et al., 1982; Ginther, 1992), we define the fetal stage as beginning on day 40 and continuing to term (days 320–340). The first significant event in the fetal period is that the expanding chorioallantois (or allantochorion) starts to interdigitate with the endometrium to form the chorioallantoic placenta and thus takes over from the regressing yolk sac placenta. The mature placenta of the mare is diffuse, epitheliochorial and microcotyledonary in structure and it gradually elongates to completely fill the body and both horns of the uterus (Samuel et al., 1974, 1976). Haemotrophic nutrition takes place within the microcotyledons but histotrophic nutrition, involving the absorption of endometrial gland secretions by columnar trophoblast cells within the inter-cotyledonary regions, also plays an important role throughout gestation. Supplied by this placental nutrition, the fetus undergoes numerous morphological changes and essential maturational events to prepare it for postnatal life. While gestation length in mares normally ranges from 320 to 340 days, it varies considerably both within and between breeds, and viable foals can be born as early as day 300 and as late as day 399 (Hintz et al., 1979; Vanderplassche, 1980; Jainudeen and Hafez, 1993). Comparing the developmental events and regulation of genes during embryogenesis and fetal development in the horse with other farm mammals can be useful (Cockett, 1997; Pomp and Geisert, 1998, Ruvinsky and Spicer, 1999). In terms of placental development, the horse is most similar to the pig and dromedary camel, both of which have an epitheliochorial placenta.

Genetic Control of Pre-implantation Development Maternal regulation of early development There is considerable evidence in lower species that very early development is controlled by maternal factors stored in or made by the oocyte (Gurdon, 1992; St Johnston and Nüsslein-Volhard, 1992; Nüsslein-Volhard, 1996). This has not been studied in the horse, but information from laboratory species has shown that morphogen gradients also exist in the mammalian oocyte which are believed to be important in specifying major polarities (Holliday, 1990; Fulka et al., 1998).

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Recent data indicate that the leptin and STAT3 proteins play critical roles in early mammalian development, and may be involved in the determination of the animal pole of the mammalian oocyte and in the differentiation of the trophoblast and ICM (Antczak and Van Blekom, 1997). A potential role for these proteins in early development is indicated at the morula stage where the ‘inner’ cells consist of blastomeres that contain little, if any, leptin/STAT3 while ‘outer’ cells contain both leptin/STAT3-rich and -poor cells (Antczak and Van Blekom, 1997). It was also shown in the mouse that tropomyosin, an actinbinding cytoskeletal protein, becomes associated both with the blastomere cortex after fertilization and with the cleavage furrow during cytokinesis. The interphase cortical association is uniform until the eight-cell stage, when tropomyosin becomes associated with the developing apical pole and is excluded from the basolateral cortex. Thus the early mouse conceptus contains a unique and specific set of tropomyosins which respond to polarizing signals (Clayton and Johnson, 1998). Increasing cell polarity was described at the eight-cell stage in both the mouse and rat (Gueth-Hallonet and Maro, 1992). Cell fate, controlled by positional information, seems reversible and provides the developing embryo with a certain degree of flexibility. In cattle, cell polarization has occurred in some blastomeres at the eight- to 16-cell stage, but typical, distinct polarity is not evident until after the 16-cell stage with approximately 40% polar cells per embryo (Koyama et al., 1994) Available data indicate a low level of embryonic gene expression in the mouse during the first few cell divisions (Davis and Schultz, 1997). It is likely, however, that replication of DNA and maintenance of the majority of cell functions during the first two or three divisions is provided by RNAs and proteins accumulated during oocyte maturation. oviducal proteins are also believed to be involved. For example, oestrus-associated glycoprotein (EGP) has been shown to influence early cleavage rate (Nancarrow and Hill, 1995). The vast majority of transcripts studied from the one-cell stage onwards show the same pattern of expression, with the number of copies per embryo declining from the one- to two-cell stage and then increasing dramatically (reviewed by Kidder, 1993).

Genome activation Electron microscopy studies suggest that activation of the horse embryonic genome commences between the two- and four-cell stage (Grondahl et al., 1993; Grondahl and Hyttel, 1996). In six- and eight-cell embryos, further activation of the genome has been traced using [3H]uridine incorporation and autoradiography (Brinsko et al. 1995; Grondahl and Hyttel, 1996). In 16-cell embryos and beyond, transcriptionally active compact fibrillo-granular nucleoli are evident (Grondahl and Hyttel, 1996), suggesting that nucleolar activation, leading to the synthesis of rRNA and other nuclear RNAs, is initiated during the fourth cell cycle. Figure 13.2 shows the poly(A) RNA profile during

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Fig. 13.2. Dynamics of poly(A)+ RNA during the pre-implantation development of the mouse embryo (from Ménézo and Renard, in Reproduction in Mammals and Man 1993, with permission of Ellipses).

pre-implantation development in the mouse embryo. A similar picture is expected for other mammalian embryos, including that of equids. The major anticipated difference between species would be in time scale. Little is known about development of the protein profile during early equine embryogenesis, but comparative data obtained in other domestic and laboratory mammals are probably applicable to the horse. Shehu et al. (1996) showed that lamin B in bovine embryos appears as a constitutive component of nuclei at all pre-implantation stages, whereas lamins A and C have a stagerelated distribution. The nuclei from the early cleavage stages contain lamins A and C which generally disappear later, with a few possible exceptions in the morula and blastocyst. Other proteins essential for morphogenetic events appear in developing bovine embryos. These include several cytoskeletal and cytoskeleton-related components such as F-actin, α-catenin and E-cadherin. These proteins first appear on day 6 and their appearance and polarized distribution are related to compaction of the morula (Shehu et al., 1996). Data from the pig suggest that several more morphogenetically important proteins appear during early cleavage and compaction, such as actin and the actin-associated proteins α-fodrin, vinculin and E-cadherin (Reima et al., 1993). These molecules are distributed evenly in blastomeres during early cleavage but then accumulate gradually in regions of intercellular contact towards the blastocyst stage (Reima et al., 1993).

Embryonic gene expression Blastocyst formation creates two different cell lineages: non-polarized inner mass cells and polarized trophectoderm or outer cells with prominent

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microvilli. Louvet et al. (1996) showed in mice that this process is accompanied by specific redistribution of the actin-associated protein ezrin, which is proposed to play a role in the formation of microvillous structures that are crucial for normal implantation. Before morula compaction, ezrin is located around the cell cortex. However, after blastocyst formation, it segregates to the outer trophectoderm cells which have microvilli. Two phosphorylated forms of ezrin are present from the ovum period throughout pre-implantation development, but they gradually decrease in amount. A third non-tyrosinephosphorylated isoform appears at the eight-cell stage and increases to the blastocyst stage (Louvet et al., 1996). Several other actin-associated proteins (α-fodrin, vinculin and E-cadherin), which are involved in cytokeratin bundles, are not observed until the early blastocyst in both the mouse and the pig (Reima et al., 1993). E-cadherin cell adhesion function is essential for the establishment and maintenance of epithelial cell morphology during embryogenesis and adulthood. Mouse embryos homozygous for a targeted mutation of the gene show severe abnormalities before implantation, because dissociation of adhesive cells of the morula has occurred shortly after compaction and their morphological polarization is then destroyed. Maternal E-cadherin is able to initiate compaction, but cannot maintain the process (Riethmacher et al., 1995). Significant defects in the cell junctional and cytoskeletal organization were found in E-cahedrin null mouse embryos, and the trophectoderm layer failed to differentiate (Oshugi et al., 1997). Although the rate of embryonic development is quite different in mice and pigs, there is a close correlation between the developmental stage and cytoskeletal organization in both species. Likewise, in the expanded bovine blastocyst, the distribution of several cytoskeletal and cytoskeleton-related proteins appeared similar (Shehu et al., 1996). Extracellular fibronectin was first detected in the early blastocyst before differentiation of the primitive endoderm, and at this stage was localized at the interface between the trophectoderm and extra-embryonic endoderm (Shehu et al., 1996). Cingulin, the tight junction peripheral membrane protein, also contributes to morphological differentiation in early mouse development and it is likely that other mammals have the same gene. Its synthesis is tissue-specific in blastocysts, is up-regulated in the trophectoderm and down-regulated in the ICM (Javed et al., 1993). It is commonly accepted that proto-oncogenes are involved in numerous processes of embryonic development and that they encode a series of nuclear transcription factors, intracellular signal transducers, growth factors and growth factor receptors. For example, activation of the c-fos and c-jun protooncogenes in sheep conceptuses occurs during the period of rapid growth and elongation (Wu, 1996), and a similar pattern probably occurs in equine embryos. These proto-oncogenes are involved in the regulation of gene expression, cell proliferation and differentiation. Information on the expression of housekeeping genes during equine embryonic development is sparse but it is likely to be similar to that observed in bovine embryos. The mRNA levels for various studied genes in bovine

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embryos remain constant or decrease slightly from the mature oocyte to the six- to eight-cell or morula stage and increase greatly in blastocysts. These changes in gene expression were significant ranging from two- to sixfold to 110- to 118-fold (Bilodeau-Goeseels and Schultz, 1997). However, caution is required when extrapolating from one species to another, particularly if the species belong to different orders. For example, the equine embryonic exon 1 complementary DNA (cDNA) sequence from cytochrome P450 aromatase has no significant homology with the corresponding region in porcine, human and bovine sequences. This finding indicates divergence in the regulatory motifs of this portion of the gene amongst mammals and suggests potential significance with regard to activation of the ‘embryonic’ promoter and/or splicing of novel aromatase 5 ′ exons in the transient production of oestrogens by pre-implantaion blastocysts (Choi et al., 1996). A developmental switch in expression from the blastocyst to endometrial/placental-type cytochrome P450 aromatase gene described in horses may function in embryo–maternal signalling at the peri-implantation stage (Choi et al., 1997). Two equine genes significantly activated between 12 and 15 days of pregnancy were cloned recently. The first of them encodes calcyclin, which belongs to the family of calcium-binding proteins. A similar protein was found in mouse decidua and trophoblast. Expression of calcyclin increases approximately 30-fold from day 12 to 15. The other gene, that for phospholipase A2 (PLA2), is involved in release of arachidonic acid needed for prostaglandin, thromoxane and leukotriene synthesis. Multiple transcripts of PLA2 were detected and they appeared to be differentially regulated in day 12 and day 15 conceptuses (Simpson et al., 1999). Many efforts have been made to try and ascertain the relative importance of internal versus external factors in controlling the development of preimplantation embryos. Most experiments with rodents show that it is unlikely that pre-implantation development is significantly dependent on external factors. Furthermore, none of the known endogenously produced factors and their receptors are essential until the blastocyst stage (Stewart and Cullinan, 1997). However, later during development, the importance of growth factors increases dramatically. It was found that some of the regulatory substances secreted by the uterus can act as growth factors. Together with a number of growth factors and their receptors produced by the embryo itself, they create the medium essential for development. A detailed review of these regulators of mammalian embryonic development can be found elsewhere (Schultz and Heyner, 1993). In equine embryos, transcripts for insulin-like growth factor 2 (IGF-2) were present at all examined stages (14–150 days). They were found predominantly in tissues of mesodermal origin, but also in the endoderm-derived liver and epithelia of the gut and lung bronchioles, and the ectoderm-derived facial mesenchyme and choroid plexus (Lennard et al., 1995a). Data suggest that the equine IGF2 gene is under developmental control, with the possible existence of several promoters (Joujou-Sisic et al., 1993).

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As in all mammals, the equine ovum and early conceptus are protected by a covering called the zona pellucida which is shed at blastocyst ‘hatching’. In addition, as mentioned above, the equine zygote lays down another covering beneath the zona pellucida, called the embryonic capsule, which is composed of mucin-like glycoproteins. After hatching from the zona pellucida, the capsule expands along with the equine conceptus but then appears to rupture at about day 23; fragments of it have been seen as late as day 25 (Enders et al., 1993). Therefore, in addition to surviving on the simple absorption of uterine secretions during the first 3 weeks of pregnancy, these nutrients must pass through the capsule. It is therefore not surprising that a number of progesterone-dependent endometrial proteins have been observed in the mare (Zavy et al., 1982; Beier-Hellwig et al., 1995; McDowell et al., 1995). One of these, a novel 19 kDa protein has been characterized and shown to be a member of the lipocalin family, most of which are transport proteins (Crossett et al., 1996). This protein was discovered originally in the capsule of early horse conceptuses (Stewart et al., 1995a) and later shown to be present in yolk sac fluid up to day 20 of gestation (Crossett et al., 1998). The protein has not yet been found in any other species, but homologues of it may exist and may only be needed in small quantities for a short period of time. The reason that it is so plentiful in the mare may be due to the presence of the capsule and the need to transport a labile, maternal factor through it.

Trophoblast gene expression Differentiation of trophoblast cells, the first differentiation event during mammalian embryonic development, provides the key tissue for development of the fetal–maternal interface during implantation and placentation. Reviews of current knowledge about the genetic control of trophoblast development and implantation (Cross et al., 1994; Rinkenberger et al., 1997; Schultz and Edwards, 1997) are based largely on studies in mice. However, many features of these processes are common for the majority of eutherian mammals and to some degree are applicable to the horse. At present, 44 loci with a range of functions have been implicated in pre- and peri-implantation events (Rinkenberger et al., 1997). A basic helix–loop–helix (bHLH) transcription factor gene, Hxt, later named Hand1, is expressed in early trophoblast and in differentiated giant cells of mouse embryos (Cross et al., 1995) and is essential for placentation (Firulli et al., 1998; Riley et al., 1998). The negative HLH regulator, Id-1, inhibited rat trophoblast (Rcho-1) stem cell differentiation and placental lactogen-I transcription. These data indicate a role for HLH factors in regulating trophoblast development and demonstrate a Hand1-positive function in promoting formation of trophoblast giant cells. A preliminary study has demonstrated very strong expression of the Hand1 gene in equine trophoblast cells, particularly the chorionic girdle cells (F. Stewart and J.C. Cross, unpublished data) and it is therefore almost certainly involved in the differentiation and/or

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growth of equine trophoblast cells. A separate gene, Hed, encodes a related protein that is expressed in maternal decidium surrounding the implantation site (Cross et al., 1995). Another transcription factor gene, Mash-2, situated in a locus homologous to the achaete/scute complex genes in Drosophila, is also essential for successful placentation in mice. Its expression begins during pre-implantation development but is restricted to the trophoblast lineage after the blastocyst stage (Nakayama et al., 1997). This murine locus belongs to the quite rare category of imprinted genes (Guillemot et al., 1995). Mouse embryos which inherit a mutant Mash-2 allele from the mother and a normal allele from the father die after implantation. The cause of death is a lack of placental spongiotrophoblast (McLaughlin et al., 1996). The MMp9 gene, which is involved in development of giant trophoblast cells in mice (Newman- Smith and Werb, 1997), is another candidate for an imprinted gene. A specific form of imprinting manifests itself in the trophoblast of all species studied whereby the paternally derived X chromosome is inactivated preferentially in the trophoblast of female embryos (reviewed by Goto and Monk, 1998). Furthermore, a recent study involving targeted disruption of the X-linked homeobox gene Esx 1, whose expression is restricted to extra-embryonic tissue, showed that expression of this gene from the maternal allele is necessary for normal trophoblast morphogenesis in mice (Li and Behringer, 1998). Genetic determination of integrin trafficking, which regulates adhesion to fibronectin during differentiation of mouse peri-implantation blastocyst, has been studied by Schultz et al. (1997). The regulation of several metalloproteinase and corresponding genes may also shed additional light on the process of implantation and further trophoblast development (Bass et al., 1997; Das et al., 1997). However, since placental development in rodents is very different from that in the horse, further research will be needed to determine if these murine trophoblast genes are relevant to equine trophoblast development. A recent study has identified an aspartic proteinase expressed by equine trophoblast cells (Green et al., 1999). It is a member of the pregnancy-associated glycoprotein (PAG) family and the horse is the first species outside the Artiodactyla order (cattle, camels and pigs) in which a PAG has been identified. Monoclonal antibodies raised against equine trophoblast cells have identified a number of potentially interesting equine trophoblast proteins (Antczak et al., 1987). Two of the antibodies have identified proteins of 115 and 66 kDa, respectively, in equine trophoblast tissue, and both antibodies stained human trophoblast cells (Oriol et al., 1991). Significant efforts have also been made to study developmental dynamics of class I and class II major histocompatibility complex (MHC) antigens in trophoblast and endometrial cells in the horse. The MHC class II antigens were not detected on any trophoblast cells, but endometrial cells expressed them. On the other hand, it was found that MHC class I antigens were expressed at high density on the surface of the trophoblast cells of the chorionic girdle at days 32–36, but these were down-regulated as the cells invaded the maternal endometrium (Donaldson et al., 1990, 1992). This phenomenon is considered to be involved in the mechanism which

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prevents maternal immune rejection of the fetal–placental unit. The expression of MHC class I genes may be controlled at the transcriptional level in horse invasive and non-invasive trophoblast cells (Maher et al., 1996). As expected, high concentrations of IGF-2 mRNA were detected not only in the embryo itself but also in the extra-embryonic mesoderm, invasive chorionic girdle and mature endometrial cup tissue (Lennard et al., 1995a). Distribution of four cytokines in the endometrium and trophoblast of the horse between days 30 and 55 of gestation showed that only tumour necrosis factor-α (TNF-α) was present in the trophoblast cells. This cytokine might have an important role in regulating trophoblast–endometrial interactions. The other three cytokines studied, interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon-γ (IFN-γ) were not detected in trophoblast (Grunig and Antczak, 1995). The terminally differentiated, trophoblast-derived, endometrial cup cells secrete large quantities of the dimeric glycoprotein hormone, eCG (see Chapter 12). There is now considerable evidence that the genes for the α- and β-subunits of this hormone start to be expressed in the progenitors of the cup cells, the chorionic girdle cells (McDowell et al., 1993; Wooding and Flint, 1994), and that their expression is linked to the cells becoming binucleate and invasive. This is analogous to the situation in primates where the CG genes are expressed in terminally differentiated syncytiotrophoblast. Equids and primates are the only species which secrete a true chorionic gonadotrophin during pregnancy, and both have a single glycoprotein hormone α-subunit gene which is expressed in the pituitary gland and the trophoblast. However, unlike primates which have evolved a separate family of trophoblast-specific β-subunit genes via duplication of an ancestral luteinizing hormone (LH) β-subunit gene (Talmadge et al., 1984), equids express their LH (pituitary) β-subunit gene in trophoblast cells (Sherman et al., 1992; Chopineau et al., 1995). Thus, strictly speaking, equids secrete a placental (chorionic) LH. Furthermore, unlike humans, and probably all primates, where CG is essential for the recognition and maintenance of pregnancy, it does not appear to be essential for pregnancy maintenance in equids (Stewart and Allen, 1995; see Chapter 12).

Gametic imprinting The fundamental assumption of Mendelian genetics is that the behaviour of an allele is identical whether it arrives in a zygote through the paternal or maternal germline pathway. Gametic imprinting phenomena discovered in mammals show limitations of this classical view. Two sources of evidence were essential to describe gametic imprinting. The first approach, based on genetic evidence, demonstrated that some maternally and paternally derived regions of certain chromosomes were not equivalent. Paternal or maternal disomy of the regions containing particular genes caused significant effects on viability and development of progeny (Lyon and Glenister, 1977; Cattanach,

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1986). The second set of data was obtained by nuclear transplantations and parthenogenetic activation of mammalian oocytes. These data showed that the contribution of parental genomes was not equivalent, and differential imprinting of nuclear genes during gametogenesis was very likely (McGrath and Solter, 1983; Surani et al., 1984). Until now, the main bulk of information regarding imprinting comes from the mouse and to a lesser extent from human studies (Barlow, 1995). The number of loci found in mice showing gametic imprinting currently is over 35 (Beechey and Cattanach, 1999). Gametic imprinting is generally viewed as a mammalian phenomenon and there are differences in imprinting patterns between species. The developmental function of gametic imprinting is still under intensive investigation, but an explanation proposed by Moore and Haig (1991) is widely spread. It is based on the idea of involvement of imprinted genes in the control of fetal growth and fetal–maternal interactions, thus keeping a balance between contradictory fetal and maternal requirements. It is therefore possible that gametic imprinting evolved in mammals to regulate intrauterine growth to ensure a safe outcome of pregnancy. Details of the molecular mechanisms responsible for gametic imprinting are not entirely understood, but in several instances it has been shown that imprinting ‘marks’ are imposed on the control regions of imprinted genes during gametogenesis by a parent-specific DNA methylation process (Shemer et al., 1996; Bartolomei and Tilghman, 1997). These marks are resistant to global demethylation during cleavage and de novo methylation after implantation and also maintain different methylation patterns in the paternal and maternal alleles of imprinted genes (Solter, 1998). Acquisition of imprints is believed to occur before fertilization and imprint propagation takes place until the morula–blastocyst stage. It seems likely, however, that primary gametic signals are not simply copied from the gametes, but rather methylation patterns typical for imprinted genes are established gradually during early development (Shemer et al., 1996). Gametic imprinting is likely to have evolved in mammals by adopting already existing epigenetic mechanisms. The latest data indicate that imprinting in mammals and gene silencing in Drosophila may have some similarities (Surani, 1998). Imprinting is a reversible phenomenon and can be achieved only if erasure of imprinting signals occurs in each consecutive developmental cycle. The erasure occurs in primordial germ cells and, soon after that, new epigenetic modifications occur at specific CpG nucleotides in imprinted genes (Goto and Monk, 1998; Surani, 1998; Ruvinsky, 1999). Data on farm animals are still limited and include some indirect evidence of gametic imprinting (reviewed by Ruvinsky, 1999). A recent comparative study of normal and parthenogenetic embryos in sheep is the first direct evidence that in sheep, as in mice and humans, the growth-related PEG1/Mest and IGF2 genes are expressed from the paternal alleles (Feil et al., 1998). Thus, there are convincing indications that gametic imprinting is a common phenomenon in mammalian species, including equids.

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A study of eCG concentration in the serum of pregnant mares and jenny donkeys carrying intraspecies and interspecies embryos indicated that production of the hormone is influenced by gametic imprinting (Allen et al., 1993). It was suggested that differential expression of maternal and paternal alleles may control the size and secretory activity of the fetal endometrial cups, the structures that secrete eCG. However, embryo bisection to create identical twin embryos and their subsequent transfer to different species showed that any such effect was overridden by the maternal uterine environment (Allen et al., 1993). Nevertheless, imprinting of, for example, the IGF2 gene, may contribute to placental and/or fetal growth effects in mules and hinnies. Similar phenomena were observed in mice (reviewed by Ruvinsky, 1999). The equine IGF2 gene recently has been cloned and characterized (Otte et al., 1998). It spans a 9 kb region, which is substantially less than the corresponding human gene. Three coding exons and three untranslated leader exons, all highly homologous to those in other species, were identified. Downstream of the polyadenylation site in exon 6, a dinucleotide repeat sequence was identified. Three putative promoters (P1–P3) were localized in the 5 ′ region of the gene. RNase protection analysis revealed two active promoters in fetal tissues, P2 and P3, whereas P3 was the only promoter active in adult tissues. This represents a transcriptional pattern different from that in humans and rodents. A novel structural element, an inverted repeat, is predicted in the 3 ′ region of the IGF2 gene. This repeat is conserved between species and located in a region which is differentially methylated in the human and mouse genes and might therefore be involved in the imprinting mechanism. The inverted repeat acquires a stem–loop structure in vitro with a hybrid A/B-DNA conformation in the stem area. In both horse and mouse, a methylation-sensitive protein binds this structure with a strong requirement for the loop area. Furthermore, the protein might be developmentally regulated (Otte et al., 1998). The IGF2 gene was mapped to ECA12q13, which appears to be homologous to human chromosome 11 (Raudsepp et al., 1997) and murine chromosome 7. Interestingly this gene tends to maintain a terminal location on the chromosome arm in a number of mammalian species. Lack of maternally or paternally derived alleles in a zygote causes, in several instances, embryonic mortality and should therefore impose strict requirements on the stability of imprinting signals. Successful cloning of mammals using somatic cells of adult individuals (Wilmut et al., 1997) is the first evidence of stability of differential imprinting signals maintained in somatic cells long after intrauterine development. It is possible that knowledge about the influence of the pathway (paternal or maternal) used by an allele to enter the next generation will be adopted sooner or later by selection programmes. Selection of modifier genes may significantly change the effect of gametic imprinting, and this information should also be taken into consideration.

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Maternal Recognition of Pregnancy and Placentation Maternal recognition of pregnancy The mare must recognize the presence of the embryo in her uterus before day 12 after ovulation in order to prevent luteolysis of the primary corpus luteum (CL). If she fails to do so, the CL will have regressed completely by day 16, thereby withdrawing the progesterone support needed for pregnancy. As in most species, the mechanism by which luteolysis is prevented in equine pregnancy involves suppression of the release of prostaglandin F2α (PGF2α) from the uterus. The embryonic signal(s) which prevent this release have not been characterized fully in the mare, but several candidate factors, based on studies in other species, have been investigated (Bazer et al., 1994). In the pig, which has a similar type of placentation to the horse, the antiluteolytic signals include embryonic oestrogens and the equine conceptus has been shown to release significant amounts of oestrogens between days 10 and 30 (Heap et al., 1982; Zavy et al., 1984). However, attempts to prolong the life span of the CL in the mare by injecting oestrogens have provided inconclusive results (Goff et al., 1993). It has now been established that conceptus-derived interferon-tau (IFN-τ) constitutes the major recognition signal in most ruminants (Bazer et al., 1994). However, repeated attempts to find an equivalent gene or protein in the horse have failed (Roberts et al., 1992; see Chapter 12) and embryonic oestrogens remain the front running, although as yet unproven, luteostatic mechanism in the pregnant mare. One reason why it has proved difficult to identify a specific embryonic factor in the mare may be the presence of the embryonic capsule. Most studies to date have excluded the capsule, yet it is this structure that is in closest contact with the maternal endometrium. The embryonic signals must pass through the capsule and this may involve interactions and/or modifications which are essential for correct presentation and/or interaction with the endometrium. Mobility of the equine conceptus within the uterine lumen is certainly important in the signalling process as restricting this movement results in pregnancy failure (McDowell et al., 1988). In addition to the primary CL, the pregnant mare develops several accessory CLs in her ovaries in response to the eCG produced by endometrial cups. However, although these help to maintain elevated maternal blood levels of progesterone until the placenta takes over the role between days 80 and 100 (Holtan et al., 1975), secondary luteal development is not absolutely necessary (Allen and Stewart, 1993; see Chapter 12).

Development of the placenta Placentation in the mare is a prolonged process, involving a transient yolk sac placenta, which becomes fully functional from about day 23 when the capsule

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has disintegrated, and the gradual development of the chorioallantoic placenta from around day 40. The time of implantation is therefore very difficult to define in the mare and is considered by some to begin at about day 25, and by others on day 40. Furthermore, since the equine conceptus does not ‘implant’ in the true sense, the term implantation is probably best avoided in this species. Equine placentation is also characterized by the development of the unique chorionic girdle and its invasion of the maternal endometrium to form the gonadotrophin-secreting endometrial cups (see Chapter 12). There have been few studies on genes that control development of the yolk sac, but gene knock-out experiments in mice have demonstrated profound effects of removing fibronectin (George et al., 1993), vascular endothelial growth factor (VEGF; Carmeliet et al., 1996; Ferrara et al., 1996), VEGF receptors (Fong et al., 1995; Shalaby et al., 1995), transforming growth factor-β1 (TGF-β1) (Dickson et al., 1995) and the GATA transcription factors (Koutsourakis et al., 1999). Although these factors have not been studied in the horse, they are very likely to be involved in establishing a functional, vascularized yolk sac placenta in this species. In eutherian mammals, the allantois fuses with the chorion to form the allantochorion. The allantois first apppears in the equine conceptus at around day 22. Unlike humans and rodents, it is fluid filled and lined with endoderm so that the equine allantochorion is composed of three layers; trophoblast, vascularized allantoic mesenchyme and endoderm. Fusion of the chorion with the allantoic mesenchyme is a crucial step in all species and, in mice, it has been shown to depend on the expression of vascular cell adhesion molecule-1 (VCAM-1) (Gurtner et al., 1995; Kwee et al., 1995) and α4 integrin (Yang et al., 1995). By day 25, the allantoic sac of the still-spherical equine conceptus is about one-third the size of the yolk sac and, by day 30, the two sacs are more or less equal in size. It is during this period that the chorionic girdle develops on the surface of the conceptus, precisely where the underlying yolk and allantoic sacs abut one another. The trophoblast cells in the girdle region multiply rapidly and pile up on one another to form a 3–4 mm white band of tissue which encircles the entire conceptus. The mechanisms that control the development of this equine-specific structure are not entirely clear, but they must depend largely on local, fetally derived mitogenic signals. The allantoic mesenchyme is the most likely source of these signals (Stewart, 1996) and expression of two such signals, IGF-2 and hepatocyte growth factor–scatter factor (HGF-SF), has indeed been identified in horse allantoic mesenchyme (Lennard et al., 1995a; Stewart et al., 1995b). HGF-SF is probably the more important of the two, since both HGF-SF and HGF-SF receptor (c-met) knock-out mice fail at around mid-gestation due to a major placental defect involving the lack of labyrinthine trophoblast (Bladt et al., 1995; Schmidt et al., 1995; Uehara et al., 1995). Maternal growth factors, such as TGF-β1 (Lennard et al., 1995b) and epidermal growth factor (EGF) (Lennard et al., 1998), may also be involved in chorionic girdle formation but are likely to play only a permissive role in the process.

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After the chorionic girdle has detached from the fetal membranes to form the endometrial cups, the allantochorion at last begins to interdigitate with the maternal endometrium to form, eventually, the very complex microvillous structure that is needed to ensure fetomaternal exchange until term. Both fetal and maternal mitogenic and morphogenic factors obviously continue to be important during this process.

Genes Involved in Control of Morphogenesis Gastrulation Gastrulation in the horse starts on days 13–14 of development (Ginther, 1992). Cell proliferation and rearrangement in the germinal disc are the main events during gastrulation in eutherian embryos. The visible indication of commenced gastrulation in the horse conceptus is formation of the primitive streak. In mammals, ‘this process begins with the production and proliferation of mesodermal progenitor cells at the proximal (allantoic) end of the primitive streak; this position marks the future caudal end of the fetus. As ectodermal cells migrate through the primitive streak, they move both laterally and distally towards the future cranial end of the embryo, extending the primitive streak towards the distal lip’ (Wilkins, 1993). A white peripheral zone, which can be seen encircling the germinal disc at day 14 in the horse, is mesoderm growing beneath the trophoblast layer (Van Niekerk and Allen, 1975; Ginther, 1992). While genetic mechanisms that are responsible for gastrulation in mammals are still mainly unknown, new data are starting to arrive (Viebahn, 1999). The next step, establishing anterior–posterior orientation, recently became the subject of intensive investigations (Beddington and Robertson, 1998). Two genes, the homeobox gene goosecoid (gsc) and the winged-helix gene hepatic nuclear factor-3β (HNF-3beta) are co-expressed in all three germ layers in the anterior primitive streak and at the rostral end of mouse embryos during gastrulation (Filosa et al., 1997). Fgf-4, a member of the fibroblast growth factor (FGF) gene family which shows expression in the primitive streak and a sequential expression in developmental pathways such as mesoderm formation and myogenesis, is believed to play a role in specific epithelial– mesenchymal interactions (Niswander and Martin, 1992). The recently discovered murine Axin gene seems to be a crucial regulator in embryonic axis formation in vertebrates. This gene inhibits the Wnt/ Wingless signalling pathway, involving several polypeptides and enzymes (Zeng et al., 1997). This pathway plays an important role not only in embryonic development but also in tumorigenesis. Interaction of axin protein with glycogen synthase kinase-3β is required for β-catenin down-regulation. β-Catenin and axin are positive and negative effectors of the Wnt signalling pathway, respectively (Nakamura et al., 1998). The T gene, which is required for extension of the posterior axis (Clements et al., 1996) and for several other essential steps in mammalian

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development, including notochord formation, is discussed below. The next step in development is the so-called ‘head process’, which gives rise to the notochord and contributes to part of the endodermal lining of the gut.

Notochord formation The notochord is a rod-shaped structure which extends along the embryo and represents the initial axial skeleton, playing an important role in induction of the neural plate, chondrogenesis and somite formation (Gomercic et al., 1991). Early development of the notochord in the equine embryo has not been studied, but is estimated to begin at about day 14 after ovulation. Its histological appearance in the embryo at day 21 is described in detail by Ewart (1915) and also in a three-dimensional reconstruction of Ewart’s embryo (Robinson and Gibson, 1915). Clearly, activation of nuclear genes responsible for basic morphogenetic rearrangements is requisite for notochord formation and development. The T gene, which was first described as the Brachyury mutation in mice 70 years ago, is an important participant in events required for differentiation of the notochord and formation of mesoderm during posterior development. The T protein is located in the cell nuclei and acts as a tissue-specific transcription factor (Kispert et al., 1995). Cloning and sequencing of the T gene led to the discovery of the T-box gene family, which is characterized by a conserved sequence, called the T-box (Bollag et al., 1994). This ancient family of transcription factors which underwent duplication around 400 million years ago is common to all vertebrates (Ruvinsky and Silver, 1997). There are indications that several murine T-box genes are essential in different mesodermal subpopulations and one is essential in early endoderm during gastrulation (Papaioannou, 1997). Involvement of the T-box genes Tbx2-–bx5 in vertebrate limb specification and development was shown recently (Gibson-Brown et al., 1998). Formation of the notochord leads to several key ontogenetic events including induction of the neural tube and development of the gut, heart and brain. A putative morphogen secreted by the floor plate and notochord, Sonic hedgehog (Shh), specifies the fate of multiple cell types in the ventral aspect of the vertebrate nervous system. Shh, in turn, induces expression of the oncogene Gli-1, which affects later development of dorsal midbrain and hindbrain (Hynes et al., 1997).

Hox genes and development of axial identity The homeotic genes, which encode transcription factors, were first described in Drosophila as the primary determinants of segment identity. They all contain a similar 180 bp DNA sequence motif named the homeobox. Comparative analysis of the Drosophila homeotic gene complex called HOM-C and the mammalian (murine) homeobox genes called the Hox complex demonstrates

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a striking case of evolutionary conservation. The Hox gene family determines a set of transcription factors crucial for development of axial identity in a wide range of animal species (Maconochie et al., 1996). Figure 13.3 shows the striking similarity and colinearity found in the molecular anatomy of the insect and mammalian (vertebrate) Hox complexes. The main difference is the number of complexes per genome. In insects, there is only one, while mammals and other vertebrates have four paralogous sets of genes. Although only two homeobox genes belonging to the H6 homeobox gene family, HMX1 and HMX2, have been studied in the horse (Stadler et al., 1995), they both show a high degree of homology to the equivalent genes in other species. This suggests that the main features of the murine Hox complexes would be typical for the homologous equine gene complexes. The Hox genes are expressed in a segmental fashion in the developing somites and central nervous system, and each Hox gene acts from a particular anterior limit in a posterior direction. The anterior and posterior limits are different for different Hox genes (Fig. 13.3). The genes located at 3 ′ end have the most anterior limit of activity. Transcription of the genes, however, moves in the usual 5 ′–3 ′ direction. The genes located at the 3 ′ end are expressed earlier and genes located at the 5 ′ end are expressed later. The process of segmentation moves along the anterior–posterior axis, but there are differences in development of segmentation between the hindbrain and the trunk (Maconochie et al., 1996). Thus, the vertebrate body is, at least partially, a result of interactions of Hox genes that provide cells with the essential positional and functional information. Signals from the Hox genes force embryonic cells to migrate to the appropriate destination and generate certain structures. Retinoids can affect the expression of Hox genes, and there is a 5 ′–3 ′ gradient in responsiveness of Hox genes to retinoids (Marshall et al., 1996). A key role for the neural crest as the source for numerous cell lineages, including sensory neurons, glial cells, melanocytes, some bone and cartilage cells, thyroid cells and smooth muscle, is well known (Le Douarin, 1982). There has been considerable progress during the past few years in identifying genes controlling development of the neural crest and associated cell migration (Anderson, 1997). Several growth factors affect the developmental fate of neural crest cells: glial growth factor (GGF), TGF-β which promotes smooth muscle differentiation, and a bone morphogenic protein (BMP2/4) involved in bone morphogenesis. Transcription factors are also important in neural crest lineage determination, including the bHLH transcription factors Mash1 and Mash2 (Anderson, 1997).

Organogenesis: T-box and Pax genes Some of the T-box genes are involved in limb morphogenesis and specification of forelimb/hindlimb identity. It was shown that Tbx5 and Tbx4 expression is restricted primarily to the developing fore- and hindlimb buds, respectively. These two genes appear to have been selected divergently in vertebrate

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Fig. 13.3. (A) Alignment of the four mouse Hox complexes with that of HOM-C from Drosophila. The vertical shaded boxes indicate related genes. The 13 paralogous groups are noted at the bottom of the alignment. The colinear properties of the Hox complexes with respect to timing of expression, anteroposterior (A-P) level, and retinoic acid (RA) response are also noted at the bottom (from Maconochie et al., 1996, with the author’s permission). (B) Summary of HOM-C and Hox-2 expression patterns. The upper part of the figure is a diagram of a 10 h Drosophila embryo with projections of expression patterns of different genes from the HOM-C complex to particular body segments. The lower part of the figure is a diagram of a 12 day mouse embryo with projections of expression patterns of different genes from the Hox-2 complex to particular body segments (from McGinnis and Krumlauf, 1992, with the author’s permission).

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evolution to play a role in the differential specification of fore- (pectoral) versus hind- (pelvic) limb identity (Gibson-Brown et al., 1998). Mutations in the human TBX3 gene cause the ulnar–mammary syndrome characterized by posterior limb deficiencies or duplications, mammary gland dysfunction and genital abnormalities. It was suggested that TBX3 and TBX5 evolved from a common ancestral gene and each has acquired specific yet complementary roles in patterning the mammalian upper limb (Bamshad et al., 1997). Pax genes are another family of developmental genes encoding nuclear transcription factors. They contain the paired domain, a conserved amino acid motif with DNA-binding activity. Pax genes are key regulators of development in organs and structures such as the kidney, eye, ear, nose, limb muscles, vertebral column and brain. Vertebrate Pax genes are involved in pattern formation possibly by determining the time and place of organ initiation or morphogenesis (Dahl et al., 1997). Pax-1, for instance, is a mediator of notochord signals during the dorsoventral specification of vertebrae (Koseki et al., 1993). The Pax-3 gene may mediate activation of MyoD and Myf-5, the myogenic regulatory factors, in response to muscle-inducing signals from either axial tissues or overlying ectoderm and may act as a regulator of somatic myogenesis (Maroto et al., 1997). Mutations in the Pax-2 gene prove involvement of this gene in eye formation, as mutations in Pax-6 result in eye malformation, known as aniridia in humans and small eye syndrome in mice, (Dahl et al., 1997). Aniridia in horses, which has been described many times in several breeds, may also be caused by changes in the homologous gene (Joyce et al., 1990; see Chapter 4). The eyes absent gene (Eya2), which is involved in eye development in several metazoan phyla, may also be relevant to horse development. Like the Pax-6 gene family, Eya2 was probably recruited for visual system formation some considerable time after its original function was established (Duncan et al., 1997). Several other genes, such as Bmp-4, Msx-1 and Msx-2, which encode bone morphogenetic proteins and are expressed before and after neural tube closure, interact with Pax-2 and Pax-3 (MonsoroBurq et al., 1996).

Muscle development Information on development of muscle tissue in the horse is also important from a practical point of view. Firulli and Olson (1997) have reviewed recent progress relating to genetic mechanisms of muscle development in mammals. Skeletal, cardiac and smooth muscle cells express overlapping sets of musclespecific genes, although some genes are only expressed in one particular muscle type. So-called genetic modules or independent cis-regulatory regions are required to direct the complete developmental pattern of expression of individual muscle-specific genes within each muscle type, and the temporospatial specificity of these regulatory modules is established by unique combinations of transcription factors (Firulli and Olson, 1997). A gene encoding an actin-modulating protein, gelsolin, from equine smooth muscle recently was

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cloned and studied (Koepf et al., 1998). Comparison with human gelsolin has shown a high degree of identity (94–95%). The same observation is true for several other compared mammalian species. It is well established that mitogens inhibit differentiation of skeletal muscle cells, but the IGFs, acting through a single receptor, stimulate both proliferation and differentiation of myoblasts. For example, an inhibitor of mitogenactivated protein (MAP) kinase inhibits IGF-stimulated proliferation of L6A1 myoblasts and associated events, such as phosphorylation of the MAP kinases and elevation of c-fos mRNA and cyclin D protein. This inhibitor caused a dramatic enhancement of differentiation, evident at both a morphological and a biochemical level. In sharp contrast, an inhibitor of phosphatidylinositol 3-kinase and p70 S6 kinase completely abolished IGF stimulation of L6A1 differentiation. These data demonstrate that the MAP kinase pathway plays a primary role in the mitogenic response and is inhibitory to the myogenic response in L6A1 myoblasts, while activation of the phosphatidylinositol 3-kinase/p70(S6k) pathway is essential for IGF-stimulated differentiation. Thus, it appears that signalling from the IGF-1 receptor utilizes two distinct pathways leading to either proliferation or differentiation of muscle cells (Coolican et al., 1997). Selection for greater muscle mass in horses may use some mutations affecting muscle development. Hyperkalaemic periodic paralysis is the result of one such mutation in the sodium channel gene, which is expressed in skeletal muscle. It is inherited as an autosomal dominant trait. The classical signs of this syndrome are muscle fasciculation, spasm, and weakness associated with hyperkalaemia (Naylor, 1994, see Chapters 4 and 8).

Developmental effects of coat colour mutations Classical coat colour genetics in mammals has acquired developmental and molecular orientation (Jackson, 1994). Most data were obtained in mice, but the high homology of mammalian genomes provides a sufficient foundation for extension to other species including the horse (see Chapter 3). Colour mutations give excellent examples of numerous pleiotropic effects. One of the reasons for this phenomenon is that mutations of several coat colour loci affect normal development of the neural crest region, which plays a key role in migration of melanoblasts, neuroblasts and other cell types (Anderson, 1997). Details about migration of melanoblasts and neuroblasts in the horse are unknown but it probably occurs within a week or so after day 18. A recent molecular genetic analysis of Overo lethal white syndrome (OLWS) in horses connected this dominant autosomal disorder with a mutation in the endothelin receptor B (EDNRB) gene and neural crest migration (Santschi et al., 1998). It was found that a substitution in codon 118 of the equine EDNRB gene changes the characteristics of the transmembrane domain 1 of a seven transmembrane domain G-protein-coupled receptor. This mutation causes not only white coat colour in homozygous foals, but also

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intestinal aganglionosis (Hultgren, 1982) which results in a poorly developed enteric nervous system and rapid death of the foal soon after birth. It is most likely that disturbances in neural crest cells caused by this mutation explain this phenomenon in horses. Similar syndromes in humans and mice have been described and induced in mice by mutation in the same gene (McCabe et al., 1990). Mutations which affect melanocyte morphology and create dilute colours are common in mammals. For example, it was shown that mutations in a myosin protein (Jackson, 1994), which may be caused by a proviral insertion (Jenkins et al., 1981), led to lack of dendrites in melanocytes and diluted coat colour. Interestingly, neuronal dendrites are not affected by these mutations. Several other mutations affect melanogenic enzymes and related proteins. Mutations in the tyrosinase gene lead to albino phenotype, while brown colours, at least in mice, are the product of mutations in a locus encoding a tyrosinase-related protein (Jackson, 1994). Different pleiotropic effects of these mutations, including decreased viability, have also been observed. Mutations of two other coat colour loci, agouti and extension, affect regulation of melanogenesis. It was shown that the ratio between black eumelanin and yellow phaeomelanin is regulated by α-melanocyte-stimulating hormone (αMSH). The product of the agouti gene is an antagonist of αMSH and the extension gene encodes the αMSH receptor. Again, there are several developmental effects of mutations in these loci including obesity and tumour formation. The chestnut coat colour in different horse breeds which is controlled by a recessive allele in the extension locus (e/e) is the result of a mutation in the MSH receptor gene (MC1R). A missense mutation at codon 83 (TCC→TTC) which causes a non-conservative substitution (Ser→Phe) in the MCIR protein is associated with the e allele (Marklund et al. 1996). This mutation probably alters the α-helix structure of the second transmembrane domain of the protein, leading to a defect in the receptor. A similar mutation has been described in humans (Valverde et al., 1995). Obviously molecular analysis of coat colour mutations provides good opportunities for a greater understanding of basic developmental processes.

Sex Determination The major steps in gonad differentiation The earliest stages of gonadal development in mammals occur at a similar stage in XX and XY embryos. Primordial germ cells, which differentiate relatively late in mammals, migrate into the gonads of either presumptive sex indiscriminately and may function even across a species barrier (McLaren, 1998, 1999). To be functional, a gonad needs both germ cells and somatic cells. Assuming that gonadal development in the horse does not deviate strongly from that of the mouse and other mammals, one may expect that a few dozen germ cells, originating from the proximal region of the embryonic

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ectoderm, start their journey inside the embryo along with the invaginating hindgut. A recent study in mice showed that expression of Bmp4 (bone morphogenetic protein 4 gene) in the trophectoderm layer which is in closest contact with the epiblast is responsible for the differentiation of both the primordial germ cells and the allantois (Lawson et al., 1999). If a similar mechanism operates in the horse, BMP4 would presumably be produced by the polar trophoblast cells which overlie the inner cell mass before the former are lost, i.e. before day 12 (Enders et al., 1993). Due to ongoing proliferation, a significant number of germ cells reach the genital ridge, which consists of a thin layer of mesenchymal cells located between the coelomic epithelium and the mesonephros. Two genes, Sf1 and Wt1, are particularly important in development of the murine genital ridge (McLaren, 1998). Eventually, four different cell lines comprise the genital ridge: primordial germ cells, somatic steroidogenic cells, supporting cells and connective tissue. The fate of each lineage depends on the sexual determination of the embryo in which they develop, and their structure, function and pattern of genetic activity is quite different in testes and ovaries. A study using alkaline phosphatase staining, to investigate the distribution of primordial germ cells in early equine embryos (Curran et al., 1997), detected only one or two positive cells in embryos at day 20 and about 3267–2424 cells at days 28–30. However, unlike other species, a large number of these positive cells (72% at day 28) were found in the vascular system and other organs with only 28% in the genital ridges, suggesting possible germ cell migration via the vascular system (Curran et al., 1997). It was known long ago that sex determination in mammals depended on the presence or absence of the Y chromosome. Embryos without a Y chromosome develop as females and those with a Y chromosome develop as males. The breakthrough in molecular understanding of sex determination and differentiation in the mouse and human (Goodfellow and Lovell-Badge, 1993) paved the way for other mammals including the horse. In humans and mice and probably other mammals, gonadal sexual differentiation starts relatively late in embryonic development, and morphological differences in XY embryos appear prior to XX embryos. In the horse, sexual differentiation of the gonads occurs by about day 40 (Table 13.1). It is likely that this differentiation starts in males several days earlier than in females (Merchant-Larios, 1979). Entry of the oocytes into meiotic prophase occurs later during gestation. The first meiotic prophase begins in equine fetal ovary cells at days 60–70. Later, at 150–200 days, oocytes in early meiotic stages fill the ovarian cortex (Deanesly, 1977). The great majority of the oocytes first involved in meiotic divisions disappear, and only a small number of them later develop into primordial follicles. Spermatocytes enter meiosis during postnatal life and it was found recently that the murine male genital ridge at about 12 days post-coitum produces a factor that inhibits entry of germ cells into meiosis (McLaren and Southee, 1997). Testicular development is a key element in establishing mammalian sex. The chromosomal constitution determines the migration of cells into gonads

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and the final differentiation into a testis or an ovary (Hunter, 1995). Testicular development in mammals is triggered by a gene on the Y chromosome encoding the testis-determining factor (TDF), or sex determining region of the Y chromosome (SRY). In genetic males, this factor induces differentiation of Sertoli cells (reviewed by McLaren, 1991) and secretion of anti-Müllerian hormone (AMH). AMH, which belongs to the TGF-β family, causes regression of Müllerian ducts, promotes development of Wolffian ducts and the differentiation of Leydig cells which secrete the male steroid hormone, testosterone (Behringer, 1995). Testosterone binds to androgen receptors, which in turn act as transcription factors. Further details about AMH and its activity in bovine development are presented elsewhere (Cate and Wilson, 1993). Differentiation of somatic cells into steroidogenic cells takes place in horse embryos very early during development. The seminiferous cords of the developing testis are completely segregated from the steroidogenic tissue by basal lamina, while in the medulla of the ovary, steroidogenic cells differentiate inside the epithelial cords which contain germ cells (Merchant-Larios, 1979; Knospe and Budras, 1992; Knospe, 1998). Details about further sexual differentiation in equids are not available, but in bovine fetuses, regression of the Müllerian ducts occurs in males between 50 and 80 days of development (Vigier et al., 1984). A whole chain of developmental events follows, and the phenotype typical for males arises. In females, Müllerian ducts develop, no Leydig cells form in the gonad, no testosterone is produced and gonad development steadily moves towards the female phenotype. The female developmental programme is therefore the ‘default’, while the male programme requires switching on of the SRY gene followed by a cascade of activation of autosomal genes. From around day 80 of gestation, the gonads of both male and female equine fetuses undergo extraordinary proliferation and growth such that, by day 250 of gestation, they may weigh as much as 50 g each and are usually much larger than the maternal ovaries (Hay and Allen, 1975). Proliferation of the interstial cells causes this enlargement and these cells secrete large quantities of C-19 steroids which are aromatized in the placenta to both the phenolic oestrogens, oestrone and oestradiol and the unique ring B unsaturated oestrogens, equilin and equilinin. Beyond day 280, the gonads begin to regress so that at birth they are of normal size and histological appearance. Bilateral fetal gonadectomy results in retarded growth of the fetus and abnormal parturition, thereby suggesting that the large quantities of oestrogens produced by the equine fetoplacental unit are involved in fetal growth and in preparing the uterus for parturition (Pashen and Allen, 1979).

SRY gene and sexual differentiation The testis-determining role of the SRY gene in mammals is widely accepted after the experiments performed in the early 1990s (reviewed by Goodfellow and Lovell-Badge, 1993). Available data also suggest that the cell-autonomous

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activity of the murine Sry gene in Sertoli cell precursors results in differentiation of Sertoli cells (Burgoyne et al., 1988). Polymerase chain reaction (PCR) products of the horse HMG box, the only region of SRY that is conserved between species (Lovell-Badge, 1993), have been sequenced (Meyers-Wallen et al., 1997) and are available from GenBank. The complete coding sequence of cDNA (1420 bp) for the equine SRY gene has been determined just recently. Contrary to the situation in mice, the equine linear RNA transcript in testicular tissue was expressed just after puberty (Hasegawa et al., 1999). After cloning of the SRY gene and the demonstration that it was a transcription factor (Ramkissoon and Goodfellow, 1996; Greenfield, 1998), several autosomal genes acting downstream of SRY were shown to be involved in the mammalian sex differentiation pathway. This set of genes includes the SRYrelated high-mobility group box (SOX) autosomal gene family, which display properties of both classical transcription factors and architectural components of chromatin (Pevny and Lovell-Badge, 1997). Sox9 has an essential function in sex determination in mammals and is critical for Sertoli cell differentiation (Morais da Silva et al., 1996). The human DAX-1 gene and its mouse homologue are located on the X chromosome and encode an unusual member of the nuclear hormone receptor superfamily. Mutations in this gene cause adrenal hypoplasia (Greenfield, 1998). The autosomal SF-1 gene produces another nuclear receptor, steroidogenic factor 1. Mutations in this gene may cause gonadal and adrenal agenesis and other disorders. These genes act at the same time (Greenfield, 1998).

Cycle of the X chromosome As proposed by Lyon (1961), it is now accepted that one of the X chromosomes in mammalian females undergoes inactivation during early embryonic development. Numerous investigations have shed light on different aspects of X chromosome behaviour, including preferential inactivation of the paternal X chromosome in trophoblast, random inactivation in the ICM, and molecular mechanisms of inactivation (Goto and Monk, 1998). These data appear to be fully applicable to the cycle of the X chromosome in the horse. Inactivation of the X chromosome in equine XX embryos begins gradually in trophoblastic cells around day 7.5 and in the embryonic disc around day 11.5. Cells with an inactive X chromosome predominate in the trophoblast by day 10.5 and in the disc by day 12.5 (Romagnano et al., 1987). In post-meiotic oocytes, the X chromosomes should become active again, as observed in mice and humans. The paternal X chromosome enters the zygote inactive but, soon after fertilization, the paternal X chromosome reactivates in XX embryos and both X chromosomes are active until trophoblast differentiation. At this time, preferential inactivation of the paternal X chromosome takes place in trophoblast cells (days 7.5–10.5). In the ICM (embryonic disc), both X chromosomes are active for some time, but later one of them undergoes random inactivation (days 11.5–12.5) (Serov et al., 1978; Romagnano et al.,

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1987). The promoter region of a key gene involved in X inactivation (XIST) was cloned recently from a horse genomic library and used, along with the equivalent human, mouse and rabbit sequences, to identify the minimal promoter region of the XIST gene (Hendrich et al., 1997). Sterility associated with a lack of one X chromosome (XO karyotype) observed in the mare (Bowling et al., 1987; Buoen et al., 1993) appears similar to the human XO disorder, but different from the mouse condition. It could be an additional indication of minor variations in the pattern of X chromosome inactivation in divergent mammalian species (Goto and Monk, 1998). Different forms of mosaicism (chimaerism) for X chromosomes described in horses cause abnormalities of sexual differentiation (Power and Leadon, 1990) and infertility (Gill et al., 1988).

Sex reversals in the horse As indicated above, the SRY locus plays a crucial role in sex differentiation and, in the normal situation, only embryos carrying an Y chromosome possess SRY. However, SRY can be non-functional or transferred from the Y chromosome to the X chromosome by a rare recombination event. These events can cause complete sex reversal whereby XY individuals become females and XX individuals become males (Cattanach et al., 1982). The XY sex reversal syndrome has been described in the domestic horse. In several cases, the progeny of stallions showed significant deviation from the expected sex ratio, as well as an increased level of female infertility (Kent et al., 1986). Also, a karyotype indistinguishable by G- or C-banding from that of a male horse (64,XY) was common in mares with development defects (Bowling et al., 1987; see also Chapter 9, pp. 188–191). Molecular analysis of an XY mare showed that at least the DNA-binding domain of the SRY gene was deleted from the Y chromosome (Pailhoux et al., 1995). An investigation of 38 mares with the XY sex reversal syndrome identified four classes with different degrees of abnormality. These include: (i) normal females, some of which were fertile; (ii) females with gonadal dysgenesis and normal Müllerian duct development; (iii) intersex with gonadal dysgenesis, enlarged clitoris and abnormal Müllerian duct development; and (iv) virilized intersex with high levels of testosterone. Usually, the two former classes were H-Y negative whereas the two later classes were H-Y positive (Kent et al., 1988a, b). The opposite situation was also described. A bilateral cryptorchid stallion with mild development of mammary glands was identified by karyotyping as an XX male. Underdeveloped accessory sex organs and hypoplastic, inguinally located testes that were deficient in spermatogonia were found in this stallion (Constant et al., 1994). The cause of this rare syndrome is unknown but it could be related to SRY abnormalities. However, SRY-negative XX true hermaphroditism in a horse also appears possible (Meyers-Wallen et al., 1997). One described Pasa Fino horse had ovotestes, no Müllerian or Wolffian duct derivatives, a blind-ending vagina and an enlarged clitoris. It was diagnosed

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SRY-negative by PCR analysis, suggesting that the masculinization effects during development were due to expression of genes downstream of SRY in the male developmental pathway.

Development of Interspecies Hybrids Description of interspecies hybrids Equids display a remarkable ability to interbreed between the member species of the genus, and the mule (female horse ×male donkey), as well as being produced for centuries as an efficient work animal, has also provided many insights into the genetics and evolution of speciation (Short, 1975). Mules are much more common than the reciprocal cross, the hinny (female donkey ×male horse) because, for some unknown reason, the latter mating is not as fertile as the former. Both mules and hinnies, although infertile, are stronger and live longer than either parental species, thus displaying ‘hybrid vigour’. This was recognized in at least 1000 years BC, which led to their extensive use in war during succeeding generations, particularly as pack animals. Mules are still used today in many parts of the world as work animals. Not only is hybridization possible between all the equine species (Gray, 1972), the transfer of embryos between the different species is also frequently successful (Allen and Short, 1997). An underlying reason for this remarkable ability of female equids to carry inter- and extra-specific conceptuses of very different genetic constitution may relate to the non-invasive nature of the equine placenta and the retention of six layers of tissue separating the maternal and fetal blood supplies (Samuel et al., 1976). It is also likely to be influenced by the immunological responses of the mother to the development of the endometrial cups and to the absence of paternally derived MHC antigens on the surface of the non-invasive trophoblast cells of the allantochorion (Allen et al., 1984; Donaldson et al., 1990).

Contribution of equine hybrids to developmental genetics In addition to being useful as work animals, equine hybrids have made a significant contribution to scientific thought, including ideas on developmental genetics (Short, 1975). The earliest example was Aristotle’s realization that, based on the physical appearance of mules, there was a more or less equal contribution from both parents to the phenotype of the offspring, rather than the female playing a purely passive role. However, it was not until the 17th century that sperm and eggs were discovered and the mechanism of sexual reproduction via fusion of the gametes was realized (Short, 1975). Equine hybrids have also contributed to thinking on gestation length and the control of parturition. Gestation lasts about 11 months in the horse and about 13 months in the donkey. Mule and hinny fetuses, on the other hand, are both

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carried for around 12 months (Allen et al., 1993), thereby indicating that the fetus, not the mother, is the major determinant of gestation length. Other areas in which equine hybrids have proved useful are those involving the importance of chromosome pairing during meiosis and the influence of viable germ cells on gonadal development. The difference in the number and structure of chromosomes present in each parent underlies the block to meiosis which occurs in hybrid offspring (Chandley et al., 1975). However, occasional oocytes have been observed in sections of mule fetal ovaries (Taylor and Short, 1975). The importance of viable germ cells for normal gonadal differentiation has been studied in male equine hybrids. The testes of three male hinnies were examined by light and transmission electron microscopy to observe the development of germ cells and to verify morphological modifications due to the hybridization (Landime Alvarenga and Bortolozzi, 1994). The hinny seminiferous epithelium contained Sertoli cells and spermatogonia with normal features, but the primary spermatocytes appeared abnormal and other cells in the spermatogenic sequence were not present. Most of the alterations began to occur in the primary spermatocytes, which showed nuclear vacuolization and deposits of amorphous material between the carioteca and the nuclear lamina to form vesicles or exaggerated chromatin condensation which resulted in pyknosis. Vacuolization and organelle destruction was also observed in the cytoplasm. The arrest of meiosis due to lack of chromosome homologies leads to germinal cell degeneration and, therefore, the arrest of spermatogenesis. This, in turn, causes a profound alteration in the morphology of the seminiferous epithelium. Equine hybrids have also been used to study the phenomenon of X chromosome inactivation. The Lyon hypothesis (Lyon, 1961, 1970) stated that only one of the two X chromosomes was active in the somatic cells of female mammals and that inactivation was a random event which took place early in embryonic life. Female mules and hinnies provided the ideal test situation and Hamerton et al. (1971) used the expression of species-specific glucose-6phosphate dehydrogenase (G6PD) to show that, in any given cell, only one of the two X chromosomes is functional. This was later examined in more detail using starch gel electrophoresis of erythrocyte G6PD recovered from 42 female and 32 male mules, 35 donkeys and ten horses (Serov et al., 1978). The quantitative expression of the parental alleles at the GPD locus varied greatly in female mules, from hemizygous expression of the maternal allele to that of the paternal, thereby confirming random inactivation in females mules. No selective advantage of a cell population with a maternally (or paternally) derived active X chromosome was found. Equine hybrids will continue to play an important role in research on developmental genetics in the future. For example, the role of gametic imprinting in placental and fetal development should be very fruitful, and progress in this area is bound to be stimulated as more and more equine developmental genes and their products are identified.

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References Allen, W.R. and Moor, R.M. (1972) The origin of the endometrial cups. 1. Production of PMSG by fetal trophoblast cells. Journal of Reproduction and Fertility 29, 313–316. Allen, W.R. and Short, R.V. (1997) Interspecific and extraspecific pregnancies in equids: anything goes. Journal of Heredity 88, 384–392. Allen, W.R. and Stewart, F. (1993) Equine chorionic gonadotrophin. In: McKinnon, A.O. and Voss, J.L. (eds), Equine Reproduction. Lea & Febiger, Philadelphia, pp. 81–96. Allen, W.R., Hughes, J.P., Jeffcott L.B., Mitchell, D., Osborne, V.E., Pickett B.W., Rossdale P.D. and Rowlands I.W. (1982) Recommendations of the working party on terminolgy in equine reproduction. Journal of Reproduction and Fertility (Suppl. 32), 647–652. Allen, W.R., Kydd, J., Miller, J.M. and Antczak, D.F. (1984) Immunological studies on fetomaternal relationships in equine pregnancy. In: Crighton, D.B. (ed.), Immunological Aspects of Reproduction in Mammals. Butterworths, London, pp. 183–193. Allen, W.R., Skidmore, J.A., Stewart, F. and Antczak, D.F. (1993) Effects of fetal genotype and uterine environment in equids. Journal of Reproduction and Fertility 97, 55–60. Amoroso, E.C., Hancock, J.L. and Rowlands, I.W. (1948) Ovarian activity in the pregnant mare. Nature 161, 355–356. Anderson, D.J. (1997) Cellular and molecular biology of neural crest cell lineage determination. Trends in Genetics 13, 276–280. Antczak, D.F., Oriol, J.G., Donaldson, W.L., Stenzler, L., Volsen, S.G. and Allen, W.R. (1987) Differentiation molecules of the equine trophoblast. Journal of Reproduction and Fertility (Suppl. 35), 371–378. Antczak, M. and Van Blekom, J. (1997) Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Journal of Molecular Human Reproduction 3, 1067–1086. Bamshad, M., Lin, R.C., Law, D.J., Watkins, W.C, Krakowiak, P.A., Moore, M.E., Franceschini, P., Lala, R., Holmes, L.B., Gebuhr, T.C., Bruneau, B.G., Schinzel, A., Seidman, C.E. and Jorde, L.B. (1997) Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nature Genetics 16, 311–315. Barlow, D. P. (1995) Gametic imprinting in mammals. Science 270, 1610–1613. Bartolomei, M.S. and Tilghman, S.M. (1997) Genomic imprinting in mammals. Annual Review of Genetics 31, 493–525 Bass, K.E., Li, H., Hawkes, S.P., Howard, E., Bullen, E., Vu, T.-K.H., McMaster, M., Janatroup, M. and Fisher, S. (1997) Tissue inhibitor of metalloproteinase-3 expression is upregulated during human cytotrophoblast invasion in vitro. Developmental Genetics 21, 61–67. Bazer, F.M., Geisert, R.D. and Zavy, M.T. (1993) Fertilization, cleavage, and implantation. In: Hafez, E.S.E. (ed.) Reproduction in Farm Animals, 6th edn. Lea & Febiger, Philadelphia, pp. 188–212. Bazer, F.W., Vallet, J.L., Roberts, R.M., Sharp, D.C. and Thatcher, W.W. (1986) Role of conceptus secretory products in establishment of pregnancy. Journal of Reproduction and Fertility 76, 841–850. Bazer, F.W., Ott, T.L. and Spencer, T.E. (1994) Pregnancy recognition in ruminants, pigs and horses. Signals from the trophoblast. Theriogenology 41, 79–94.

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Genetic Resources and Their Conservation D.P. Sponenberg Genetic 14 D.P. Resources Sponenberg and Their Conservation Department of Biomedical Sciences and Pathobiology, Virginia–Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USA Introduction Reasons for Horse Conservation Genetic insurance Historical and cultural aspects of conservation Scientific value Characteristics of Candidate Populations Wild horses Domesticated horses Feral horses Genetic Erosion in Horses Causes of Genetic Erosion Establishing Priorities for Conservation Defining Populations of Conservation Interest Geographic and Political Aspects of Breed Conservation Herd Book Management Importance of Type in Conservation and Selection Philosophy Theoretical Aspects of Conservation Assisted and Expanded Reproduction Gamete Preservation Organized Conservation Efforts Governmental efforts Non-governmental organizations Private efforts Future Directions References

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Introduction Horses are a component of global biodiversity. Along with most other taxa, they currently face significant erosion of genetic variation. The conservation situation for horses is complicated by the near extinction of the wild species during the last few centuries. The identity of the wild ancestor of the horse has been controversial in the past, but is currently generally held to be the extinct tarpan rather than the takh (Przewalski’s horse) (Bökönyi, 1987; see Chapter 2). Due to the fate of these two (wild tarpan extinct, takh endangered), the vast majority of the species’ genome currently resides in domesticated horses. Conservation efforts for most species have focused on wild taxa. Only recently have domesticated animals been recognized as key components of biodiversity. Horses comprise an intricate web of breeds, wild species and production niches which makes their conservation complicated. Significant genetic erosion can occur easily in the face of very high global horse populations unless the relationships of various horse populations are understood and appreciated. Failure to conserve domesticated genetic resources will ensure that a large portion of the horse genome is lost. The domesticated horse is organized into subpopulations in ways distinct from those typical of wild taxa as well as from those of most other domestic species. Conservation efforts must account for these distinctions in order to be effective. Breeds are the basic units of genetic resources in domesticated species. Breeds differ from one another because each has an array of genetic combinations that is distinct and reasonably repeatable throughout the breed population. Breed identity usually arises from a specific combination of relatively few phenotypic traits that are caused by the underlying genotype. The essence of breeds is the repeatability and predictability of these genetic combinations, and therein lies much of their production utility as well as their importance as reservoirs of genetic variation.

Reasons for Horse Conservation Compelling reasons for conserving genetic resources of domestic livestock, including horses, can be classified into a few categories. These include genetic insurance for the species and for agriculture, historical and cultural concerns, and concerns for the extinction of variants that might have scientific value.

Genetic insurance Maintaining diverse breeds as distinct entities for possible future use is one reason for conservation. This philosophy views conservation as a ‘genetic insurance policy’ so that conserved variation can be used to meet future changes in demand for types and uses of horses. Horse breeding in industrialized societies has recently changed from an essential component of economic

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production to a leisure activity, and with that change the ‘genetic insurance policy’ rationale for breed conservation becomes weaker. The trend in industrialized societies is increasingly toward a general-purpose riding horse, in contrast to previous production for several distinct and specialized breeds for riding, agriculture and transportation. It is difficult for many observers to imagine that horses might regain their previous importance for agriculture and transportation. The recent upsurge of demand for agricultural and forestry draught horses in the USA and elsewhere points to the futility of predicting future trends, and supports the need to conserve a variety of specialized types to be ready for whatever changes the future may hold. The current trend in horse breeding is for a few general-purpose riding breeds to become predominant while large numbers of more specialized breeds decline. Future trends are difficult to predict, but if these include demand for multiple breeds and types, then the breeds need to be conserved now against any potential future changes in demand. Conservation can be the bridge ensuring survival of breeds through periods of low demand that occur between periods of higher demand. In non-industrialized settings, horses still play many essential roles. Maintaining a range of breeds with different characteristics is important in these situations, so that each human generation is able to select out what is most useful for its time period and production goals. Loss of breeds results in lost choices for future generations, and runs counter to the historical trend over millennia when large numbers of breeds and types were maintained from generation to generation, although with varying frequencies as dictated by prevailing popularity. The fact that the wild progenitor of the domestic horse is extinct has implications if conservation is viewed as genetic insurance. The domestic form is now the only extant representation of horse biodiversity. This makes conservation of the domestic horse genetic diversity more compelling than if the wild ancestor were still extant. Saving the entire genetic variation of the horse, albeit in domesticated form, is saving genetic diversity that ecosystems may one day need should it be possible to return ecosystems to wild or nearly wild status. Locked into the genome of each breed are the results of adapting to both natural and agricultural selection pressures. Having survived these selection pressures, the horse breeds of today are each adapted successfully to distinct habitat of uses, demands and pressures. Stefan Adalsteinsson (personal communication, Edinburgh, 1990) refers to extant breeds as having the ‘genetic heritage of survival’. This is an important concept: genomes of the various breeds contain the information needed for survival and adaptation to an array of human and natural pressures that vary among habitats. When one breed, or other genetic resource, becomes extinct through fashion, so too does the genetic combination that succeeded in a specific setting. The ‘genetic heritage of survival’ also has implications for the methods of breed conservation. For this heritage to continue to be shaped, it is best if the population is conserved in its original setting in an in situ programme.

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A second best approach is an ex situ programme that still involves live animals or a combination of live animals with occasional introduction of frozen gametes or embryos from a founder population gathered from the original setting. A third approach is cryopreservation of gametes and embryos, which can save the population as a snapshot in time but fails to provide opportunities for ongoing expression and selection for the genetic heritage of survival.

Historical and cultural aspects of conservation Historical and cultural reasons for conserving genetic resources are compelling, although frequently overlooked because biological reasons for conservation are usually held to be more valid. Horses were integral to the distribution of peoples in the world today, having contributed to their mobility and to their ability to exploit various regions of the world in ways that would otherwise have been impossible (Bökönyi, 1987). The conservation of the various breeds, in their traditional forms, allows them to serve as reminders of the history and culture of various human groups. Horses are an extension of the identity of humans, both as groups and as a whole, and are an important aspect of the human experience. Conservation of historical genetic resources is no less compelling than is the maintenance of historical buildings.

Scientific value The scientific rationale for maintaining genetic resources is that different populations hold different genetic variants. These hold important keys to unravelling a variety of biological processes. Should these variants become extinct, the opportunity for scientific advancement is also gone. In some cases, the benefit will be to horses directly, in other cases the horse serves as a model for another species. An example is the lethal white foals related to the frame overo pattern, which are a good model of colonic aganglionosis in people (Hultgren, 1982; see Chapter 4). Variants such as the frame overo may become extinct if spotting is discouraged as is typical of many breed registries. Extinction of this pattern would eliminate a model of a debilitating human disease. Choosing among the three rationales for the one that is the most compelling is unnecessary. All three – genetic insurance, cultural record and scientific resource – are important reasons to conserve horse genetic resources. How to conserve horse genetic resources effectively is a difficult issue, and conservation strategies vary due to differences in conservation philosophies among those doing the work. All conservation programmes should be guided by some appreciation of how the species genome of the horse is organized, so that efforts can target populations most likely to hold unique variants and unique combinations. The horse genome today survives mainly in domesticated and feral situations, each of which contributes to overall diversity within the species. Truly wild horses are perched precariously on the edge

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of extinction, and while their contribution to the horse genome is critically important it is also very small.

Characteristics of Candidate Populations Wild horses The conservation of the few remaining truly wild horses is critically important to the horse. The takh is the target of an ambitious and carefully planned programme to reintroduce it as a free-living species in the Asian steppelands of Mongolia (Hendricks, 1995). This species had become extinct in the wild, and experienced a few generations of captive breeding. The reasonably rapid return of this animal to its original habitat engenders hope that the species will be maintained as a viable population with ongoing natural selection. This is essential because the takh species is the only truly wild horse remaining. The need for effective conservation of this species in its original environment is in no way diminished by the fact that it has made only minimal genetic contributions to domesticated horse populations. The tarpan was the other wild horse, and can now only be saved as a reconstruction of the extinct species. The strategy that has been used for the tarpan has been the conservation management of feral horses considered to be closest to the original tarpan (Hendricks, 1995; refer to Chapter 2 for a photograph). As a biological endeavour, this raises a host of questions. Among those are whether the wild phenotype and genotype can ever be regained once lost, which appears not to be the case (Hemmer and Beckhaus, 1990). Maintaining a primitive domesticated type under feral conditions does indeed provide an environment in which natural selection works upon something close to the original equine genome, and this conservation programme is of great value in maintaining an important and irreplaceable piece of the genetic legacy of the horse.

Domesticated horses Domesticated horses are associated integrally with the mobility of people throughout the ages of civilization (Bökönyi, 1987). Indeed, domestication and development of horses are so tightly associated with Eurasian civilization that it is difficult to imagine one without the other. Mobility has caused the genome of the horse to be organized into breeds and strains differently to the genomes of the other common domesticated species. The histories of development and maintenance of different horse breeds, and other populations of interest, needs to be appreciated if meaningful conservation is to occur. The evolution of most domesticated genetic resources is one of breed formation. The concept of ‘breed’ is important, and varies among cultures and time periods. A useful definition of breed involves a genetic basis, and

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stipulates that the population in question have a phenotype that is consistent and readily distinguished as unique among other populations, and that when interbred reproduces the original phenotype. This definition implies sufficient genetic uniformity to allow for predictable reproduction of the conformational and functional type of the population (Clutton-Brock, 1987). A key aspect of breeds is that each has an array of reasonably repeatable genetic (and therefore phenotypic) traits. Breed conservation is viewed usefully as the conservation of these genomic combinations being as important as the conservation of individual genes. Different breeds contain different portions of the genetic variability of a species, and breed variation is estimated to be responsible for 50% of the overall genetic variation in most species (Oldenbroek, 1999). Genetic uniformity results in consistency and predictability, and is always going to pull in a direction opposite to genetic variability and nonpredictability. At one extreme will be tightly inbred populations with minimal variability, and at the other will be populations so variable that most members are generally heterozygous, variable in phenotype and unpredictable in production. Populations vary along this continuum, and at some point become uniform enough for predictability, without sacrificing viability which could suffer when animals are entirely homozygous.

Landraces Throughout horse breeding’s long history, several pathways have been used to form breeds. One mechanism for breed formation includes a landrace stage (Sponenberg and Christman, 1995). Landraces are local types which have become uniform through a combination of founder effect, long isolation from other populations and selection within a local environment. Founder effect is frequently a mere accident of history – certain animals end up going to a new location, while others do not. Iberian horses going to the Western Hemisphere in the 1500s are a good example, as are Norse horses going to Iceland much earlier (Hendricks, 1995). These founders were not chosen deliberately; they were simply the horses that were available at the point of departure. Following a founder event, long isolation from other populations allows a landrace population to become relatively uniform genetically, as well as adapted to its environment. The relative genetic uniformity of landraces results from both moderate inbreeding and from selection. Selection is, in this case, a combination of natural factors and human factors, all acting in an agricultural or pastoral setting. Landraces with long periods of isolation from other horse populations are rare among horse breeds. This is because horses were the means of human mobility. Horses accompanied humans on their travels, arriving in new places to serve as migrants into local populations. A few island populations are good examples of isolated landraces, including those of Shetland, Iceland, Greece and Indonesia (Hendricks, 1995). New World populations based on the Colonial Spanish horse also fit here. Landraces attain their uniformity (and, therefore, breed type and character) more by default than by design, being

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shaped by some rather arbitrary forces (founders, isolation, selection) rather than deliberate and unified breeding decisions of a breed association. Standardized breeds Standardized breeds are maintained and fostered more deliberately than are landraces. A standardized breed is generally forged by design rather than by default, with breeders striving to achieve some pre-conceived and formal notion of an ideal animal upon which groups of breeders have agreed (Sponenberg and Christman, 1995). In most situations, the genetic isolation of a standardized breed is contrived, so that matings are kept within the breed regardless of geographic location. Standardized breeds tend to be genetically isolated due to conscious human choice rather than by geographic isolation. Many international breeds, such as the Thoroughbred and Arabian, are typical of standardized breeds which have achieved high levels of selection accompanied by arbitrary reproductive isolation imposed by the breeders. The cultural environment of horse breed formation and use is determined largely by human choice. For many populations, including a great many registered breeds, the tendency has been routinely to introduce horses from other breeds into the breeding population. This acts to decrease uniformity, and introduction coupled with selection can eventually change a breed to resemble the introduced type more than the original type. This phenomenon occurs due to strong preferences of modern riders and users of horses for relatively few horse types. These types and styles have changed throughout history, and with these changes in popularity have risen and fallen the fates of a variety of genetically based breeds. The current trend in horse breeding has been to emphasize a relatively few types, and the result has been the convergence of many breeds, both phenotypically and genetically. Breeds widely used for crossing with local horses include the Arabian for many saddlehorses and ponies, the Thoroughbred for warmbloods and Western stock horses (Quarter Horse, Paint and Appaloosa), and the Belgian, Percheron and Clydesdale for draught horses (Hendricks, 1995). A multiplicity of breed names still persists, but the genetic packages represented by these have become less and less distinct as crossbreeding and selection for only a few types pervades international horse breeding. The designation of breeds can become arbitrary, especially when considered from the point of view of genetic resource conservation. Standing in contrast to the internationally popular and somewhat homogenized types are the few pockets of unique genetic resources among horses. These are usually distinct by conformational type, function and use. Most of these only poorly fit the mould of the current international type, if they fit it at all. These tend to have a precarious future because their niche is small and shrinking. A few such breeds have achieved security through a local preference for horses of their type and ability, but most lack such security. Examples of unique local resources include the Caspian, Akhal Teke, Exmoor, Icelandic, American Cream Draft, Florida Cracker and a host of other breeds worldwide.

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Breed development has resulted in numerous breeds. The geography of this is outlined in Table 14.1, which illustrates the great diversity of breeds in Europe and Asia which were the cradle of horse domestication and development. Other regions of the world have less diversity. The Food and Agriculture Organization (FAO) (Scherf, 1995) databases contain information on 384 horse breeds, of which 277 have population data. Of these, 120 are listed as either critical or endangered, which is nearly half of those breeds for which data are available. Feral horses Feral horses are domesticated horses that have returned to a free-living state where human selection is minimal and natural selection once again becomes the major selection pressure. Most ‘wild’ horse populations are not truly wild, but feral, and it is doubtful that a wild genotype or phenotype could ever be recovered from a feral population (Hemmer and Beckhaus, 1990). Some feral populations have long histories and are important components of horse diversity. Many of these are unique due to founder effect, including some Japanese populations such as the Misaki pony (Obata et al., 1994), and populations of Colonial Spanish origin in the USA (Pryor Mountain, Sulphur, Cerbat and Kiger), and the coldblood Sable Island feral horses of Canada. These, and others of similarly old origin, not only survive in a feral selection environment, but also descend from foundation strains otherwise rare or extinct. Feral populations, whatever their origin, provide an opportunity for the horse genome to respond once again to natural selection pressures. Many of these populations are introduced into ecosystems that have had no exposure to the aggressive and selective grazing pressure typical of horses, resulting in degraded native flora. In such cases, the conservation of horse genetic resources and native wild biota are in conflict, and each case must be analysed carefully to provide for the wisest conservation of biodiversity. Those feral horse populations that are of unique or otherwise extinct breed origins do deserve special consideration for conservation, for once gone, their portion of horse biodiversity is irrevocably lost. The case for feral populations of recent, Table 14.1.

Number of breeds of horses by geographic region.

Region Africa Asia Europe Latin America/Caribbean Pacific Islands USA/Canada Total

No. of breeds

% of total breeds

60 148 209 32 20 58 527

11 28 40 6 4 11 100

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crossbred origin is not as compelling, including most populations in the USA and the Australian brumby.

Genetic Erosion in Horses Erosion of genetic variation in horse breeds is obvious when populations become extinct. More subtle erosion occurs when breed distinctions are blurred, either from genetic erosion within breeds, or from erosion among breeds and types. In any of these situations, useful or rare variants are likely to be lost, as are useful and interesting genomic combinations. Erosion within a breed results from both deliberate and non-deliberate mechanisms. In small populations, it is likely that rare variants will simply fail to be transmitted to the next generation by the chance which governs the passage of any gene from generation to generation. This is genetic drift, and occurs when a variant is found in only a few individuals and these fail to reproduce, or fail to transmit the genetic variant if they do reproduce. Breeds can also lose genetic material through conscious selection for or against certain types or other traits. An obvious, if trivial, example is the loss of certain colour variants in a number of breeds over time, usually through human selection. Spotting patterns are especially likely to be lost, as has occurred in the Morgan horse, Welsh pony and cob, and other breeds in the 20th century. Some changes within breeds are very substantive, including the selection for type which pulls the breed away from its original to some newer style. This phenomenon occurs in many breeds where a modern and traditional type both occur. In some instances, these types are strongly divergent, and the loss of traditional type within a breed means loss of the genetic foundation of the traditional type. It is common for the traditional type to be unique, with the modern type resembling one of a few internationally popular types. Loss of the original type indicates some loss of the breed’s genetic uniqueness. A second process of genetic erosion occurs simultaneously among many breeds and types, and involves selection of different breeds toward a single type, style and function. This is an insidious loss, and is pervasive throughout many breed groups of horses. This across-breed selection usually is related to the prevailing use of horses in a culture, as well as the prevailing concept of equine beauty. As riders and performers selectively favour a uniform type and style, the conformational types that vary from that type and are unique to certain breeds tend to become more and more rare. Convergence of type across breeds blurs the distinctions among breeds and, as a result, blurs their uniqueness as genomic resources. Although convergent selection towards a few types across several different breeds can reduce breed type distinctions, many underlying biochemical or antigenic traits may still remain distinct, and may still provide the population with enough distinctiveness to be considered a genetic resource. This is only true if selection occurs from within the original breed, and the change in type is not accomplished through crossbreeding. Selection does necessarily change

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gene frequencies, and convergent selection across breeds still increases the risk of elimination of some genetic variants as well as genomic combinations. However, genetic resources are certainly better protected by maintaining distinct purebreeding populations with convergent selection than they are by crossbreeding, or by the extinction of currently unpopular purebred populations unable to change toward modern types. Genetic erosion among types and breeds can occur from selection within a breed population, and also occurs through crossbreeding. Many breed associations permit the use of breeding animals with ancestry from outside the breed. This practice is usually limited to animals of other specified breeds, and so is different from allowing a purely open herd book. Examples include use of the Thoroughbred in the Quarter Horse breed, and inclusion of these two in Paint and Appaloosa breeding. These breeding practices do not make the resulting population sufficiently diverse to lose its character as a breed, but they are outcrosses in only one direction. Such outcrossing tends to pull the type of a breed away from an originally distinct type, and towards a more modern, homogenized phenotype in which colour determines more about breed identity than does underlying type. Similar phenomena occur in many warmblood breeds, with similar consequences for the breed’s genome.

Causes of Genetic Erosion Genetic erosion usually occurs through selection and breeding practices, and can also occur as an accident of history when a unique population with limited distribution is destroyed by a single disaster. Most genetic erosion occurs because breeds lose their habitats, and conserving breeds in the absence of defined habitats is very difficult. As horses become used increasingly for recreational purposes, many breeds and types that had specific agricultural or other draught uses have become rare or extinct, simply because their agricultural habitat is gone. The same is true of hardy riding horses historically used in subsistence settings. As this habitat declines, so does the type of horse shaped by it. Examples of breeds losing their habitat are numerous. In the USA, several large draught horse breeds have gone from agricultural use to showing, recreational driving and parade work. These tasks call for different horses, and the American Belgian has responded by changing from a relatively stocky horse to a taller more linear horse with flashier action than the more historic farm horse. The same era has witnessed the increasing use of many European draught breeds for meat production, so their ability to work has become secondary to meat characteristics. This has altered the type of some of these breeds. As riding has gone from a necessary means of transport to a recreational pursuit, many important riding horse types have been changed to more modern, larger types. Subtle changes in uses can drastically affect horse populations. The southeast USA during the 1930s underwent prolonged drought, and as part of

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drought relief cattle were brought from the southern plains into Florida. These cattle introduced screw worms into the region, and cattle managers needed to rope and hold cattle for treatment. Before this time, cattle were rarely roped and held, and cattlemen preferred the small, gaited Florida Cracker horse of Iberian heritage. Following the introduction of the screw worm, the Quarter Horse gained popularity as a larger horse better able to hold cattle once they were roped. Thus, the habitat for working cattle horses changed, and with it a once common breed approached extinction (Warner, 1980). Another important cause of genetic erosion for some breeds is their excellence in crossbreeding. This is paradoxical, but some breeds produce crossbred foals of greater value than their purebred foals. This is the case for Irish Draught, Hackney and Cleveland Bay horses. Crossbred foals by Thoroughbred stallions are avidly sought, and consistently are worth more than purebred foals. Incentive programmes are developing so that farmers can afford to produce purebred foals. This effort is aimed at retaining sufficient numbers in these breeds to sustain their important function as dams of crossbred performance horses.

Establishing Priorities for Conservation Conservation needs must be ranked by priority because financial and other resources are limited and must be focused where they are most likely to succeed. Different entities will establish priorities differently, and a diversity of conservation approaches is likely to contribute to overall success rather than being detrimental. Priorities must be chosen carefully, and involve both numerical and genetic aspects of a population. Conservation efforts ideally should target the most unique populations that have the lowest numbers of individuals (Ruane, 1999). Consciously selecting breeds that are divergent from most other breeds by history and phenotype greatly increases the chances of conserving significant variants as well as unique combinations of variants that are adapted predictably to unusual or extreme habitats. This approach is effective, but also problematic because most such populations are rare for the very reason that they do not conform to the prevailing fashion in horses. Lack of popularity in no way diminishes the importance of such populations as important reservoirs of the genetic heritage of the horse.

Defining Populations of Conservation Interest Defining populations of conservation interest is a difficult endeavour. It is important for conservation efforts to focus on unique, genetically consistent populations, and many of these survive outside the framework of a herd book and a breeders’ organization. Useful steps for evaluating candidate populations include investigations on numbers, history, phenotype and either blood types

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or DNA fingerprints. Establishment of herd books where herd books are not in use can also be beneficial, but this approach will only succeed in cultural situations where herd books are likely to include all members of the population of conservation interest. Table 14.2 outlines the numerical considerations for helping establish conservation priorities that are used by the FAO, the Rare Breeds Survival Trust (RBST) and the American Livestock Breeds Conservancy (ALBC). The FAO and RBST methods estimate total populations of breeding age animals. The ALBC has chosen an approach that monitors the levels of purebred replacement as reflected by annual registration activity. The ALBC approach has special merit in horse breed conservation because many mares are not used for breeding or are used for crossbreeding rather than purebreeding. These tend to reduce the level of purebreeding below that expected from a total population number. Annual purebred registration figures accurately reflect the level of purebred breeding. Additionally, the registration figures usually are obtained more easily than are accurate total numbers of breeding age animals. Numerical factors are important in establishing priorities, as are factors affecting the status of a population as a genetic resource. All of these factors must be evaluated if conservation efforts are to be targeted to those populations that not only have low numbers, but also are genetically uniform enough to serve as useful reservoirs of predictable genetic combinations. History, phenotype and blood typing or DNA fingerprinting are useful in assessing this dimension of candidate populations (see Chapters 5 and 6). Historical investigations should evaluate any information available on founders, migrants to the population, present breeding practices, degree of isolation and traditional selection procedures. Documenting the history of a population is usually inexpensive, and can help to eliminate rapidly any recently formed crossbred populations from conservation programmes. Populations that withstand historical scrutiny can then be evaluated for external phenotype. This is a relatively subjective endeavour, but when

Table 14.2. Conservation categories from American Livestock Breeds Conservancy, Rare Breeds Survival Trust and Food and Agricultural Organization. ALBC Critical Rare Watch Study

RBST < 200 annual Critical registrations < 1000 annual Endangered registrations < 2500 annual Vulnerable registrations At risk Imported Feral Native

FAO 200 total animals

Critical

333 total animals

Endangered

600 total animals

< 100 females < 5 males < 1000 females < 20 males

1000 total animals No minimum No minimum No minimum

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accomplished by experienced observers can usually contribute greatly to the final decisions concerning a population. External phenotype can be evaluated for agreement with the alleged history. Overall type of the population can be considered, as well as variability, which might indicate significant crossbreeding even in the absence of historical confirmation. A final investigation can include blood typing or DNA fingerprinting. These are generally expensive investigations, in terms of both sample collection and sample processing. These techniques all depend on comparative analysis, and results must be compared with results for related breeds and types. A consequence of the history of horse breeding, with frequent interchange among many populations, is that variants that are unique to certain populations are rare. Final decisions on status usually depend on all four parameters (numbers, history, phenotype, and blood type or DNA fingerprint), with no single parameter able clearly to define a population as deserving a high priority. Conserving landrace horse populations in the USA has had to depend very much on these techniques. Some of these populations include Colonial Spanish horses, which have a fairly consistent and distinctive external phenotype. In recent years, a few previously overlooked populations have been discovered, and their histories have been investigated along with their external phenotype. Those populations which have passed historical and phenotypic screening have then undergone blood typing. The Pryor Mountain feral horses have survived all three, and are included now in the breed group of Colonial Spanish horses in the USA. Florida Cracker horses likewise have been admitted to this group as an important component due to their geographic location in the extreme south-east of the range of this breed group. The Nokota horse of traditional type, in contrast, was found to have been resegregating from a recently crossbred population (E.G. Cothran, personal communication, 1995, Blacksburg). These, descendants of the horses of Native American Chief Sitting Bull, have been removed as a high priority for conservation because their status as a genetic resource had been greatly compromised.

Geographic and Political Aspects of Breed Conservation Many rare breeds persist as isolated pockets of animals, usually in remote regions having poor communication. Others persist in more privileged regions, but remain peripheral to the agricultural and hippoligical mainstream. These two groups of breeds are local and isolated, and need national or regional programmes in order to be conserved effectively. Advantages of local distribution include the fact that the conservation programmes usually have ready access to all individuals within the breed. An example of a local type that is rare is the Pantaneiro horse of Brazil (da Silva Mariante, 1992). This horse population is a ‘criollo’, or Iberian-based, population that has been shaped by an environment with a pronounced wet–dry cycle. During the wet season, these horses survive in swamps. This,

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coupled with the colonial Iberian foundation and the largely feral existence, has resulted in a distinctive and environmentally adapted population of horses. Local rarity and an extreme environment combine to expose the Pantaneiro to risks of extinction by local disaster. Another example of isolated, local populations with very distinctive characteristics are the several Chinese breeds of Miniature Horses (Chen and Wang, 1996). These few breeds of very small horses appear to have developed separately from most other Chinese as well as non-Chinese breeds, and persist as local resources only poorly recognized outside their small ranges. They have been used over several centuries for local packing and draught needs, as well as serving as items of tribute from provinces to the central government. One strain was called the ‘under-fruit-trees’ horses in recognition of their small size. A second group of rare breeds in need of conservation includes some breeds that are internationally represented, but rare wherever they occur. Some of these breeds were once popular and numerically important internationally, and then fell out of favour. Heavy draught breeds are among these. Some draught breeds, such as the Clydesdale, Shire and Suffolk, are reasonably rare in several countries. Conservation can proceed for these breeds by monitoring the status of each breed in each country, and allowing for occasional exchange of breeding stock among the various populations. Some of these ‘international rarities’ face several challenges. One of these is mutual acceptance of breeding stock by all herd books. The Suffolk horse is rare in Britain as well as in the USA, and is a unique, practical type of farm draught horse. The population in the USA includes some horses that were upgraded from non-Suffolk horses, and as such the British do not accept them as purebred. The result is fragmentation of the breed, and although the USA population accepts British horses, the migration of breeding material in just one direction limits the recovery of this breed genetically. Lipizzaner horses are another good example of an international rarity, with six strains usually accepted by all countries, and two additional strains accepted by only some countries. Recent upheavals in the Balkan states have put some strains of this breed in acute peril, and demonstrate the need for close monitoring of international breeds and relative uniformity of herd book practices as to inclusion or exclusion of portions of many breeds. In the case of both the Lipizzaner and the Suffolk, the pertinent question of ‘what is a breed’ has bearing on the actions and philosophies that guide conservation. Breed purity is an important and defining aspect of breeds as genetic resources, yet can be taken to extremes where the breed can become doomed by exclusion of graded animals that are bona fide members of the genomic package represented by the breed. The conservation goal should be to include all animals that are purebred (including graded animals), while excluding all of those that are crossbred. This frequently is a difficult line to draw, especially in those populations lacking herd books. Unfortunately, the poorly documented populations frequently are those of most genetic uniqueness and therefore of highest conservation priority.

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Herd Book Management Breed conservation can hit some obstacles in breeding practices and the culture of various herd books. In some instances, herd books are so open to outside horses that the resulting population can barely be considered to be a breed in the genetic sense. Even relatively minor introductions can have pervasive consequences if selection then disseminates the introduced type throughout the breed. In a few instances, however, levels of inbreeding may require that occasional outside animals of appropriate type and background be allowed into a population as a last effort to restore vitality to a failing population. In some situations, rare breeds are constrained by a strictly closed book that excludes some outside individuals that are as purebred as those within the herd book. One example is international Akhal Teke breeding. Akhal Teke horses are required to descend only from horses entered into Soviet herd books. Iranian members of this breed are not registered in this way, and currently are excluded from international efforts to ensure continuation of this unique and ancient genetic resource. The philosophy of herd book management and its effects on breed conservation are complicated. In most instances, it is important for the managers of the breed to be aware of the cultural and historic background of the breed. Some breeds, such as the Icelandic horse, have been isolated for so many centuries that they represent truly isolated genomic resources. Most other breeds are much more recent, or much more closely related to their cousin breeds. In these cases, it is appropriate to allow some upgrading, or the inclusion of appropriate outside individuals that are genetically members of the breed of concern and can boost its numbers as well as increase its genetic vigour. Horse breeding sometimes has tended to focus on some trivial aspects of colour or other superficial characteristics instead of focusing on underlying type. For example, the American Cream Draft horse (Fig. 14.1) should be a light champagne colour with pink skin and amber eyes. This colour phenotype is achieved through a single gene, which has caused confusion of the colour with the breed. Breed type is distinct, and blood typing studies have confirmed that the American Cream Draft horse is in fact a genetic breed. This implies that sorrel individuals produced in Cream breeding programmes can be used within the breed, rather than outcrosses of other breeds, to ensure that the entire genomic resource underlying the distinctive colour is not lost in this rare breed.

Importance of Type in Conservation and Selection Philosophy A very important issue in breed conservation is the issue of breed type. Breed type is what makes breeds visually distinct from one another, and is usually a combination of conformational and behavioural traits. Many horse breeds are distinctive by looks alone, some are also distinctive by gaits, temperament or

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Fig. 14.1. The American Cream Draft horse is a landrace that has been documented as a unique genetic resource whose conservation now seems ensured.

other behaviours. Distinct breed type is a reflection of a distinct breed genome. Because current international horse breeding favours fewer types, an insidious loss has occurred such that breeds that were once distinctive remain so in name only. The case of the Morgan horse is informative. The Morgan originally was a short, stocky multipurpose breed that was used for agricultural and general draught, riding and racing. In the early 1900s, the draught potential of this breed became unimportant, and breeders began to prefer a more refined, and taller, saddlehorse type. Change in the type was achieved both through matings to other breeds and from selection within the Morgan breed. The result has been a divergence of type within the Morgan, so that the modern saddle type coexists (uneasily) with the older, stouter, traditional type. Breeding practices have blurred the original distinctiveness of the Morgan, but have multiplied the potential settings for the breed, because the multiple types appeal to multiple users. The example of the Morgan exemplifies one of the conundrums of breed conservation. Evolution of breed type may well contribute to its commercial success, and therefore to its survival as a genetic resource, but fails to conserve some of the original uniqueness of the breed as a repeatable and unique genome. Similar divergences of type and breeding have occurred in many other breeds, including the American Quarter Horse and the Appaloosa. Type is highly diverse in the American Quarter Horse, a breed having a large population. One benefit of diversity is that the breed finds appeal and acceptance with a wide range of horse owners and users because the type they personally prefer can be found somewhere within the breed. The breed, by virtue of

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encompassing a range of types, has allowed its uniformity and predictability to diminish, and has also supplanted other breeds by overwhelming their habitats. The Quarter Horse can serve as an example of a breed evolving away from its foundation type, generally in response to pressures from the showring for a change in type. This phenomenon has sparked a reaction among many breeders who have organized to identify, conserve and actively promote the foundation type. Similar efforts are preserving traditional type in a number of breeds worldwide, notably the Appaloosa and some European warmblood breeds. Breed type is certainly going to evolve over time in many breeds, but the genetic consequences need to be acknowledged. Mechanisms for conserving traditional types vary. In many European countries, governments have breeding organizations that control the licensing of stallions. In these situations, the officials have great influence in channelling the direction of a number of breeds. In some instances, these seem to be instrumental in conserving the traditional type in a number of breeds. Noriker horses in Austria (Fig. 14.2) are notable as an old breed still very close to its traditional type. In other instances, selection appears to deviate towards a more uniform, modern type in many breeds, especially warmbloods. One successful conservation of traditional type has been the Saxony warmblood, a heavy coach and agricultural horse from which the modern Oldenburg largely descends (Sprenger, 1994). Photographs of stallions from the late 1800s and from modern horses are remarkably similar, and demonstrate that progression of years does not necessarily mean loss of traditional type within a breed.

Fig. 14.2. The Noriker horse is maintaining its traditional type due to actions by both governmental agencies and private breeders.

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Theoretical Aspects of Conservation Conservation theory is an important part of horse conservation programmes. One important concept is that of effective population size. Effective population size is a complicated computation in populations where breeding groups differ in size, animals enter the reproductive population at different ages and generations overlap. A very simplified approximation is that of effective population size (Ne) = 4mf/(m + f), where m = number of males and f = number of females (Nicholas, 1987). Effective population size reflects the importance of the less numerous sex in constraining matings and contributing to inbreeding. As populations dwindle, it is important to understand that small populations need to be managed carefully in order to minimize inbreeding where possible. Table 14.3 outlines the consequences of various numbers of males and females on effective population size, and readily demonstrates that increasing the number of the rarer sex (males in most horse populations) has a profound impact on effective population size and the increase in relationship expected per generation. Effective population size can also be reduced insidiously by selection practices. This reduction can occur despite inclusion of high numbers of sires and dams through the effects of selecting only certain offspring for reproduction. It is best to ensure that all breeding animals in every generation provide replacements in the succeeding generation, so that all provide genetic material rather than just a few. With small populations, it becomes important to constrain matings to avoid increasing inbreeding. Different populations have differing evolutionary histories and appear to resist inbreeding depression to different degrees. For rare genetic resources, inbreeding is a gamble that may damage a population, and should therefore be avoided where possible. A general rule is to avoid increasing average relationship by more than 1% per year, since levels of inbreeding will rise following any rise in average relationship. It is worth noting that linebreeding and inbreeding have successfully produced many outstanding horses in a number of breeds, but must be managed carefully and should not involve entire breed populations lest inbreeding depression then eliminate the entire breed.

Table 14.3.

Effect of varying numbers of males and females on effective population size.

No. of males

No. of females

Total number

Effective population size

Increase in average relationship per generation (%)

1 3 9 30 1 1

30 30 30 30 60 90

31 33 39 60 61 91

3.87 10.9 27.6 60.69 3.9 3.96

12.9 4.6 1.8 0.82 12.7 12.6

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Assisted and Expanded Reproduction Assisted and expanded reproduction can either help or hurt breed conservation, depending on how these tools are used. It is increasingly possible to help subfertile horses to reproduce, whether stallions or mares. In some instances, these technologies can be used to reproduce animals that would otherwise be lost from the gene pool. The reasons for infertility must be considered, however, to avoid perpetuating genetic tendencies for subfertility. Expanded reproduction through embryo transfer and artificial insemination can be important in rescuing populations that are on the brink of extinction. They can also, if used in more numerous breeds, shrink the effective population size by limiting reproduction to only a small portion of the breed rather than sampling the breed more widely at each generational step. One assisted reproduction technique that can contribute greatly to conservation breeding is the transfer of embryos from performance mares into surrogates. This allows the performance mare to continue campaigning, while also allowing her to contribute to purebred breeding. This technique must be managed so that certain horses do not overwhelm a breed’s population and thereby reduce the effective population size (see Chapter 12).

Gamete Preservation Gamete preservation is potentially useful for conservation of genetic resources. Cryopreservation has been perfected only recently for horses, and offers the potential for literally freezing a genetic resource as a snapshot in time. This can be very helpful in providing future generations with a chance to reintroduce genetic material from individuals long dead. Gamete preservation freezes the genome as a static entity, and therefore does take away the opportunity for genetic resources to continue to evolve. This is risky if that evolution is for resistance to various pathogens. A frozen store potentially could be reconstituted to enter an environment containing threats to which it could not respond. A related issue is that the frozen store is not being shaped by human selection, and this can be either good or bad depending on whether the human selection is maintaining the genetic resource, consolidating it or changing it from its original type. Gamete preservation can be a very powerful tool for conservation when used in concert with other strategies. One such strategy is periodically to reintroduce frozen material back into a population. This acts to increase effective population size, and can also reintroduce foundation genotypes into an ongoing breeding programme where they can then be subjected to selection pressures.

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Organized Conservation Efforts Many different approaches have been used for the conservation of horse breeds. Many of these vary country by country. Some efforts are accomplished by various governments, others are undertaken by non-governmental entities. Each of these has strengths and weaknesses, and no single model seems to be a perfect fit for all conservation challenges.

Governmental efforts In some countries, governments are largely responsible for organizing horse breeding and, consequently, are responsible for most rare breed conservation as well. The Polish tarpan conservation effort is governmental, consisting of two separate populations in forest reserves. Selection in this case favours horses as similar to the primitive original as can be produced (Hendricks, 1995). Many European breeds, especially warmbloods, are controlled by governmental or paragovernmental organizations (Hendricks, 1995). In the USA, the Bureau of Land Management is responsible for management of feral horse populations on public land. These horses are usually of recent crossbred origin, and are managed in defined geographic areas as a cultural resource. A few areas still have Colonial Spanish-type horses, and in these areas management is tailored to conserve this unique resource. In the Pryor Mountain herd management area, the horses are studied closely as to behaviour, phenotype and genotype. The horses are assessed as to relative degree of Colonial Spanish phenotype, and when horses are removed the goal is to ensure that those remaining are of this type. Other concerns, such as age profiles within the herd and retention of rare colour variants, also enter into management decisions. This is an important population which is exposed to natural selection in its feral state, but also to human selection pressure as excess horses must be removed periodically to preserve vitality of their range.

Non-governmental organizations Effective conservation also occurs by action of non-governmental organizations. Many of these are national or regional in scope, and tend to be interested in all agricultural species rather than horses only. Examples include the ALBC in the USA, the RBST in the UK, and the Safeguard for Agricultural Varieties in Europe (SAVE), which works mostly in the Danube Basin. Approaches in these organizations vary. The ALBC serves mainly as a central coordination and networking point for private breeders and for breed associations. The ALBC works extensively in the documentation of unregistered populations to determine if they qualify as genetic breeds. It then facilitates the development of breeder organizations, and monitors the progress of the various populations. The ALBC does not own animals, and has an underlying

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philosophy that recognizes that breeders developed breeds, and they are the best hope for their conservation. Very rarely the ALBC will step in directly to rescue endangered populations of interest, such as the Wilbur–Cruce strain of Colonial Spanish horse, but tries quickly to distribute the rescued animals to private breeders. Breeding programmes are loosely monitored, and technical support is available for breeders as well as breed organizations. The ALBC approach of working through breed organizations has helped such breeds as the American Cream Draft horse. This is an agricultural draught breed, and is the only one developed in the USA. The breed history of this horse indicated that it might be a crossbred composite of various European breeds, although the type is reasonably consistent and the colour is unique. Blood typing studies demonstrate that indeed the American Cream is genetically consistent enough to be considered a true breed (E.G. Cothran, personal communication, Blacksburg, 1995), and that it is distinct from other heavy draught breeds in the USA. These findings and an educational endeavour to demonstrate that the breed is much more than the colour have helped to inspire the breeders actively to conserve the American Cream as a purebred genetic resource. The RBST has a successful approach that varies somewhat from that of the ALBC. The RBST works closely with breeds developed in the UK, and classifies other rarities in a separate category. The UK is the site of development of many important horse and pony breeds, several of which are now rare. The UK has also become an important refuge for some endangered breeds originating in other countries, such as the Caspian pony. As a holder of the largest population of this breed, it is important to expand active conservation efforts beyond only native breeds to ensure that populations of rare breeds that are internationally important are not neglected. The efforts of SAVE are especially important, and their regional focus is going to become an increasingly useful model for rare breed conservation. SAVE actively pursues the discovery and recovery of small landrace populations that have been mistakenly thought extinct, or have failed to be described in the past. Their inclusion of all species of animals as well as plants gives them a good general overview and an inclusive philosophy under which many populations are being rescued from extinction and brought to more sustainable numbers. One recent effort involved the blood typing of the Croatian Posavina horse in an effort to better define and conserve this breed (Anonymous, 1996).

Private efforts Private efforts have been essential in the conservation of some horse breeds, and in a few cases certain individuals have stood between a breed and extinction. The work of Ruy D’Andrade with the Sorraia horse in Portugal is one good example of an individual private effort ensuring the survival of a unique and endangered genetic resource (Hendricks, 1995). Louise Firouz and her

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work in Iran with the ancient and pivotal Caspian pony is also in this category (Hendricks, 1995). Bryant and Darlene Rickman in the USA have diligently assembled the remnants of the Choctaw tribal strain of the Colonial Spanish horse (Fig. 14.3), and without their involvement this historic and important strain would almost certainly been lost as a discrete entity. Similarly, Marye Ann Thompson has assembled the remnants of the unique Colonial Spanish horse strain from the Cerbat Mountains in the USA. Most breeds worldwide depend on private breeders at some level, and for most breeds this includes several different private breeders. The importance of private breeders caring for and propagating their breed of choice cannot be overemphasized as essential for the long-term viability of rare breeds. As long as breeders are well informed, it helps to have many different private breeders each making slightly different breeding decisions to ensure the long-term genetic health of a breed. An added benefit is that many private breeders are traditionalists, and diligently guard and conserve traditional type even in the face of changing fashions.

Future Directions Conservation efforts over the next few decades are going to be critically important to the survival of much horse biodiversity. The 1900s saw great advances

Fig. 14.3. The Choctaw strain of the Colonial Spanish horse in the USA is an example of a once numerous strain now conserved through the efforts of a few private breeders.

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in communication and travel, and these have brought increased homogenization of global horse breeding. In the 21st century, significant new efforts must be made. Distinctive populations of horses need to be sought and monitored actively. These efforts need to target both standardized breeds and landraces, although landraces are much more difficult to define and conserve. Powerful biotechnical tools such as DNA fingerprinting need to be pursued actively in order to help document the interrelationships among populations, and can also contribute to the genetic management of conservation populations. Technology and tradition must be equal partners in generating forward-looking and inclusive conservation programmes which will succeed in saving the majority of horse biodiversity. Conservation organizations have made great strides in forming philosophical and conceptual frameworks which drive active conservation programmes, and their experiences need to be sought out and duplicated. If conservation is passive rather than active, then it is certain that many distinctive populations will fail to survive.

References Anonymous (1996) Posavina Horse Breeder Society. SAVE – Report Summer, 17. Bökönyi, S. (1987) History of horse domestication. Animal Genetic Resources Information 6, 27–32. Chen, Y.-C. and Wang, T. (1996) Mini-horses in China. Animal Genetic Resources Information 18, 25–30. Clutton-Brock, J. (1987) A Natural History of Domesticated Mammals. Cambridge University Press, London. da Silva Mariante, A. (1992) Animal genetic resources conservation programme in Brazil. Animal Genetic Resources Information 10, 9–14. Hemmer, H. and Beckhaus, N. (1990) Domestication, the Decline of Environmental Appreciation, 2nd edn. Cambridge University Press, Cambridge. Hendricks, B.L. (1995) International Encyclopedia of Horse Breeds. University of Oklahoma Press, Norman. Hultgren, B.D. (1982) Ileocolonic aganglionosis in white progeny of overo spotted horses. Journal of the American Veterinary Medical Association 180, 289–292. Nicholas, F.W. (1987) Veterinary Genetics. Clarendon Press, Oxford. Obata, T., Takeda, H. and Oishi, T. (1994) Japanese native livestock breeds. Animal Genetic Resource Information 13, 13–24. Oldenbroek, J.K. (ed.) (1999) Genebanks and the Conservation of Farm Animal Genetic Resources. DLO Institute for Animal Science and Health, Lelystad. Rare Breeds Survival Trust (1998) Conservation categories. The Ark 26, 108c. Ruane, J. (1999) Selecting breeds for conservation. In: Oldenbroek, J.K. (ed.), Genebanks and the Conservation of Farm Animal Genetic Resources. DLO Institute for Animal Science and Health, Lelystad, pp. 59–73. Scherf, B. (1995) World Watch List for Domestic Animal Diversity, 2nd edn. FAO, Rome. Sponenberg, D.P. and Christman, C.J. (1995) A Conservation Breeding Handbook. American Livestock Breeds Conservancy, Pittsboro, North Carolina.

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D.P. Sponenberg Sprenger, K.-U. (1994) The German Coach Horse: ‘Saxony Warmblood’. Animal Genetic Resources Information 14, 107–114. Warner, J.G. (1980) Biscuits and ‘Taters. Great Outdoors, St Petersburg, Florida.

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Genetics of Performance Traits 1, E.Traits A. RicardA. Genetics 15 of etRicard Performance al. Bruns2 and E.P. Cunningham3 1Institut National de la Recherche Agronomique, Station de Génétique Quantitative et Appliquée, 78352 Jouy en Josas, France; 2Institut für Tierzucht und Haustiergenetic, Universität Göttingen, Albrecht-Thaer-weg 3, 37075 Göttingen, Germany; 3Department of Genetics, Trinity College, Dublin 2, Ireland

Introduction Thoroughbreds Introduction Heritability of track performance Rate of genetic improvement Limits to performance? Horses for courses Trotters Speed and quality of trotter Earnings Are there still true precocious performances? A new way to measure success and career The problem of non-recorded horses Conclusion Sport Horses Measurements of performance Heritability of performance traits Genetic relationships between performance traits Concluding remarks References

411 412 412 412 414 414 417 418 418 419 422 423 426 426 427 427 429 432 434 434

Introduction Horses are used for a much more diverse range of functions than is generally appreciated. This chapter deals with performance traits of race and sport horses. The first two sections of the chapter consider performance traits in two of the most common groups of racehorses, Thoroughbreds and trotters. ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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Interestingly, there were 114,026 recorded births worldwide in Thoroughbreds in 1996, against 47,795 in trotters. The third section analyses the performance traits in sport horses.

Thoroughbreds Introduction The selection and breeding process in Thoroughbreds is founded on the belief that racing performance is inherited. Attempts to analyse the genetics of performance in a systematic way have involved some distinguished names (Galton, 1898). However, it is only in recent decades that good estimates of heritability of performance, based on adequate data, have been produced (see reviews by Hintz, 1980; Langlois, 1980; Tolley et al., 1985; Klemetsdal, 1990). A specific question concerns the choice of measure of performance. The simplest and, for many people, the most relevant measure is earnings. However, earnings are very non-linear, and many horses have no earnings at all. Racing time is an obvious measure, and is recorded routinely for all horses in the shorter American and Japanese races. It is a less useful measure in longer races, and is often recorded only for the winner. Handicap ratings involve a lot of subjective judgement, but are well tested and generally available for all horses. All of these measures can be converted into ranks, or subjected to a variety of transformations. These issues are reviewed by Ricard (1998).

Heritability of track performance The most comprehensive assembly of published results on heritability of racing performance is that of Tolley et al. (1985). They summarized results from over 40 studies, about half of which related to analyses in Thoroughbreds, and the remainder to trotters, pacers and Standardbreds. For the Thoroughbred studies, they presented the results separately for earnings, handicap and racing time measures. As might be expected, the reported estimates vary depending on the analytical model used, the scale and quality of the data, and the particular measure of performance involved. For the five studies reporting heritabilities of earnings (generally logs) or normalized ranks, the values ranged from 0.23 to 0.56 (if we focus on 3-year-olds and take account of all races), with most values being in the lower end of this range. Eight studies reported heritabilities for handicap measures. In these studies, there was more variability in analytical method, and a general tendency for paternal half-sib and regression on sire methods to give very high estimates. Most authors discounted these on the basis of expected, but unquantifiable, correlations between environment of offspring and phenotype of sire. The remaining estimates (mainly regression on dam or on midparent) fell in the range 0.24–0.61, with most grouped in the range 0.30–0.40.

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Ten studies were given in which heritability of racing time was estimated. Several of the estimates were based on quite small data sets. For those with reasonable amounts of data, estimates ranged from 0.09 to 0.78, but most values were in the range 0.10–0.20. Since the review of Tolley et al. (1985), a number of studies with large volumes of data have been completed. Gaffney and Cunningham (1988) published estimates of heritability of Timeform (handicap) ratings based on full year 3-year-old ratings of 31,263 horses from 2087 sires. The estimates ranged from 0.39 ± 0.01 (regression of offspring on dam) to 0.76 ± 0.02 (regression of offspring on sire). As in earlier analyses of similar data, the authors concluded that sire-based estimates were likely to be biased upwards. Oki et al. (1995) published estimates of heritability of racing time for various race lengths, based on performances of over 25,000 horses. The data were approximately equally recorded on dirt and turf. The values on dirt and turf were broadly similar, but there was a clear tendency for heritability values to be lower as the length of race increased over the range 1000–2000 m (see Table 15.1). In keeping with the earlier results summarized by Tolley et al. (1985), the heritability estimates for racing time in this study were lower than for the other measures of performance reported by other authors. Williamson and Beilharz (1998) carried out an analysis of almost 0.5 million individual race records from Australia. They used three measures: lengths (distances between horses at the end of a race), ranks and a function of earnings. By an approximately linear adjustment for length of race, they derived a speed rating and a stamina rating for each horse. They then calculated heritabilities for all of these measures. The resulting estimates were generally higher than those from other studies. Most estimates were above 0.5, with values for the stamina rating being consistently higher than those for speed (approximate average of 0.65 against 0.49). Langlois (1996) has discussed the difficulties in obtaining reliable heritability estimates in Thoroughbreds. He concluded that racing speed is not a useful criterion because it has low heritability and is not, on its own, a major determinant of success. He favoured ranks. He also presented results showing a consistently higher heritability of log earnings calculated by regression of offspring on dam than by regression on sire. This effect was attributed to non-genetic maternal contributions to the offspring, as well as to the lower selection and, therefore, greater genetic variability in female parents. The overall picture emerging from these numerous studies is a reasonably consistent one. It shows that the heritability depends on the measure used. Table 15.1.

Heritability of racing time for races of different lengths (Oki et al., 1995). Race length (m)

Type of track

1000

1200

1400

1600

1800

2000

Turf Dirt

0.25 0.19

0.16 0.22

0.10 0.12

0.12 0.09

0.09 0.17

0.08

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Time measures generally had a heritability in the region 0.1–0.2, with the higher values for shorter races. For handicap and earnings measures, reported heritabilities were generally higher, frequently in the range of 0.3–0.4.

Rate of genetic improvement The rate of genetic improvement in a population under selection can be predicted if the following four factors are known: (i) genetic variance for the trait under selection; (ii) its heritability; (iii) the intensity of selection for this criterion; and (iv) the generation interval in both males and females. Genetic change can also be measured retrospectively by appropriate analyses. Gaffney and Cunningham (1988) predicted the rate of genetic gain in the British Thoroughbred for Timeform rating at 0.92 Timeform units per year. This was based on generation intervals of 11.2 and 9.7 years, and selection intensities of 6 and 52% in males and females, respectively, and a heritability value of 0.36. They then estimated the rate of genetic change by a sire model best linear unbiased prediction (BLUP) analysis involving a total of 516 stallions born in the years 1952–1977, and performances of their 11,328 progeny between 1961 and 1985. The estimated change averaged 0.94 ± 0.13 Timeform units per year. This result is paralleled by the results of a large analysis of Quarter Horse data from the USA (Buttram et al., 1988). Quarter Horse races are run over much shorter distances, most commonly 320 m, with a mean finishing time of 18.7 s. From an analysis of finishing time data on over 1 million racing records, they estimated genetic trends in the population between 1960 and 1983. They found a consistent improvement, averaging 0.47, 0.43 and 0.16% per year, respectively, for the distances 320, 366 and 402 m. This genetic trend accounted for one-third of total improvement in finishing times over the period. In contrast to these studies, Preisinger et al. (1990) and Chico (1994) were unable to demonstrate genetic change in the German and Spanish Thoroughbred populations.

Limits to performance? In contrast to the results showing steady genetic improvement in Timeform ratings, Gaffney and Cunningham (1988) observed that winning times of Classic races have not been improving for a long time. In fact, the three English Classics have improving winning times from the 1840s onwards to about 1910, and from then seem to have been static (Fig. 15.1). If steady genetic improvement is being achieved, how can we interpret the static winning times in Classic races? James (1990) has argued that the heritability values, and hence the calculated improvement, are overestimated. One possibility is that there is some kind of physiological ceiling to performance, particularly in longer races. There is some support for this idea in the fact that

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Trends in winning times for the English Classics. Each point is an average of 10

winning times are continuing to improve in the shorter races, common in USA, while the plateau is most evident in longer races such as the St Leger (Fig. 15.1). Thus, the physiological limit might come into play only beyond a certain threshold of effort. If this hypothesis is true, then it should be possible to identify the physiological elements which set a limit to performance. The probable limiting factors will depend on the length of the race. The energy required for muscle contraction is derived from glucose (or its storage form glycogen). Energy release from glucose comes in two stages. The first, anaerobic, stage releases less than 10% of the energy available and produces lactic acid which accumulates in muscle tissue. The second, aerobic, stage requires oxygen, and releases the energy stored in glycogen. The oxygen supply has the additional function of removing lactic acid. In theory, either of these factors – the ultilization of oxygen and glycogen for aerobic metabolism during prolonged events, and the production and clearance of lactic acid following anaerobic metabolism during short-term events – could be limiting factors. In every race, both kinds of muscle metabolism are involved. Although the flow of blood to the muscles, and hence the supply of oxygen, increases dramatically during exercise, maximum effort could not be sustained by aerobic contraction alone. Anaerobic contraction may be limited by glucose availability and lactic acid accumulation. The balance of these two sources of energy supply to muscular activity depends on many factors, and particularly on the amount of activity involved, i.e. the length of the race. The 30 s sprint-type races of American Quarter Horses are mainly anaerobic. The 2.5 min required to run the English Classics shifts the balance to the point where most of the energy supply is aerobic. This difference in the energy supply route may

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indeed be part of the explanation for the apparent limits to performance for the longer races, while records continue to be broken in the sprints. The physiology of blood circulation and exercise in horses has been reviewed by Fregin and Thomas (1983). Figures 15.2 and 15.3 present two important sets of data from their report. They measured a number of metabolic indicators as the work effort was increased in a number of horses. With increasing effort, heart rate increased steadily from a resting figure of about 40 beats min−1 to a maximal rate close to 200. At the same time, blood volume per beat increased, also linearly. The result is a dramatic linear increase in blood circulation with increasing effort. At full stretch, a 500 kg horse is pumping 250 l of blood through their system each minute, equivalent to ten times his blood volume (Fig. 15.2). The important point is that blood circulation and oxygen uptake increased linearly over the range. Figure 15.3 shows blood lactic acid levels over the same range of effort. Here, it is evident that lactic acid clearance is not keeping pace with increasing effort, and is indeed becoming critical at maximum effort. What this suggests is that the limiting factor in performance may well be lactic acid accumulation. These conclusions are supported by the results of Evans et al. (1993), who found a strong relationship between blood lactate concentration after a strenuous treadmill test, and Timeform rating, in 14 Thoroughbred horses. The area is reviewed by Ronéus (1996) who also found a significant correlation between plasma lactate levels and speed over 1600 metres in Standardbred trotters.

Fig. 15.2. Cardiac output (Q) and heart rate (HR) at rest and during a five stage treadmill exercise test on an 11.5% gradient in five sedentary horses (redrawn from Fregin and Thomas, 1983).

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Fig. 15.3. Relationship between lactic acid concentration and work effort (redrawn from Fregin and Thomas, 1983).

Horses for courses A recent study from Japan (Oki et al., 1997) adds weight to the view that the genetics of performance depend very much on the length of the race. They calculated genetic correlations between performances at different race lengths, on both dirt and turf, from over 20,000 performance records. Race lengths varied from 1000 to 2000 m. As the difference in the race lengths increased, the genetic correlation decreased. At a difference of 400 m, the correlation was 0.91; at 600 m difference it was 0.78, and at 800 m the correlation was 0.68. This pattern was very much the same on turf and dirt tracks. The genetic correlations between performance on dirt and turf are even lower, and depended on the length of the race. At 1200 m, the correlation was 0.69, while at 1800 m it was 0.31. Thus if a horse is evaluated on dirt, the effectiveness of selection for performance on turf is about half what it would be if comparable data from its turf performances had been used. This corresponds to an average correlation between performance in these two environments of 51%. The loss of effectiveness is not so great at short distances as it is at long distances: at 1200 m the reduction is only 31%, while at 1800 m, it is 69%. Within any racing environment, turf or dirt, selection at one length for performance at another becomes progressively less effective as the distance between the two lengths increases. If the difference between the two lengths is 200 m, about 5% of effectiveness is lost. At 400 m, this is about 9%, at 600 m difference it is 22%, and at 800 m difference the loss in effectiveness is 32%.

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However, the effect of different lengths is in general much smaller than the effect of turf versus dirt. These results indicate that, to a considerable degree, different genetic factors are involved in performance in longer or shorter races and on different kinds of track. They could have serious implications for the evaluation of animals based on American data (short races, dirt tracks) for performance in European (longer races, turf tracks) races. It means that new weight (and discount) should be applied to data generated in a different racing environment. The net conclusion is to reinforce the old notion of ‘horses for courses’.

Trotters Performance comparison in trotters deals with the general problem of measuring performance in races with additional complexity due to the type of exercise and breed. Performances in trotters, in contrast to Thoroughbreds, are characterized by qualifying tests before entering races; greater longevity in their racing career; relatively shorter term of selection for trotting speed (since early this century); and the inclusion of more than one breed, i.e. Standardbred, French trotter and Nordic local breeds. Firstly, classical measures (speed and earnings) will be analysed and then new methodology introduced, to give a better evaluation of performance. Performance for non-recorded horses will be considered, and the general trend to a precocious racer will be discussed.

Speed and quality of trotter A natural choice to measure a performance trait for a racing horse is obviously to measure his ability to run fast. Speed is a traditional measure used as a reference for breeding. Results are summarized by time for a given distance (often 1 km) and then by best time for this distance (in the year or the lifetime). For this kind of record, it is important to take into account variability due to racing distance (often under 2000 m, near 2000 m and >2000 m), kind of start (flying start, volt-start, others) and eventually track type and racecourse. The use of repeated times or best times leads to different strategies. Heritability of such a trait is moderate and homogenous across country, with few exceptions. In Finland, Saastamoinen and Nylander (1996a) estimated heritabilities for best time in career at 0.28 for Standardbred and 0.08 for Finnhorse. Heritability of time in particular races, i.e. first qualifying, passed qualifying and first race, is higher (from 0.36 to 0.53 at 3 years old) but perhaps overestimated because it does not take racing conditions into account. In Belgium, Leroy et al. (1989) found a heritability of 0.11 for best time in life for a mixed trotter population (European and American origin). In France, Langlois (1984) found a heritability of between 0.26 and 0.43 for the best time at different ages (2–6 years old). In Germany, Katona and Distl (1989) found values of 0.35 for average time and 0.50 for average time performed at 2–3 years old. In Norway,

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Klemetsdal (1989) found a value of 0.03 for repeated racing time for Standardbred and 0.18 for Norwegian trotter. In Arnason et al. (1989), heritability for best racing time ranges from 0.45 to 0.26 according to the limit age of the record (3–13 years old); a reasonable mean seems to be 0.29 (Swedish Standardbred) and 0.25 (North Swedish trotter). In Italy, for repeated annual best time (up to 6 years old), Silvestrelli et al. (1995) found a value of 0.37. No clear rules exist regarding over- or underestimation of heritability when models do not take into account racing conditions or when best record is used instead of all recording times. According to recent selection for speed in trot, this trait remains heritable and genetic increase is observed in most countries. There is no doubt of the possibility to select for this trait, but the search for other traits to record performances nevertheless is necessary in order to report the complete aptitude of a horse to win a race. Speed is only one quality of a good racehorse, its capacity to adapt to race conditions and to win at a certain pace is the complete goal.

Earnings At the end of a race, money is allocated to the horses according to their ranking in the race. Most recent authors measure performances in trotter horses by their earnings. The choice of the measure linked to earnings reveals different aspects of the aptitude of the horse. Differences are principally in the mathematical transformations of earnings to reach a normal distribution in the population and in the choice between cumulated trait (year, life) or repeated trait per race, including the method to take into account the number of starts. As pointed out by Langlois (1975), a transformation is necessary in most cases. For the betting, or simply for the spirit of the exhibition, riders must have the will to win. Thus, earnings are distributed exponentially as a function of the ranks in a course or an event: a rider would only really want to be first if the difference in prize money is significant between the winner and the second placed horse. This raw performance scale is attractive for competition but inadequate for genetic evaluation because it emphasizes the differences in quality between ranked horses. Transformations are now commonly used and give a reasonable normal distribution of the trait. Earnings = a exp (b rank)

⇒

Log (earnings) = Log (a) + b rank

The following are only the most recent references available to see the generalization of this concept: in Italy, Silvestrelli et al. (1995) used log10 (annual earning + 1/number of starts); in Finland, Saastamoinen and Nylander (1996b) used (annual earnings)1/4; in Norway, Klemetsdal (1994) used (cumulated earnings)0.20 standardized within birth year; in Sweden, Arnason et al. (1982) , used (lifetime earnings)1/2, (lifetime earnings)1/4, earnin s n starts Log10 earnin s n . According to the initial distribution of earnstarts 1 ings in the population, due to rules of distribution of money in one event and

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Fig. 15.4.

Distribution of measures based on earnings – example of French trotters in 1994.

the programme of endowment of races, these different transformations lead to a more or less normal distribution in each country (Fig. 15.4) Money is distributed in every race, but the quality of a horse must be measured on its entire career. It may be appreciated as a repetition of each race or as the cumulated success over a year or a life. Choice of a unit of measurement depends on the objectives of the measure but also on the recorded results. When you use a cumulated performance over a year or a lifetime, this is a combination of regularity, longevity and level of races won which is included in the measure. If only placed races (as repeated earnings without non-placed results or earnings divided by number of places) are used, the maximum level of the horse is taken into account but not its capacity to repeat this maximum. If cumulated earnings divided by starts are used with no regression on the number of starts, the mean level where a horse may be placed, and the agreement between the race programme proposed for the horse and its true quality are estimated but the results are not weighted with the number of tests and so give an advantage to horses with few races. Whatever these differences, correlations between these measures are very high, especially genetic correlations. For example, Arnason et al. (1989) found genetic correlation between total earnings and total earnings divided by the number of starts of 0.95 (5 years

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old), and Langlois (1984) found genetic correlations of 0.92–0.98 between annual earnings and annual earnings divided by number of starts according to age (2–6 years old). However, high genetic correlations do not take into account a few special cases which may be of great importance when selecting stallions on their own performance. Thus, it is better to distinguish the different traits which are responsible for a complete successful career. We have the advantage of being able to consider success event by event and then to consider regularity and longevity as two additional traits for a good horse. This is what will be discussed in the next section about future methods of evaluation. In many cases, heritability of ‘earnings’ is moderate and is sufficient to facilitate selection. A summary of these heritabilities for Standardbred, French trotter and Nordic trotters is given in Table 15.2. Correlations between earnings and times are negative and high, and so are favourable (Table 15.3). Therefore, Table 15.2.

Heritability of criteria based on earnings.

Breed/Age

Reference

French trotter Standardbred Standardbred up to 3 years Dutch trotter 2–8 years Standardbred 2–6 years Norwegian trotter 3–6 years Standardbred 3–6 years North Swedish trotter Standardbred Standardbred Finnhorse trotter

1 2 2 3 3 4 5 6 6 6 6 7 8 8

Trait related to earnings

Heritability

Log(annual earnings/starts) Total earnings Total earnings Total earnings (Total earnings)1/2 (Total earnings)0.20 (Total earnings)0.20 (Total earnings)1/4 Log10(earnings1/2/starts + 1) (Total earnings)1/4 Log10(earnings1/2/starts + 1) Log10(annual earning + 1/starts) (Total earnings)1/4 (Total earnings)1/4

0.26 0.21 0.22 0.20/0.36 0.23/0.40 0.14/0.22 0.15/0.25 0.22/0.36 0.29/0.38 0.28 0.19 0.10 0.36 0.17

References: 1, Tavernier (1989); 2, Katona and Distl (1989); 3, Minkema (1989); 4, Klemetsdal (1993); 5, Klemetsdal (1989); 6, Arnason et al. (1989); 7, Silvestrelli et al. (1995); 8, Saastamoinen and Nylander (1996a). Table 15.3.

Correlation between criteria based on earnings and time.

Breed/age French trotter 3–6 years Norwegian trotter 3–6 years Standardbred North Swedish Standardbred Finnhorse trotter

Reference

Genetic correlation

Phenotypic correlation

1 2 3 3 4 4

−0.86/−0.95 −0.93/−1.00 −0.88/−0.91 −0.93 −0.98 −0.98

−0.59/−0.69 −0.81/−0.89 −0.37/−0.46 −0.68 −0.81 −0.88

References: 1, Langlois (1984); 2, Klemetsdal (1989); 3, Arnason et al. (1989); 4, Saastamoinen and Nylander (1996b).

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selection based on times and earnings is quite effective. A multiple trait approach avoids potential biases of one particular measure, even if the objective of all traits is much the same. It seems to be the better choice, as proposed initially by Arnason et al. (1982). Are there still true precocious performances? In the preceding, performance is supposed to play the same role throughout the career of the horse. According to the race programme and the strategy of the race business, the question of which breed and which type of trotter is important. Local breeds are often preserved by a particular programme of races as they are often slower and less precocious. However, the tendency to precocity is very often a tool for rapid economic profitability. From a genetic viewpoint, it is also obligatory information in order to select early even for a mature horse. Therefore, several authors studied the capacity of early performances and the relationship between early performance and mature performances. Early performances Age at first start in trotters was analysed by Saastamoinen and Nylander (1996a, b). Heritability estimates for age at first qualifying start, passed qualifying start and first race were low (0.04–0.16) and lower for the local breed (Finnhorse trotter) than for the Standardbred. In the absence of other results, these heritability estimates suggest that early start of a career depends mainly on environment. Relationship to mature performances Normally, only results with a correct model, i.e. taking into account the influence of selection between ages in the estimation of parameters, should be reported. This requires the use of a methodology which includes horses with only one performance in the analysis of correlation. However, this methodology is too recent for comparison with old studies on trotters and so results may be biased. No general trend is found regarding the level of heritability of early performances compared with mature performances. A recent estimation with a complete model for annual earning per start for French trotters showed a rather similar heritability from 2 year olds to 4 year olds: 0.24, 0.26 and 0.27, respectively (A. Ricard, unpublished). Arnason et al. (1989) and Saastamoinen and Ojala (1991) found a decrease of heritability with age for Standardbred: from 0.38 to 0.29 for earnings and from 0.45 to 0.34 for best time criteria (3 and 4 years old in contrast to cumulated before 6 years old) in Arnason et al. and from 0.38 to 0.19 for annual earnings and from 0.52 to 0.26 for best time (from 3 to 5 years old) in Saastamoinen and Ojala. The decrease is accentuated for time criteria. For Norwegian trotters, Klemetsdal and Stubsjoen (1996) found the opposite result, with an increase of heritability for cumulated results from 0.14 to 0.22. It is interesting than the most precocious breed (Standardbred)

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still expresses higher heritability for early performance. Genetic correlations between early and mature performances were always very high, particularly for cumulated performance, partially due to autocorrelation of the same trait. Correlations between different ages were estimated in Sweden by Arnason et al. (1989). They were found to be high for performances measured as earnings or earnings per start (0.76–1) between horses of 3, 4 and 5 years old. In Norway, for cumulative earnings in Norwegian trotters, genetic correlations were very high (0.97–0.99) between 3–6 years old (Klemetsdal, 1994). For age-based trait analysis, in French trotters these correlations remain high: 0.89 between 2 and 3 years old, 0.96 between 3 and 4 years old and 0.77 between 2 and 4 years old. Actually it is difficult to model a general explanation of early performances. If entrance in races seems to depend on environmental factors (as criteria based on qualifying races), the success at such an age is already heritable. Early performance seems to be a good predictor for selection for a mature horse, with little specificity for very early ones (genetic correlations near 0.80).

A new way to measure success and career In future, measuring performance for trotters must be able to describe what happens in one event and then model the repetition of this trait over life with more subtlety than these global measures. Some ways have begun to be investigated. Underlying performance To measure one performance in one race, the problem is to evaluate the level of the race and the level of one rank in this race. With regard to earnings, the measure of the level of the race is supposed to be the total endowment, and level of one rank is a linear function of it (after transformations). However, the level of the race is only the level of competitors because the only reason a horse is ranked at a given place is because the horse ranked before has beaten it and it has beaten the horse ranked behind. The level of a rank cannot be a linear scale independent of the number of starters; this is only the difference between one performance of a horse and those of the others. To explain the final ranking of a race, without introducing any subjectivity, let us imagine that all horses in the race performed a physical effort. This effort may be designed by a classical model with a normal distribution and expectation based on the influence of environmental and gene effects. The apparent result of the race, the ranks, is the expression of the hierarchy between these efforts. Thus, to estimate the effects of the model, inference will be based on the probability of obtaining the ranking of underlying performances instead of the probability of obtaining a measured performance. Figure 15.5 illustrates a race with three horses (A, B and C) with three different expectations. Table 15.4 contains the probability of each possible result of the race, i.e. different ranking, according the difference of the mean value of the horses.

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Fig. 15.5. ranking.

Example of a race with three horses – underlying performance and probability of

Table 15.4. Probability of ranking for the sample race (deviation between mean value of performance of A and B: 1.2, B and C: 0.6 in unit deviation). Ranking

Probability (%)

A–B–C A–C–B B–A–C B–C–A C–A–B C–B–A

49 27 15 3 5 2

The likelihood of ranking may be written as: +∞ +∞

P ( y (1) > y ( 2) >K> y (t ) >K> y ( n )) =∫

+∞

∫K ∫

−∞ y ( n )

y (t

+ 1)

+∞

K∫

n

∏ ϕ ( y (i ) −µ (i ) )dy (i )

y (1 ) i = 1

with (1).. (n) the ranks of horses in the event, y(t) the underlying performance of the horses ranked t, ϕ the normal density, µ(i) the location parameter of the horse ranked (i) (equal to environmental and gene effects). This model is actually applied for jumping, dressage and 3-day eventing in France. It will also be applied in the next few years in trotters and has already been tested on a single year file (Tavernier, 1994b). The file contains 9228 races run in 1989 by 13,065 horses and represents 129,379 starts. The model includes sex, age, distance of backing (0, 25 m, 50 m) and optionally trainer effect. The repeatability was estimated to be 0.26. The validity of the model was measured by the posterior probability of races calculated with the

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estimations of the effects. The probability of the races was multiplied by 11 on average by comparing it with the probability of each possible combination when all horses are equal. Only 1% of race results are unexpected: the probability of the ranking calculated from estimations is smaller than the probability when horses are equal. More than 12% of races have their equal probability multiplied by 20. A good presentation of results is the computing of elementary results for match between two horses. A horse estimated at 1 SD of the distribution of evaluations on this file has a probability of 74% of winning against a horse estimated at −1 SD. Between the better and the worse horse, the probability that the former one wins is 99%. This view of the result of a race has an attractive aspect for horsemen as it corresponds to traditional use. Trainer effect All cumulated measures are unable to take into account a trainer or driver effect. This effect also is often missing from time records. With this new measure of performance, it is possible to add this type of major environmental effect. With the previous French file, a model with and without a trainer effect was tested. A total of 529 different trainers were identified, with grouping of trainers with less than ten horses. The variance of horse effect was reduced but the validity of the model was better: the posterior probability of each race is multiplied by 17 comparing it with random instead of 11 with no trainer effect. The correlation between horse effect in the model without a trainer effect and that with a trainer effect is 0.88. The correlation between trainer effect and horse effect in the model with a trainer effect is −0.05; without a trainer effect it is 0.38. Here again, the new method seems to be more adaptable to the true situation than other measures. Random regression The model of underlying variables solves the problem of measuring success in one race. The possibility remains to measure the evolution of such a performance during life. A repeatability model suggests a constant mean and only a residual variation throughout life around it. A new development will be the design of underlying performance with time. Random regression models, which explain performance as a function of time, provide new methods in genetics. Unlike usual production (lactation or growth), there are no reference curves for the natural career. Thus different regressions must be tested with different visions of time. Time may be appreciated as starts or days, days from birthday or from the first race or days between races (rate of use). No applications are yet available for trotters but work has begun in jumping horses with an example model as follows: y i =bi 1 + bi 1 t + ai 0 + ai 1 t + pi 0 + pi 1 t + ei with yijk, underlying performance responsible for ranks; bi, environmental fixed effects i; aij, genetic effect for the horse j; pij, permanent environmental

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effect common to all performances of the horse; eijk, residual. The degree 0 refers to an effect independent of time; 1 refers to regression terms as a function of time t.

The problem of non-recorded horses The preceding discussions suggest that all horses have some available performances. In fact, there are two possible scenarios: some horses never come on to a racecourse and some would never be ranked or earn some money. In the second case – the starter but non-winner horses – different strategies have been used, depending on the trait. When log (earning) is used (by year or event), the usual choice is to discard starts without earnings (Langlois, 1984). Some authors add 1 to calculate the logarithm (Silvestrelli et al., 1995). Cutting off the data introduces selection and therefore cannot be recommended. Applying the same value to non-winner horses (Arnason et al., 1989; Katona and Distl, 1989; Minkema, 1989; Saastamoinen and Nylander, 1996b) without any reference to the level of the race is also a mistake. The strategy of the new model based on ranks makes it possible to take into account unplaced horses as they were beaten by the last-placed horse. Therefore, the value of unplaced horses is modulated at each event by the value of horses which participated into the event. This solves the problem of identical value. For the first case – horses which never enter a race – no information is available about this lack of data: they may be very good horses which have been exported to another country or perhaps they are just poor trotters. Klemetsdal (1992) simulated a population which would be pre-selected before entering a trotter race on a hypothetical trait correlated with future earnings. He proved that genetic trend was underestimated when ignoring this fact and that applying a zero to non-starters is a better way than cut-off to estimate genetic trend. Arnason (1996) suggested a multiple trait approach with a variable defining the status of the horse (starter or not) and proved that it is an easy and good method to avoid biases in genetic progress and an aid to limit inbreeding.

Conclusion Performance in trotters is actually recorded as best times per kilometre and transformed earnings. These traits express heritability (0.20–0.40) adapted to an effective selection. Breeding evaluations from these records commonly are used in most European countries. New developments will be able to improve the estimation of the true level of each race, the evolution of performance with age, time and the rate of use, and the adequacy of genetic models by adding better environmental components such as trainers and drivers. The ideal is to obtain a horse economically better adapted to race and, according to race programmes, with a better precocity or longevity.

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Sport Horses Measurements of performance In sport horses, breeding objectives consist of a number of different variables measured at different ages of horses. The ultimate goal is to improve dressage and jumping ability of horses in competitions. The overall breeding objective is a combination of performance traits relating to horses’ dressage and jumping ability which are measured at different times and in various testing schemes. For breeding horses which are used primarily as leisure horses, the definition of the overall breeding objective leads to different weighting of performance variables, such as giving more weight to non-physical variables such as character and temperament. For sport horses, performance traits are measured in various systems of performance testing which can be differentiated due to test location, age and sex of horses. In some European countries, young stallions and mares, possible candidates for breeding, are being tested either at station with a common training period (stallions) from 30 to 100 days or in 1-day field tests (mares) without any common training. Horses participating in those performance tests are pre-selected by their owners or breeding organizations, e.g. stallions coming to licensing or thereafter to performance testing at station are at least the better quarter of each annual group whereas in mares the pre-selection is less intensive. Stallions and mares having passed the performance tests normally go for breeding, but some, as well as geldings, participate in dressage or jumping competitions which are grouped according to quality level and age of horses. Horses participating in competitions start only if they have a real chance of winning, whereby this type of selection becomes stronger for older horses and for high-level competitions. In some countries, station or field tests are obligatory, in others no station or field tests exist and participating in competitions is the only requirement for breeding stock horses. In general, the system of performance testing implemented in each country affects the number and quality of horses participating in the tests and finally the distribution and the estimates of genetic parameters of variables recorded in the performance tests. Performance testing at station and in field tests Most variables recorded in station or field tests refer to conformation, basic gaits, riding and jumping ability, and their scores given by one or more judges vary between 0 and 10. Licensing of young stallions at an age of 2.5 years is based mainly on scoring conformation variables, gaits judged in hand and jumping ability as scored in free jumping. Similar measurements are taken during registering young mares, excluding jumping ability. Performance testing of 3-year-old stallions and mares focuses on variables scored under saddle. Riding and jumping ability are measured and again basic gaits are scored. For stallions participating in the test at station, variables of character and temperament are judged. The basic problem with most variables is the limited

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degree of objectivity due to the subjective scoring. Introducing a higher degree of repeatability and objectivity is achieved through the use of several judges and test riders (e.g. for scoring riding ability) for one horse, changing the rider during the common training, and measuring performance without any rider (e.g. free jumping). Objective measurements of gaits such as length of steps and jumps are rarely used. The distribution of scores tends to follow the normal distribution with a mean between 6.0 and 8.0 and a standard deviation of about 1.0 (Table 15.5). In some countries, e.g. in Switzerland (Hascher, 1999) and The Netherlands (Koenen et al., 1995), conformation variables and walking and trotting in hand are described based on a linear scale. Such traits show a more centred distribution with a mean and standard deviation close to the values expected from the scale used (Table 15.5). Performance testing in competitions In competitions, performance of sport horses is measured by their earnings or points allocated to their ranks in events. Some countries record all the horses started in competitions, some others record on average only the top 30% of horses ranked. The measurement used for later analyses is either the earning or rank of each horse started or being placed in an event or the annually cumulated earnings or points or a derivative of the annual sum (Table 15.6). In most cases, earnings show a skewed distribution, with few very high earnings for horses ranked first or second. Transformations are now commonly used and give a reasonable normal distribution of the trait and equalize variances between events. Table 15.6 shows the most recent references for transformations of performance data in sport horses. When measuring the performance in competitions, the problem is to evaluate the level of the competition or event and the level of a rank in this event (Ricard, 1998). Several suggestions have been made, such as transforming ranks by (1 – (place – 1)/number of starters + constant for the level of event) (Bruns, 1981), (place1/2) (Hassenstein, 1998) or by the normalized rank score Table 15.5.

Selected performance data of sport horses measured in station or field tests.

Test location

Variable (scores)

Mean

Standard deviation

Stallions’ licensinga

Walk Trot Character Riding ability Jumping ability Walk, trot, gallop Free jumping Walk (lin.) Trot (lin.)

6.8 7.0 8.0 6.9 7.2 6.8 7.1 6.1 6.1

1.8 1.4 0.9 1.1 1.1 0.9 1.2 1.2 1.3

Stallions’ testb Mares’ testb Mares’ registeringc aSchade

(1996); bvon Velsen-Zerweck (1999); cHascher (1999).

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Genetics of Performance Traits Table 15.6.

429

Transformations of performance data in sport horse competitions.

Variable Cumulated points per year in show jumping Highest level during life time in dressage and show jumping Annual earnings in show jumping Earning at each place in dressage and show jumping

Transformation Log10 Square root Log10 Ln

Reference Foran et al. (1994) Huizinga and van der Meij (1989) Silvestrelli et al. (1995) Meinardus and Bruns (1989)

(Foran et al., 1995). The level of event depends on the quality of horses started in one competition. One solution to account for differences in the quality of competitions is to include competition as a fixed effect in the model describing the data for estimating genetic parameters. Tavernier (1991, 1994b) proposes an appropriate interpretation of the rank as the probability of ranks which may solve the problem of the mean level of each event as well as of the variability of competitors (Ricard, 1998). Another obstacle in working with competition data is the availability of data. Some horses never come to a competition, they are used in breeding or have little chance of winning, and other horses are never ranked and never earn money. Recording systems which ignore horses without earnings (Meinardus and Bruns, 1989) introduce selection and biases. Adding a constant to unplaced horses is recommended by Ricard (1998) if the level of competition is taken into account. Finally, the quality of the rider influences the ranking within events as well. The confounding of the genetic effects of horses with the quality of riders and level of events is a problem in estimating breeding values of horses which is tackled through the definition of variables and appropriate linear models.

Heritability of performance traits Performance testing at station and in field tests In some European countries, performance testing of sport horses at station or in 1-day tests has been carried out in a comparable and systematic way over the last 10–20 years so that genetic analyses were carried out on reasonably large data sets with thousands of individual horses sired by at least 100 stallions. The methods used for estimating genetic parameters are based on maximum likelihood techniques applying single or multiple trait animal models. In Table 15.7, the latest estimates of heritabilities from several countries are summarized. The individual estimates of heritabilities are based on data from warmblood riding horses in Germany, The Netherlands, Sweden and Switzerland. The estimates of heritabilities across countries as indicated by the average heritability allow comparisons between performance testing schemes and variables within tests:

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Heritability estimates of performance traits of young horses tested at station or in Heritability

Test location

Variable

Stallions’ licensing/ Mares’ registering Stallions’ test

Walk Trot Character/temperament Walk Trot Gallop Riding ability Free jumping Parcours jumping Walk Trot Gallop Riding ability Free jumping Parcours jumping

Mares’ test

Average

Individual

Reference

0.23 0.34 0.41 0.55 0.58 0.56 0.44 0.56 0.40 0.25 0.30 0.27 0.20 0.24 0.12

0.21, 0.19, 0.21, 0.30 0.36, 0.28, 0.38 0.41, 0.24a 0.43, 0.66, 0.33a 0.50, 0.66, 0.49a 0.47, 0.66, 0.39a 0.52, 0.36, 0.43a 0.47, 0.65, 0.47a 0.38, 0.43, 0.39 0.15, 0.27, 0.22, 0.35 0.35, 0.36, 0.14, 0.35 0.18, 0.35, 0.18, 0.35 0.26, 0.30, 0.03 0.27, 0.35, 0.15, 0.20 0.04, 0.20

1, 5, 6, 7 5, 6, 7 2, 4 2, 4, 9 2, 4, 9 2, 4, 9 2, 4, 10 2, 4, 9 4, 9, 10 1, 2, 3, 8 1, 2, 3, 8 1, 2, 3, 8 1, 2, 3 1, 2, 3, 8 1, 5

aNot

included in average heritability since data sets 3 and 5 are identical. References: 1, Hascher (1999); 2, Von Velsen-Zerweck (1999); 3, Huizinga (1991); 4, Brockmann (1999); 5, Christmann (1996); 6, Schade (1996); 7, Gerber et al. (1997a); 8, Gerber et al. (1997b); 9, Gerber et al. (1996); 10, Van Veldhuizen (1997).

• • • •

Basic gaits, riding and jumping ability are subjectively scored variables with sufficiently high heritabilities. Performance testing at station leads to variables with higher heritabilities than field testing, i.e. environmental effects are controlled and eliminated to a higher degree when testing at station. Scores for parcours jumping show lower heritabilities than scores for free jumping, i.e. in free jumping, external effects affecting the horse’s performance through the rider are excluded. Multiple trait analyses (data set 3) lead to higher estimates of heritabilities than single trait analyses (data set 5), i.e. combining data from the stallions’ and mares’ test allows partial adjustment for the selection of stallions joining the stationary performance test.

Performance testing in competitions In most European countries, performance testing of sport horses in competitions has been carried out in a standardized way over many years so that genetic analyses used very large data sets of thousands of individual horses sired by hundreds of stallions. The methods used for estimating genetic parameters are based on maximum likelihood techniques applying single or multiple trait animal models. In Table 15.8, the latest estimates of heritabilities from several countries are summarized.

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Genetics of Performance Traits Table 15.8.

431

Heritability estimates of performance traits of sport horses tested in competitions.

Variable Jumping competitions Rank (transformed Blom score) Rank (transformed Blom score, selected data) Highest level during life time Rank (transformed 1/2) Earning (transformed log) Earning (transformed log) Cumulative lifetime points (transformed log) Score Rank Error points Dressage competitions Highest level during life time Rank (transformed 1/2) Earning (transformed log) Earning (transformed log) Score

Heritability

Repeatability

0.09 0.02 0.16 0.05 0.05 0.10 0.28 0.14 0.25 0.09

0.25 0.09

0.11 0.11 0.10 0.11 0.35

0.12

0.45 0.29 0.27

Reference 5 5 4 6 6 3 7 2 8 1 4 6 6 3 2

References: 1, Hascher (1999); 2 Brockmann (1999); 3, Schade (1996); 4, Van Veldhuizen (1997); 5, Janssens et al. (1997); 6, Hassenstein (1998); 7, Foran et al. (1994); 8, Tavernier (1990).

The estimates of heritabilities and repeatabilities are based on data from warmblood riding horses in Belgium, France, Germany, Ireland, The Netherlands and Switzerland. The estimated heritabilities of variables measured in competitions as compared with those from station or field tests indicate the following:

• • • • •

Heritabilities and repeatabilities of competition variables are about 0.10 and 0.30 for jumping and dressage. Heritabilities of performance traits measured in competitions, in mares’ field tests and in stallions’ station tests are about 0.10, 0.25, and 0.50, respectively. Low heritability estimates for competition data are also caused by the selection of the data: horses at an higher age are selected, unplaced horses are either not recorded or incorrectly handled. Definition of performance in competitions through earnings or ranks insufficiently reflects the performance of a horse. Other unknown environmental effects influence the performance of horses in competitions and cause low heritabilities.

When working with competition data, estimates of heritabilities depend very much on the type of data. The definition of performance in competition and the system of performance testing in competitions as organized by the national federations affect genetic analyses. One of the greatest problems is

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selection in the data. Janssens et al. (1997) demonstrate the effect of ignoring unplaced horses which substantially decreased the estimates of heritabilities from 0.09 to 0.02. Similarly, when estimating heritabilities based on older, selected horses, reduced estimates can be found depending on the data set used (Huizinga and Van der Meij, 1989; Van Veldhuizen, 1997). However, selection also takes place between events and between quality groups of events; competitions might be grouped according to quality, training and age of horses in A- (the lowest), L-, M- and S- (the highest) classes. Defining performance data within those quality groups as separate traits and then estimating heritabilities by applying multiple trait models can account for the selection in the data. In this way, Hassenstein (1998) estimated heritabilities in jumping competitions as 0.14, 0.07, 0.06 and 0.06, and in dressage competitions as 0.17, 0.10, 0.11 and 0.13 for the four quality groups, respectively. However, Aldridge et al. (1999) found an opposite trend in the estimated heritabilities as 0.07 for the low level, 0.08 for the medium level and 0.10 for the high-level jumping competitions. Defining the performance of horses in competitions through earnings or ranks seems to be insufficient; most estimates of heritabilities based on earnings and ranks, even after transformation, are around 0.10. However, Brockmann (1999) analysed data from quality events for young horses, used scores for describing performance and estimated heritabilities of 0.14 and 0.35 for jumping and dressage. The higher estimates of heritabilities by Foran et al. (1994) and Tavernier (1990) raise questions about the system of performance testing which should lead to testing large and non-pre-selected progeny groups of stallions.

Genetic relationships between performance traits Due to the definition of the overall breeding goal in sport horses, genetic relationships between performance traits are essential in defining breeding objectives. Genetic correlations are estimated based on data from the stallions’ and mares’ tests. Again, only the latest studies are presented in Table 15.9. The studies on genetic correlations can be summarized as:

• • •

Positive genetic correlations exist between variables describing character/ temperament, basic gaits and riding ability of sport horses. The correlations are moderate. High positive genetic relationships exist between basic gaits and riding ability independently of the type of performance test. Between jumping ability and all other variables, no genetic correlations are found. The estimates were slightly positive or negative and seem to depend on the type of performance test since mostly negative correlations were found when variables are measured in the stallions’ test. Similar estimates were obtained based on competition data (Huizinga and Van der Meij, 1989), whereby the estimates were between −0.06 and −0.27 based

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Table 15.9. Estimates of genetic correlations between performance traits within testing schemes (stallions’ test above the diagonal, mares’ test below the diagonal)a.

Character, temperament Walk Trot Gallop Riding ability Jumping ability

Walk

Trot

Gallop

Riding ability

Jumping ability

0.30(4) 0.47(2)

0.39(4) 0.24(2) 0.75(4) 0.92(2)

0.24(4) 0.36(2) 0.74(4) 0.88(2) 0.87(4) 0.93(2)

0.51(4) 0.42(2) 0.79(4) 0.97(2) 0.84(4) 0.95(2) 0.80(4) 0.94(2)

−0.04(4)− 0.67(2) −0.21(4)− 0.13(2) −0.17(4)− 0.18(2) 0.03(4) 0.18(2) −0.04(4)− 0.12(2)

0.48(1) 0.52(3) 0.67(1) 0.60(3) 0.48(1) 0.68(3) 0.08(1) −0.10(3)− 0.42(5)

0.82(1) 0.82(3) 0.89(1) 0.83(3) 0.44(1) 0.02(3) 0.42(5)

0.82(1) 0.73(3) 0.59(1) 0.11(3) 0.42(5)

0.35(1) 0.06(3)

aReferences

are given in parentheses: 1, Hascher (1999); 2, Huizinga (1991); 3, Christmann (1996); 4, Schade (1996); 5, Gerber et al. (1997b).

on data from young stallions, but based on data from older stallions the estimates were between −0.04 and +0.10. Since the breeding objective is to improve the performance of sport horses in competitions, the genetic relationships between the various testing schemes are of paramount importance. Although the general principle in breeding programmes is to test potential candidates for breeding as early as possible, the testing schemes designed for young stallions and mares differ from the normal situation in competitions. The indirect selection based on the stallions’ and mares’ test is only reasonable if heritabilities and genetic correlations are high. Table 15.10 summarizes estimates of genetic correlations between corresponding performance variables in different testing schemes. The estimates indicate:

• • • •

The corresponding performance traits measured in stallions’ or mares’ tests are highly correlated. The genetic relationships between corresponding variables measuring either riding ability/dressage or jumping ability in stallions’ tests and competitions are highly positive (> 0.80). The genetic relationships between corresponding variables measuring either riding ability/dressage or jumping ability in mares’ tests and competitions are also highly positive (> 0.65). Performance traits measured in testing schemes as designed for young stallions and mares are good predictors for the performance of horses in dressage or jumping competitions.

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A. Ricard et al. Table 15.10. Estimates of genetic correlations between corresponding performance traits of different testing schemesa. Stallions’ test : mares’ test Walk Trot Gallop Riding ability Jumping ability

0.78(1) 0.95(5) 0.85(1) 0.95(5) 0.74(1) 0.95(5) 0.90(1) 0.90(6) 0.88(1) 0.95(5) 0.95(5)

Stallions’ test : competitions

Mares’ test : competitions

0.88(4) 0.68(6) 0.83(2) 0.79(4) 0.90(6) 0.90(2)

0.68(3) 0.83(2) 0.64(3) 0.48(2)

aReferences

are given in parentheses: 1, Von Velsen-Zerweck (1999); 2, Huizinga (1991); 3, Brockmann (1999); 4, Schade (1996); 5, Gerber et al. (1996); 6, Van Veldhuizen (1997).

Concluding remarks The genetic analyses of performance data of sport horses show that the variables measured in the stallions’ test at station are highly heritable and highly correlated with the corresponding variables in competitions. The variables measured in the 1-day field test of mares show similar, but lower results. On the other hand, variables describing the performance in competitions have generally low heritabilities. Model calculations were done to optimize breeding programmes in sport horses, also considering artificial insemination as a common technique in horses (Koerhuis et al., 1994; Bruns and Schade, 1998; Brockmann, 1999). Genetic progress in sport horse breeding can be increased when selection is based on multiple-trait genetic evaluations of stallions and mares and carried out at sequential steps combining information from the tests of stallions and mares and competitions. Such integrated systems of genetic evaluation of sport horses as shown by Von Velsen-Zerweck (1999) have great prospects in horse breeding.

References Aldridge, L.I., Kelleher, D.L., Reilly, M. and Brophy, P.O. (1999) Estimation of the genetic correlation between performances at different levels of show jumping competitions in Ireland. Journal of Animal Breeding and Genetics, in press. Arnason, T. (1996) Selection criterion for increased long term response in Nordic trotters. 47th Annual Meeting of the European Association for Animal Production. Lillehammer, Norway, 25–29 August.

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Arnason, T., Darenius, A. and Philipsson, J. (1982) Genetic selection indices for Swedish trotter broodmares. Livestock Production Science 8, 557–565. Arnason, T., Bendroth, M., Philipsson, J., Henriksson, K. and Darenius, A. (1989) Genetic evaluations of Swedish trotters. In: State of Breeding Evaluation in Trotters. EAAP Publication, Pudoc, Wageningen, pp. 106–130. Brockmann, A. (1999) Entwicklung einer Eigenleistungsprüfung im Feld für Hengste unter Berücksichtigung der Turniersportprüfung. Dissertation Göttingen, FN-Verlag Warendorf. Bruns, E. (1981) Estimation of the breeding value of stallions from the tournament performance of their offspring. Livestock Production Science 8, 465–473. Bruns, E. and Schade, W. (1998) Genetic value of various performance test schemes of young riding horses. 6th World Congress on Genetics Applied to Livestock Production. Armidale, NSW, Australia, 11–16 January. Buttram, S.T., Willham, R.L., Wilson, D.E., Heird, J.C., Hookstra, J.A. and Lueche, G.R. (1988) Genetics of racing performance in the American quarter horse I, II, III & IV. Journal of Animal Science 66, 2791–2825. Chico, M.D. (1994) Genetic analysis of thoroughbred racing performance in Spain. Annales de Zootechnie 43, 393–397. Christmann, L. (1996) Zuchtwertschätzung für Merkmale der Stutbuchaufnahme und der Stutenleistungsprüfung im Zuchtgebiet Hannover. Dissertation Göttingen, Cuvillier Verlag, Göttingen. Evans, D.L., Harris, R.C. and Snow, D.H. (1993) Correlation of racing performance with blood lactate and heart rate after exercise in Thoroughbred horses. Equine Veterinary Journal 25, 441–445. Foran, M.K., Cromie, A.R., Reilly, M.P., Kelleher, D.L. and Brophy, P.O. (1994) Analysis of show jumping data in the Irish sport horse population. 45th Annual Meeting of the European Association for Animal Production. Edinburgh, UK, 5–8 September. Foran, M.K., Reilly, M.P., Kelleher, D.L., Langan, K.W. and Brophy, P.O. (1995) Genetic evaluation of show jumping horses in Ireland using ranks in competition. 46th Annual Meeting of the European Association for Animal Production. Prague, Czech Republic, 4–7 September. Fregin, G.F. and Thomas, D.P. (1983) Cardiovascular response to exercise in the horse: a review. Proceedings of the First International Conference on Equine Exercise Physiology, Oxford, 1982. Gaffney, B. and Cunningham, E.P. (1988) Estimation of genetic trend in racing performance of Thoroughbred horses. Nature 332, 722–723. Galton, F. (1898) An examination into the registered speeds of American trotting horses, with remarks on their value as hereditary data. Proceedings of the Royal Society of London 62, 310. Gerber, E., Arnason, Th., Stalhammar, H. and Philipsson, J. (1996) Correlations between traits at performance tests of Swedish Warmblood stallions and field record traits. 47th Annual Meeting of the European Association for Animal Production. Lillehammer, Norway, 25–29 August. Gerber, E., Näsholm, A. and Philipsson, J. (1997a) Genetic parameters for conformation traits of warmblood horses in Sweden. 48th Annual Meeting of the European Association for Animal Production. Vienna, Austria, 25–28 August. Gerber, E., Arnason, Th. and Philipsson, J. (1997b) Procedures for genetic evaluation of conformation and performance of riding horses in Sweden. 48th Annual Meeting of the European Association for Animal Production. Vienna, Austria, 25–28 August.

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A. Ricard et al. Hascher, H. (1999) Schätzung von Populationsparametern mittels Exterieur- und Leistungsdaten für Haflinger, Freiberger und CH Warmblut. Dissertation ETH Zürich Nr. 12653. Hassenstein, C. (1998) Genetisch–statistische Analyse von neuentwickelten Merkmalen aus Turniersportprüfungen für Reitpferde. Dissertation Kiel, Heft 103. Hintz, R.L. (1980) Genetics of performance in the horse. Journal of Animal Science 51, 582–594. Huizinga, H.A. (1991) Genetic studies on performance of the Dutch Warmblood Riding horse. Dissertation Utrecht. Huizinga, H.A. and Van der Meij, G.J.W. (1989) Estimated parameters of performance in jumping and dressage competitions of the Dutch Warmblood Horse. Livestock Production Science 21, 333–345. James, J. (1990) Selection limits in thoroughbred horses. Proceedings of the 4th World Congress on Genetics Applied to Livestock Production 16, 221–228. Janssens, S., Geysen, D. and Vandepitte, W. (1997) Genetic parameters for show jumping in Belgian sporthorses. 48th Annual Meeting of the European Association for Animal Production. Vienna, Austria, 25–28 August. Katona, O. and Distl, O. (1989) Sire evaluation in German trotter (Standardbred) population. In: State of Breeding Evaluation in Trotters. EAAP Publication. Pudoc, Wageningen, pp. 55–61. Klemetsdal, G. (1989) Norwegian trotter breeding and estimation of breeding values. In: State of Breeding Evaluation in Trotters. EAAP Publication. Pudoc, Wageningen, pp. 95–105. Klemetsdal, G. (1990) Breeding for performance in horses – a review. Proceedings of the 4th World Congress on Genetics Applied to Livestock Production 16, 184–193. Klemetsdal, G. (1992) Estimation of genetic trend in racehorse breeding. Acta Agriculturae Scandinavica, Section A Animal Science 42, 226–231. Klemetsdal, G. (1993) Sire selection strategies in North-Swedish and Norwegian trotters. 44th Meeting of the European Association for Animal Production, Aarhus, Denmark, 16–19 August. Klemetsdal, G. (1994) Application of standardized, accumulated transformed earnings in breeding of Norwegian trotters. Livestock Production Science 38, 245–253. Klemetsdal, G. and Stubsjoen, L. (1996) Effect of inbreeding on racing performance in Norwegian trotters. 47th Meeting of the European Association for Animal Production, Lillehammer, Norway, 25–29 August. Koenen, E.P.C., Van Veldhuizen, A.E. and Brascamp, E.W. (1995) Genetic parameters of linear scored conformation traits and their relation to dressage and show jumping performance in the Dutch Warmblood Riding Horse population. Livestock Production Science 43, 85–94. Koerhuis, A.N.M. and Van der Werf, J.H.J. (1994) Uni- and bivariate breeding value estimation in a simulated horse population under sequential selection. Livestock Production Science 40, 207–213. Langlois, B. (1975) Analyse statistique des gains des pur sang de trois ans dans les courses plates françaises. Annales de Génétique et de Sélection Animale 7, 387–408. Langlois, B. (1980) Heritability of racing ability in thoroughbreds – a review. Livestock Production Science 7, 591. Langlois, B. (1984) Héritabilité et corrélations génétiques des temps records et des gains établis par les trotteurs Français de 2 à 6 ans. 35th Annual Meeting of the European Association for Animal Production. The Hague, The Netherlands, 6–9 August.

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Langlois, B. (1996) A consideration of the genetic aspects of some current practices in thoroughbred horse breeding. Annales de Zootechnie 45, 41–51. Leroy, P.L., Kafidi, N. and Bassleer, E. (1989) Estimation of breeding values of Belgian trotters using an animal model. In: State of Breeding Evaluation in Trotters. EAAP Publication. Pudoc, Wageningen, pp. 3–17. Meinardus, H. and Bruns, E. (1989) Züchterische Nutzung der Turniersportprüfung für Reitpferde. 1. Mitteilung: Selektionsintensität und genetische Parameter. Züchtungskunde 61, 85–99. Minkema, D. (1989) Breeding value estimation of trotters in the Netherlands. In: State of Breeding Evaluation in Trotters. EAAP Publication. Pudoc, Wageningen, pp. 82–94. Oki, H., Sasaki, Y. and Willham, R.L. (1995) Genetic parameter estimates for racing time by restricted maximum likelihood in the thoroughbred horse of Japan. Journal of Animal Breeding and Genetics 112, 146–150. Oki, H., Sasaki, Y. and Willham, R.L. (1997) Estimation of genetic correlations between racing times recorded at different racing distance by restricted maximum likelihood in thoroughbred racehorses. Journal of Animal Breeding and Genetics 114, 185–189. Preisinger, R., Wilkens, J. and Kalm, E. (1990) Breeding values and estimation of genetic trends in German thoroughbred horses. Proceedings of the 4th World Congress on Genetics Applied to Livestock Production 16, 217–220. Ricard, A. (1998) Developments in the genetic evaluation of performance traits in horses. 6th World Congress on Genetics Applied to Livestock Production. Armidale, NSW, Australia, 11–16 January, 24, 388–395. Ronéus, N. (1996) Muscle metabolic response to track exercise in standardbred trotters. Swedish University of Agricultural Sciences. Acta Veterinaria 6, 12–42. Saastamoinen, M.T. and Ojala, M.J. (1991) Estimates of genetic and phenotypic parameters for racing performance in young trotters. Acta Agriculturae Scandinavica 41, 427–436. Saastamoinen, M.T. and Nylander, A. (1996a) Genetic and phenotypic parameters for age and speed at the beginning of racing career in trotters. Acta Agriculturae Scandinavica Section A Animal Science 46, 39–45. Saastamoinen, M.T. and Nylander, A. (1996b) Genetic and phenotypic parameters for age at starting to race and racing performance during early career in trotters. Livestock Production Science 45, 63–68. Schade, W. (1996) Entwicklung eines Besamungszuchtprogramms für die Hannoversche Warmblutzucht. Dissertation Göttingen. Silvestrelli, M., Pieramati, C., Cavalucci, C. and Bonanzinga, M. (1995) The current breeding plans for saddle horse, trotter and thoroughbred in Italy. 46th Annual Meeting of the European Association for Animal Production. Prague, Czech Republic, 4–7 September. Tavernier, A. (1989) Breeding evaluation of French trotters according to their race earnings. 2. Prospects. In: State of Breeding Evaluation in Trotters. EAAP Publication. Pudoc, Wageningen, pp. 95–105. Tavernier, A. (1990) Estimation of breeding value of jumping horses from their ranks. Livestock Production Science 26, 277–290. Tavernier, A. (1991) Genetic evaluation of horses based on ranks in competition. Genetics, Selection and Evolution 23, 159–173.

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A. Ricard et al. Tavernier, A. (1994a) Special problems in genetic evaluation of performances traits in horse. Proceedings of the 5th World Congress on Genetics Applied to Livestock Production 17, 450–457. Tavernier, A. (1994b) 5th World Congress on Genetics Applied to Livestock Production. Armidale, NSW, Australia, 11–16 January. Tolley, E.S., Notter, D.R. and Marlowe, T.J. (1985) A review of the inheritance of racing performance in horses. Animal Breeding Abstracts 53, 163–185. Van Veldhuizen, A.E. (1997) Breeding value estimation for riding horses in the Netherlands. 48th Annual Meeting of the European Association for Animal Production. Vienna, Austria, 25–28 August. Von Velsen-Zerweck, A. (1999) Integrierte Zuchtwertschätzung für Zuchtpferde. Dissertation Göttingen, FNVerlag Warendorf. Williamson, S.A. and Beilharz, R.G. (1998). The inheritance of speed, stamina and other racing performance characteristics in the Australian thoroughbred. Journal of Animal Breeding and Genetics 115, 1–16.

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Genetics of Conformation, Locomotion and Physiological Traits 1 and M. Saastamoinen Conformation, 16 Markku Locomotion and T. Saastamoinen E. Barrey and Physiological Traits Eric Barrey2 1Agricultural Research Centre, Institute of Animal Production, Equine Research, Varsanojantie 63, Ypäjä, FIN-32100, Finland; 2National Institute of Agricultural Research (INRA), Quantitative and Applied Genetics, F-78352 Jouy-en-Josas, France

Introduction Conformation and Physiological Traits: Their Evaluation and Heritability Conformation traits Physiological traits Conformation Related to Performance Conformation Related to Soundness and Durability Locomotor and Physiological Traits Related to Performance Harness racing Thoroughbred racing Three-day eventing and endurance riding Show-jumping Dressage Use of Conformation, Gait and Physiological Traits in Breeding References

439 441 441 447 452 457 458 458 460 461 461 462 463 464

Introduction Conformation of a horse can be defined as its form or outline. The conformation of today’s horse is a result of both nature’s selection and selection by man for various purposes. The conformation of the body varies among different breeds and even between different subpopulations and lines of the same breed. The first treatise ‘Hippike’ dealing with the body conformation and the military use of horses was written by Xenophon (445–354 BC). A quantitative approach to conformation traits was undertaken in the 18th century by Bourgelat (1754) who studied linear measurements of the body segment of the baroque horses (Fig. 16.1). In the 19th and early 20th century, other scientific studies developed hippometric methods and took into account the joint ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

439

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M. Saastamoinen and E. Barrey

Fig. 16.1.

Study of linear measurements ratios in the Spanish baroque horse from Bourgelat (1754).

angles and inclinations of the limb segments (arthrogoniometry). Nowadays, descriptions of a horse’s conformation and its details are presented in many handbooks. Judging of conformation has long-standing traditions in horse breeding. Although selection of horses is based mainly on their performance results, conformation and gaits still have an important role in judging horses at stud book shows. However, today’s horse is mainly an athlete or a working animal, and its value is determined largely by its exercise ability and the health of its limbs. Thus, more emphasis has been put on a functional conformation and gaits, instead of certain exterior details, during recent years in order to select horses for athletic disciplines. Conformation judging is used as an indicator of, and to select horses with, better soundness and less risk of developing lameness. The breeders, trainers and buyers can save time by avoiding horses whose potential is limited by certain serious conformation defects and physical handicaps. Furthermore, correct conformation and good movements are important factors for obtaining high prices for horses. However, the horse may have good conformation from one view and poor conformation from another. Criteria for and the description of the basic and ideal conformation of the horse are presented in many publications. With the development of equine physiology studies, some standardized techniques to test the exercise ability of race and saddle horses have been established. As a consequence of this, interest in the value and significance of some physiological traits and biochemical markers as possible early indicators of performance, and their use in early selection, has arisen during the last few years.

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Conformation and Physiological Traits: Their Evaluation and Heritability Conformation largely determines the general appearance of the horse. Body and leg conformation are known to be highly heritable. It is also known that the subjective scoring of conformation is influenced by several nongenetic factors (Arnason, 1984; Preisinger et al., 1991; Saastamoinen, 1993; Saastamoinen et al., 1998a). These include, for example, the judging team, sex, body condition and management of the horse, month and year of judging and, concerning growing horses, the age of the foal. All these factors should be taken into account when the conformation results and data are used in selection of horses and in genetic analyses. Conformation traits The conformation traits include body measures and angles, leg stances, hoof quality, movements (their length, elasticity and regularity) and teeth structure. The traits can be divided into scored and measured (objective) traits. The first includes subjectively evaluated traits, such as body and leg conformation, gaits and orthopaedic traits. Verbal evaluation and description of the conformation is also used in many countries. The objective traits are measured objectively (e.g. body measures, angles) and evaluated using, for example, photography or video techniques. Scored traits Scored traits usually are evaluated by giving points for their correctness. The collection of conformational details included in the evaluation is large and varies between countries and breeds or stud books. The most typical subjects of the judging are type, head, neck, over line, leg stances (limb alignment), quality of hooves and gaits/movements (their length, elasticity and regularity). The overall balance, harmony and symmetry of the body usually are also evaluated. The heritabilities for scored traits vary from low to moderate (0.20–0.50), thus being lower than those for measured traits. The heritability of leg stances generally is low, ranging, on average, from 0.10 to 0.20 (Table 16.1). This implies the difficulty in judging the conformation of legs. In addition, for all scored traits, the judging team members do not use the whole scale of scores, which reduces the variation between evaluated animals. Heritability estimates of individual defects recorded for leg stances are between 0.00 and 0.65 (Klemetsdal et al., 1986; Saastamoinen et al., 1998b; Dolvik and Klemekdal, 1999). In some horse populations, for example, in Dutch warmblood riding horses (Koenen et al., 1995) and Thoroughbreds (Mawdsley et al., 1996), the linear assessment trait evaluation system has been proven and used in conformation evaluation. The aim of this system is to describe where the individual being assessed lies between the biological extremes for a particular

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442 Table 16.1.

M. Saastamoinen and E. Barrey Heritability estimates for scored conformation traits.

Trait

Heritability

Breed

Reference

Type

0.29 0.41 0.28–0.31 0.37

Swedish warmblood riding horse Arab Trakehner Bavarian coldblood

0.34 0.28 0.25–0.36 0.20 0.36 0.23 0.24 0.39 0.19 0.51 0.21 0.12–0.14 0.17–0.18 0.43

Arab Hannover Trakehner Holsteiner (foals) Holsteiner Hannover Hannover Swedish warmblood riding horse Swedish warmblood riding horse Arab Icelandic horse Warmblood riding horse Trakehner Bavarian coldblood

0.52 0.23–0.26 0.21 0.51 0.29 0.21 0.23 0.16

Arab Trakehner Hannover Swedish warmblood riding horse Swedish warmblood riding horse Swedish warmblood riding horse Swedish warmblood riding horse Bavarian coldblood

0.14–0.17 0.45 0.13 0.15–0.17 0.09 0.14 0.09 0.18 0.30 0.13–0.48 0.17 0.20–0.27 0.21 0.30

Trakehner Arab Hannover Trakehner Holsteiner Hannover Hannover Finnhorse trotter Swedish warmblood riding horse Warmblood riding horse Hannover Hannover Hannover Swedish warmblood riding horse

Thafvelin et al. (1980) Ström and Philipsson (1981) Preisinger et al. (1991) Grosshauser and von Butler-Wemken (1991) Seidlitz et al. (1991) Weymann et al. (1991) von Butler-Wemken et al. (1992) Kühl et al. (1994) Kühl et al. (1994) Christmann et al. (1995) Schade (1996) Gerber et al. (1997a) Thafvelin et al. (1980) Ström and Philipsson (1981) Arnason (1984) Meinardus et al. (1986) Preisinger et al. (1991) Grosshauser and von Butler-Wemken (1991) Seidlitz et al. (1991) von Butler-Wemken et al. (1992) Schade (1996) Gerber et al. (1997a) Gerber et al. (1997b) Thafvelin et al. (1980) Gerber et al. (1997a) Grosshauser and von Butler-Wemken (1991) Preisinger et al. (1991) Seidlitz et al. (1991) Weymann et al. (1991) von Butler-Wemken et al. (1992) Kühl et al. (1994) Christmann et al. (1995) Schade (1996) Saastamoinen et al. (1998b) Thafvelin et al. (1980) Meinardus et al. (1986) Weymann et al. (1991) Christmann et al. (1995) Schade (1996) Gerber et al. (1997a)

Conformation

Head–neck–body Regulation of gaits and movements

Walk

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Conformation, Locomotion and Physiological Traits Table 16.1.

443

Continued.

Trait

Heritability

Breed

Reference

Trot

0.20 0.26 0.22 0.10–0.21 0.36–0.42 0.37 0.16 0.20 0.16

Icelandic horse Swedish warmblood riding horse Icelandic horse Warmblood riding horse Hannover Swedish warmblood riding horse Icelandic horse Icelandic horse Bavarian coldblood

0.16–0.18 0.09 0.10

Hannover Swedish warmblood Finnhorse trotter

Arnason (1979) Thafvelin et al. (1980) Arnason (1984) Meinardus et al. (1986) Christmann et al. (1995) Gerber et al. (1997a) Arnason (1979) Arnason (1984) Grosshauser and von Butler-Wemken (1991) Christmann et al. (1995) Gerber et al. (1997a) Saastamoinen et al. (1998b)

Correctness of legs Leg stances

Table 16.2.

Heritability estimates for linear type conformation traits.

Trait

Heritability

Breed

Reference

Type Gaits

0.30 0.27–0.41 0.14–0.22 0.26 0.28 0.36 0.07–0.21 0.14–0.23 0.10–0.17

Haflinger Shetland pony Dutch warmblood riding horse Haflinger Warmblood riding horse Haflinger Shetland pony Dutch warmblood riding horse Haflinger

Miglior et al. (1998) van Bergen and van Arendonk (1993) Koenen et al. (1995) Miglior et al. (1998) Hartmann and Schwark (1991) Miglior et al. (1998) van Bergen and van Arendonk (1993) Koenen et al. (1995) Samore et al. (1997)

Harmony Leg stances

conformational trait, and is a consequence of interest in putting more weight on a functional conformation of the horse. The choice of the traits used is based on their importance for movements and performance, economic value and also their heritability. The heritabilities for linear traits are of the same magnitude as those for subjectively scored conformation traits (Table 16.2). Evaluation of orthopaedic health is also a part of conformation judging. Locomotor problems and lameness are shown to be the most common reasons for culling of horses, and for non-starting and training failure in galloping, trotting and equestrian sports (Bergtsen, 1980; Bendroth, 1981; Jeffcott et al., 1982; Klemetsdal et al., 1985; Saastamoinen, 1991; Lindner and Dingerkus, 1993; Philipsson et al., 1998). Examination of the orthopaedic status of a horse is normally included in the stud book evaluation, and is also an important part of Riding Horse Quality Events for 4-year-old horses in Sweden (Gerber et al., 1997a) and some other countries. As for other health traits in animals, the heritability of the orthopaedic status of horses has been reported to be low, around 0.10 (Thafvelin et al., 1980).

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The scored traits (points given for the traits) are genetically mutually moderately or quite highly correlated (Table 16.3). The strongest correlations have been reported for scores for regularity of the gaits with points for type and conformation of the horse. Measured traits Various body measurements are usually taken of horses at stud book and judging shows. These include, among others, height at withers and/or croup, Table 16.3.

Genetic correlations between conformation traits.

Traits

Breed

Reference

Withers height×heart girth 0.38 0.30–0.60 0.84

Trakehner Half-bred riding horse Finnhorse trotter

Kaiser et al. (1991) Kapron et al. (1994) Saastamoinen et al. (1998a)

Withers height×cannon bone circumference 0.52 0.54 0.46–0.66 0.76

Bavarian warmblood Trakehner Half-bred riding horse Finnhorse trotter

von Butler (1986) Kaiser et al. (1991) Kapron et al. (1994) Saastamoinen et al. (1998a)

Heart girth×cannon bone circumference 0.56 0.48 0.77

Bavarian warmblood Bavarian coldblood Finnhorse trotter

von Butler (1986) Grosshauser and von Butler-Wemken (1991) Saastamoinen et al. (1998a)

Type×conformation 0.78–0.83 0.37 0.60–0.85

Trakehner Bavarian coldblood Trakehner

Preisinger et al. (1991) Grosshauser and von Butler-Wemken (1991) von Butler-Wemken et al. (1992)

Bavarian coldblood Swedish warmblood riding horse

Grosshauser and von Butler-Wemken (1991) Gerber et al. (1997a)

Trakehner Bavarian coldblood Holsteiner Hannover

Preisinger et al. (1991) Grosshauser and von Butler-Wemken (1991) Kühl et al. (1994) Schade (1996)

Swedish warmblood riding horse

Gerber et al. (1997a)

Conformation×regularity of gaits 0.65–0.85 0.95 0.30–0.81

Trakehner Bavarian coldblood Trakehner

Preisinger et al. (1991) Grosshauser and von Butler-Wemken (1991) von Butler-Wemken et al. (1992)

Movements×correctness of legs 0.32

Finnhorse trotter

Saastamoinen et al. (1998a)

Type×correctness of legs 0.90 0.47 Type×regularity of gaits 0.58–0.63 0.89 0.93 0.48 Type×trot 0.68

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body length, heart girth, and circumference of the cannon bone. Body measures are moderately or highly (h2 = 0.25–0.90) heritable (Table 16.4). Concerning body measures in growing horses, their heritabilities increase with the age of the horse (Hintz et al., 1978; Saastamoinen, 1990).

Table 16.4.

Heritability estimates for body measures.

Trait

Heritability

Breed

Reference

Height at withers

0.33–0.88 0.77 0.60 0.25 0.25 0.73 0.25–0.71 0.48 0.25 0.55 0.89 0.40–0.46 0.67–0.81 0.59 0.78 0.79 0.34 0.06–0.52 0.70–0.84 0.77 0.13–0.65 0.64 0.71 0.55 0.24 0.27 0.27 0.18–0.99 0.06 0.31 0.30 0.56–0.63 0.12–0.77 0.50 0.39 0.53 0.47 0.55 0.30–0.55 0.44 0.51 0.34 0.36–0.62 0.65 0.13–0.90 0.27–0.88

Thoroughbred (growing) Icelandic horse Icelandic horse Bavarian warmblood Warmblood riding horse Norwegian coldblood Finnhorse (growing) Arab Trakehner Bavarian coldblood Shetland pony Half-bred riding horse Finnhorse (young) Swedish warmblood riding horse Finnhorse trotter Haflinger Thoroughbred Finnhorse (growing) Finnhorse Finnhorse trotter Finnhorse (growing) Finnhorse trotter Icelandic horse Icelandic horse Warmblood riding horse Norwegian coldblood Bavarian warmblood Finnhorse (growing) Bavarian coldblood Arab Trakehner Half-bred riding horse Thoroughbred (growing) Icelandic horse Icelandic horse Norwegian coldblood Bavarian warmblood Warmblood riding horse Finnhorse (growing) Bavarian coldblood Arab Trakehner Half-bred riding horse Finnhorse trotter Thoroughbred (growing) Finnhorse (growing)

Hintz et al. (1978) Arnason (1979) Arnason (1984) von Butler (1986) von Butler and Krollikowsky (1986) Klemetsdal et al. (1986) Saastamoinen (1990) Seidlitz et al. (1991) Kaiser et al. (1991) Grosshauser and von Butler-Wemken (1991) van Bergen and van Arendonk (1993) Kapron et al. (1994) Thuneberg (1995) Gerber et al. (1997a) Saastamoinen et al. (1998b) Miglior et al. (1998) Biedermann and Schmucker (1989) Saastamoinen (1990) Thuneberg (1995) Saastamoinen et al. (1998b) Saastamoinen (1990) Saastamoinen et al. (1998b) Arnason (1979) Arnason (1984) von Butler and Krollikowsky (1986) Klemetsdal et al. (1986) von Butler (1986) Saastamoinen (1990) Grosshauser and von Butler-Wemken (1991) Seidlitz et al. (1991) Kaiser et al. (1991) Kapron et al. (1994) Hintz et al. (1978) Arnason (1979) Arnason (1984) Klemetsdal et al. (1986) von Butler (1986) von Butler and Krollikowsky (1986) Saastamoinen (1990) Grosshauser and von Butler-Wemken (1991) Seidlitz et al. (1991) Kaiser et al. (1991) Kapron et al. (1994) Saastamoinen et al. (1998b) Hintz et al. (1978) Saastamoinen (1990)

Height at croup

Body length Heart girth

Circumference of cannon bone

Body weight

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Body measures are usually genetically strongly (0.80–1.00) correlated with each other. This means that it is possible to minimize the number of measures to be taken from horses at stud book shows, to include only those having the largest influence on performance and usefulness of the horse. The largest average genetic correlations (0.89–0.99) have been found between height at withers and height at croup (von Butler et al., 1986; Biedermann and Schmucker, 1989; Saastamoinen et al., 1998b). Other objective (quantitative) methods have also been developed for evaluating the conformation of the horse. Langlois et al. (1978) used an objective method using photographs of horses. Nowadays, this photometric method has been improved by using digital cameras and image analysis applications (Fig. 16.2). Magnusson (1985) reported a method for measuring and describing the conformation on live horses from photographs with the assistance of 25 reference points in Standardbred trotters. The same method, which is based on measuring of angles and length of bones, has also been applied to riding horses (Holmström et al., 1990). Determining the reference points has to meet certain criteria: they should be easy to palpate and situated primarily on the skeleton. The points and the lines between them should coincide with outlines and axes commonly used when judging the conformation subjectively. It is also important to standardize the position of the horse. Videotaping and a computerized gait analysis system have been used in several studies, but they have not been applied routinely (e.g. Holmström et al., 1990; Deuel, 1995). In such studies, conformational details of so-called

Fig. 16.2. Photographical method for conformation measurements using anatomical markers (Langlois et al., 1978).

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elite horses in different disciplines have been described. A gait analysis system (Equimetrix) has been designed for early performance evaluation in trotters and show-jumping horses in France (see p. 452). Further, in some countries, radiographs from certain joints are taken in connection with conformation evaluation in order to determine the DOD status of a horse. No genetic data are available for the conformation traits obtained by the quantative methods described above.

Physiological traits With the development of equestrian sports and the racing industry, more functional traits have been studied to be used for early evaluation of horses. During exercise activity, several systems are linked functionally to produce movement (Fig. 16.3). The nervous system is responsible for the control and regulation of the other systems. The cardiovascular and respiratory systems provide nutrients and oxygen to muscle which then transforms biochemical energy into mechanical energy during muscle twitching. The locomotor apparatus under neurosensorial control makes it possible to produce well coordinated gaits. To date, genetic studies in equine exercise physiology have provided only a few heritability estimates because the measuring methods could not be applied in a large-scale study. Nervous system and behaviour Quiet and balanced horses are preferred for instruction and leisure activities, for example. Conversely, more reactivity is required for race and sport activities. The influence of the nervous system is very complex to analyse because it is responsible for many aspects of regulation and sensorial input. Its influence can be evaluated indirectly by carrying out behavioural tests and measuring physiological variables such as heart rate. Several types of behavioural tests have been proposed to quantify learning capacity, emotivity, excitability and sociability (Budzynski and Wawiorko,

Fig. 16.3.

Functional relationships between the organic systems involved in exercise activity.

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Table 16.5.

Examples of tests for quantifying emotionality and learning capacity.

Behavioural test Method

Measure

Sound test

Time elapsed between stopping eating and resuming of feeding Stereotype Time to walk through Behaviour Distance from the object Time to walk through Time of passage

Timidity test Emotivity test Labyrinth test Box test

Reaction of the horse to an electric bell sound during feeding Walking between two rotating screens

Alone in arena Unknown object Walking over a bridge Evaluation of remembering a sensorimotor process and speed of learning after five trials Behaviour and time before opening the box Opening a box to find some feed inside

1992; Wolff and Hausberger, 1992) (Table 16.5), and a great interindividual variability has been observed. In most of the test results, there are significant differences due to age, sex and breed of the horse. The more emotional horses had less ability to learn a procedure such as opening a box for feeding. The emotional response during the timidity test was greater in half-bred Thoroughbred than in half-bred Anglo-Arabian and saddle horses. Unfortunately, there are limited genetic data available concerning behavioural traits; Cape and Van Vleck (1981) reported low heritability (0.10) for trainability (training score), which, in turn, is associated with learning ability and emotionality (Fiske and Potter, 1979; Mader and Price, 1980). Further, Mader and Price (1980) found differences between breeds (Thoroughbreds and Quarter Horses) in learning ability. There is one genetic study about nervous system steadiness in Thoroughbreds at five age periods between the third and 24th month (Budzynski, 1987). The heritability of the behavioural trait for all age groups was 0.14. It has been shown that the mean heart rate at rest is sensitive to behavioural events (McCann, 1988). Emotionality can be investigated indirectly by analysing heart rate fluctuations because it depends on regulation of the nervous system (parasympathetic and sympathetic system) (Clément and Barrey, 1995a, b). It was demonstrated that there was a great individual variability of the cardiac dynamics and that younger horses and mares exhibited a greater heart rate variability. There was no difference between the two saddle horse breeds Selle Français and Anglo-Arabians. Energetics With the development of exercise physiology studies, several standardized techniques for testing the exercise ability of racehorses have been established (Rose and Hodgson, 1994). The cardiac, respiratory and metabolic function can be investigated during standardized exercise tests which are performed on a treadmill in laboratory or on a track in outdoor conditions. The cardiac and the aerobic capacity can be evaluated by measuring the heart rate and blood lactate concentration during an exercise test at increased speed (Dubreucq

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Fig. 16.4. Determination of the specific velocities V200 and VLa4 which estimated the cardiac capacity by measuring heart rate (HR), and the anaerobic threshold by measuring the blood lactate concentration, respectively.

et al., 1995). Two physiological exercise variables were computed by interpolation using the linear relationship between speed and heart rate, and the exponential relationship between speed and lactate, respectively (Fig. 16.4):

• •

V200 is the velocity corresponding to a heart rate of 200 beats min−1. This velocity characterized the heart rate response to a submaximal exercise intensity. It is strongly correlated with the maximum oxygen consumption (VO2 max) (Persson, 1983). A good trotter should have a high V200 value. VLa4 is the velocity corresponding to a blood lactate concentration of 4 mmol l−1. This velocity estimated the exercise intensity of the anaerobic threshold. Above this particular velocity, the anaerobic metabolism of the muscular fibres becomes increasingly intense to cover the energy expenditure. When the horse improves its aerobic capacity after a training period, its VLa4 increases, thereby delaying the onset of fatigue. A fit race trotter should have as high a VLa4 as possible.

Using the data collected in a training centre (1210 tests performed by 447 French trotters), it was possible to calculate a first estimate of the heritability of these traits (Barrey et al., 1996). The cardiac capacity (V200) had a heritability of h2 = 0.46 (SE 0.11) which was greater than the heritability of the anaerobic threshold velocity h2 = 0.10 (SE 0.06). The genetic correlations were poorly determined except for the anaerobic threshold velocity which seemed to be positively correlated with the performance index (rg = 0.89, SE 0.31). The repeatability of the exercise parameters V200 and VLa4 were high for the cardiac capacity (r = 0.71) and moderate for the anaerobic threshold (r = 0.39). Some heritability estimates have been obtained by variance analysis between relatives in humans. In these studies, the genetic factors explained 72–86% of the individual variation in maximal heart rate (Klissouras, 1971; Bouchard, 1986). However, the cardiac structural changes (wall thickness, diastolic volume) after training were poorly dependent on genetic factors (Bielen et al., 1991). For maximum aerobic capacity, the genetic effect was estimated

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at 20–45% (Bouchard, 1986). For the latter trait, a maternal effect was suspected because of the inheritance of maternal mitochondrial DNA and the stronger correlation of the childrens’ values with those of their mother than with those of their father (Lesage et al., 1985). The increase in aerobic capacity after a period of training was found to be genotype dependent with a high or low adaptative response (Bouchard, 1986). Muscle characteristics For sprint, endurance and jumping exercises, the twitching characteristics of the main propelling muscles could be interesting traits for early selection. Three types of muscle fibres were identified by histochemistry and then by immunohistology and electrophoresis: one slow twitch fibre (type 1) and two fast twitch fibres (types 2A and 2B or 2X). The ATPase activity of the three types of myosin heavy chains determines the twitching velocity of the muscle fibre. The type 2B fibres reach the fastest twitching velocity but the performance of contraction cannot be sustained a long time. The muscle fibre composition depends on other factors such as breed, sex and training status of the horse. The effect of breed on the percentages of muscle fibre type revealed genetic variability among horse populations (Gunn, 1978; Snow and Guy, 1980; Valberg, 1987; Rivero et al., 1989) (Fig. 16.5). Comparison of breeds also shows the influence of genetic factors. It was demonstrated in Andalusian and Arabian horses that there was a maternal effect on the percentage of type 1 and 2B fibres, and a paternal effect on the percentage of type 2A fibres (Rivero et al., 1996). In Quarter Horses, families with a higher percentage of Thoroughbred pedigree had a higher percentage of fast twitch fibres (Wood et al., 1988).

Fig. 16.5. Muscle fibre composition of the gluteus muscle in different breeds (Snow and Guy, 1980; Rivero et al., 1989).

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Many genes are involved in the synthesis and regulation of the skeletal contractile proteins (Buckingham, 1989, 1992; Periasamy et al., 1989). A polygenic mode of inheritance has been demonstrated in mice (Nimmo et al., 1985). Consequently, the muscle fibre type composition appeared as a continuous quantitative trait and its heritability can be estimated. The composition of the total fast myosin heavy chains in the gluteus medius and biceps femoris muscle was studied in Anglo-Arabians by an immunoenzymatic method in order to calculate a first heritability estimate of this trait: h2 = 0.13 (SE 0.10) (Barrey et al., 1999a). The first heritability estimate of the percentage of fast myosin heavy chains in propelling muscles indicated that there was low genetic variability in the French Anglo-Arabian population. However, the high percentage of fast myosin heavy chains in the gluteus medius and biceps femoris muscle could be considered as a genetically determined breed characteristic. The low heritability found could be explained by a reduction in genetic variability that occurred after many generations selected for racing ability for middle and long distance (> 3000 m). The French Anglo-Arabian breed was created in the early 19th century by crossing two breeds (Thoroughbred ×Arabian). The high percentage of Thoroughbred genes in the measured horses could also explain the high fast myosin heavy chain percentage in the muscles of the French Anglo-Arabian horses. Heritability of the percentage of fast myosin heavy chains found in horses can be compared with other values found for other equine traits and for muscular traits in other species (e.g. human, cattle, pig, sheep and mouse). However, any comparison of values should take into account the analytical technique, the method of calculation, the animal population and its breeding history. There is less genetic variability in the horse than in other species, probably because this muscular trait already has been selected indirectly in conjunction with racing ability. The heritability (SE) of the percentage of myosin heavy chain 1 in Limousin bulls was estimated at 0.35 (0.27) in the semitendinosus and 0.41 (0.28) in the longissimus thoracis muscle (Renand et al., 1995). Using an ATPase histological technique, a heritability of 0.24 was found for the same muscle in cattle (Andersen et al., 1977). In sheep, the heritability of the fibre type composition in the ear muscle (scuto-auricularis), which was not selected for meat production, ranged from 0.27 to 0.46 (Vigneron et al., 1986). In human studies, the correlation of muscular fibre percentages between monozygous twins was higher than between dizygous twins (Komi et al., 1973, 1977; Prudhomme et al., 1984; Simoneau et al., 1986). A higher genetic influence was found for the fast and slow twitch fibre ratio than for fast twitch fibre ratio (2A/2B). These studies on human twins revealed the genetic effect on muscular characteristics but overestimated the heritabilities. Gaits During the last 30 years, the increase of equine business for racing and riding activities has stimulated scientific research in equine locomotion. For breeding purposes, the gaits of young horses are usually observed by experts (judging

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team) that evaluate, by scores, various aspects of locomotion: stride amplitude, gait elasticity, ability for dressage or jumping style. This type of procedure is used in most performance tests. The main limitations are:

• • • •

the lack of quantification because the judge uses only a limited number of score levels (3–5) even if the scale is larger; the human eye can see only slow frequency motions because the limit of visual performance is about 18 images s−1; the eye or the camera can observe the displacement but cannot estimate the forces which generate the movements; the lack of standardization in the methods of scoring introduces variability between judges and events.

The great advantage of expert judgement is the quickness of the evaluation and the ability to evaluate a complex trait such as rideability, harmony of the conformation or behaviour of the horse. With improvement of the scoring system, it should be possible to increase the efficiency of the performance tests for breeding. Objective measurements of the gaits were introduced into the young horse evaluation procedure over the last few years. Dusek et al. (1970) were pioneers in this area by measuring stride frequency and stride length at the three gaits in Czech horses. Nowadays, objective measurements increasingly are proposed by research teams to test young horses in several countries. However, the connection between breeding plans and performance test evaluation is more developed in Europe for saddle horses. The technology used for motion analysis was developed mainly for human biomechanics in the laboratory and cannot be used easily in field conditions for large-scale studies. More specific gait analysis systems have been designed for horses running in outdoor conditions (Barrey, 1999). Acceleration measurements are very convenient for measuring gaits in field conditions and provide the basic stride characteristics. A gait analysis system (Equimétrix) has been designed for early performance evaluation in racing trotters, show-jumping horses and dressage horses (Barrey et al., 1994, 1995; Barrey and Galloux, 1997). After recording the acceleration in dorsoventral, longitudinal and lateral axes of the horse, the signal could be treated by many signal analysis procedures in order to extract locomotor variables (Table 16.6). At present, there is no heritability of gait characteristics measured directly on the tested horses. However, there are some heritability estimates of locomotion traits judged by experts (Koenen et al., 1995; Gerber et al., 1996) and measured on the track (Dusek, 1971) (Table 16.7). The heritabilities obtained for the jump, canter and gallop seemed to be higher than for the trot and walk.

Conformation related to performance Clear proof of the relationship between conformation and performance is the different body conformation and type of horses used for different purposes,

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Locomotor variables that can be measured by the accelerometric gait recorder.

Riding aspects

Gait measurements

Cadence Amplitude Rythmn Regularity Symmetry Impulsion Gait transitions Elasticity

Stride frequency Stride length Time elapsed between footfalls Stride frequency fluctuation Correlation right/left Forward acceleration Wavelet analysis of transit strides Vertical displacement Table 16.7.

Heritabilities of locomotion characteristics.

Locomotion traits Walk Stride Suppleness Symmetry Stride length Trot Stride Elasticity Propulsion Stride length Canter–gallop Stride length Jump Free jump Jump with a rider

Heritability 0.12 0.14 0.21 0.61 0.22 0.20 0.20 0.63 0.67 0.65 0.43

Dusek, 1971; Koenen et al., 1995; Gerber et al., 1996.

e.g. Thoroughbred racehorses, trotters, draught horses, riding horses, etc. However, evidence of the importance of the conformation on the performance capacity within each discipline is not so strong. In many handbooks, and books dealing with different horse breeds, lists of beneficial conformational details are given, but objective data concerning the relationship between conformation and performance are few. Correct movements and good action are important for sports horses in various disciplines. There are certain conformational details and body dimensions that have been reported to be desired and advantageous to the performance in particular disciplines (Langlois et al., 1978; Holmström et al., 1990; Holmström and Philipsson, 1993; Deuel, 1995; Back et al., 1996; Dolvik and Klemetsdal, 1999). Good action, for example, is determined largely by leg and feet stances, slope of the shoulders and pasterns, and some other conformation details. Holmström (1994), for example, gives some main conformational details

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related to performance in dressage, e.g. femur inclination and length, pelvis inclination, stifle joint angle, and length of femur and humerus, the femur being the most important quantitative conformation measurement (Holmström and Philipsson, 1993). The details associated with jumping ability, are, for example, inclination of the pelvis, scapula and femur (Langlois et al., 1978). For trotters, the beneficial details are, among others, ‘normal’ shape of croups, and large shoulder and stifle angles (Magnusson, 1985; Klemetsdal et al., 1986). Long and wide crupper, wide loins, deep chest as well as deep and wide shoulders are desired conformational details for horses used for logging (e.g. Sidbäck, 1993). There are, consequently, no common contributions to so-called good bodied or good gaited horses, and the desired characteristics vary between breeds and disciplines. In the literature, several gait defects associated with incorrect limb stances and conformational details are reported (e.g. Stashak, 1987). On the other hand, many of the defects in movements and locomotion are known to be related to impaired performance capacity. However, only 8–10% of the racing performance in trotters can be explained by conformation (Magnusson, 1985; Klemetsdal et al., 1986; von Butler et al., 1986; Dolvik and Klemetsdal, 1999). With regard to riding horses, from 4 to at least more than 20% of the performance capacity may be explained by the conformation (Langlois et al., 1978; Holmström and Philipsson, 1993). In their study, Holmström and Philipsson (1993) found that walk, trot, canter and total score for gaits showed positive regressions on conformation scores. Genetic correlations between conformation traits and performance are generally low (Table 16.8). Scored traits concerning type, head, neck and body are those most weakly correlated with the performance capacity of the horse. Body measures and other objective conformation traits are also actually poorly associated with performance traits but, in general, a large withers height has a positive effect on performance. These findings have been obtained for various disciplines, e.g. trotting, equestrian and galloping, and for different horse breeds as well (e.g. Arnason, 1984; Magnusson and Thafvelin, 1989; Biedermann and Schmucker, 1989; Holmström and Philipsson, 1993; Saastamoinen et al., 1998b; Dolvik and Klemetsdal, 1999). Saastamoinen et al. (1998b) reported low positive genetic correlations between conformation traits and racing results in trotters, and found, for example, quite a high genetic correlation (0.46) between score for hardness of legs and number of starts. Of the conformational details, tied-in at the knees and the hocks, heavy front cannons and large heart girth had a negative influence on performance in 4-year-old Standardbred trotters (Magnusson, 1985). The multiple correlation between conformation and performance traits was 0.22–0.31. In their study, Dolvik and Klemetsdal (1999) reported decreased performance results for trotters with curby and straight hocks as well as for cow-hocked horses. Saastamoinen (unpublished) found best racing times for trotters with straight hocks and toe-in in forelimbs. As to saddle horses, dressage traits in general are more correlated favourably with conformation traits than jumping ability (Table 16.8). Arnason

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Genetic correlations between conformation and performance traits.

Traits Withers height×handicap weight −0.61 ×best racing time −0.14 ×earnings −0.13 ×performance −0.00 Type×jumping −0.20 −0.18 ×rideability −0.67 −0.40 Leg stances×best racing time −0.15 ×earnings −0.05 ×no. of starts −0.18 ×disq. races −0.11 ×jumping −0.03–0.08 ×rideability −0.24–0.35 ×performance −0.02 Hardness of legs×best racing time −0.17 ×earnings −0.02 ×no. of starts −0.46 ×disq. races −0.17 Conformation×performance −0.22 Body score×jumping −0.28 ×rideability −0.22 Movements×best racing time −0.49 ×earnings −0.41 ×no. of starts −0.33 ×disq. races −0.53

Breed

Reference

Thoroughbred (galloping)

Biedermann and Schmucker (1989)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

Icelandic horse

Arnason (1984)

Hannover Hannover

Christmann et al. (1995) Schade (1996)

Hannover Hannover

Christmann et al. (1995) Schade (1996)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

Hannover

Christmann et al. (1995)

Hannover

Christmann et al. (1995)

Icelandic horse

Arnason (1984)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

Icelandic horse

Arnason (1984)

Hannover

Schade (1996)

Hannover

Schade (1996)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

Finnhorse trotter

Saastamoinen et al. (1998b)

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(1994) and Gerber et al. (1997b) reported low (0.07–0.10) genetic correlations between conformation score and jumping traits, but quite high correlations (0.50–0.72) with gait under rider in Swedish warmblood riding horses. Christmann et al. (1995) and Schade (1996) reported even negative genetic correlations between conformation and jumping ability, but positive correlation between dressage traits and conformation. Further, conformation has been reported to be genetically moderately correlated with gait traits (walk, trot) in riding horses (e.g. Christmann et al., 1995; Gerber et al., 1997a). The studies show that traditional conformational evaluation prefers dressagetalented horses. It is also not so easy to combine excellent conformation, dressage ability and jumping characteristics in one population. Thus, it is more relevant to breed and select for different disciplines than try to combine the selection of these two disciplines. In addition, in order to evaluate and improve the performance capacity of the horse by judging the conformation alone is of rather limited value. In fact, Schade (1996) reported that selection based only on conformation reduces genetic trend in performance traits of riding horses by 70% as compared with selection based on data from the stationary performance test. However, according to Holmström and Philipsson (1993), inclusion of selected quantitative conformational measurements would improve the traditional judging of conformation as a means of predicting performance. To determine and select ‘an ideal horse’ or ‘ideal conformation’ from the point of view of performance is rather complicated. For example, Magnusson (1985) concluded that the ‘normal’ Swedish Standardbred trotter may have outwardly rotated limbs in contrast to the ‘desired’ limb conformation, and the frequences of certain conformational defects and deviations vary between different breeds (Table 16.9). Generalizations, for example, concerning gaits related to stance, breed or type are not, therefore, possible. Further, for example, a horse’s leg flight is influenced by many factors, and lateral gait defects can affect a pair of legs or a single limb. Some horses can also compensate for certain defects with other conformational details, and some conformation variables can be beneficial and disadvantageous at the same time. Moreover, training, shoeing, physical condition, muscularity, etc. may influence the incidence of conformational details and related defects in locomotion. Table 16.9.

Frequences (%) of certain defects of leg stances (forelimbs) in some horse populations.

Defect

Swedish Standardbred trotter

Finnish Standardbred trotter

Finnhorse trotter

Swedish warmblood riding horse

Norwegian coldblood

4.0% 2.0% 13.2% Magnusson (1985)

5.2% 33.4% 0.5% Saastamoinen (unpublished)

5.0% 25.0% 10.0% Saastamoinen et al. (1998a)

46.2–48.5% 1.0–7.1% 7.1–24.0% Holmström et al. (1990)

25.8% 43.9% 36.8% Dolvik and Klemetsdal (1999)

Toe-in Toe-out Calf-knee Reference

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Conformation Related to Soundness and Durability According to Stashak (1987), limb conformation is the major factor in soundness of the limbs. Because the forelimbs bear 60–65% of the weight of the horse, their conformation should have the largest importance in the evaluation. However, faulty limb conformation is not an unsoundness in itself, but it may be considered a warning or sign of weakness, and it prediposes the horse to many types of lameness (Stashak, 1987). Locomotor problems and lameness are shown to be the most common reasons for culling of horses, and for training failure in all disciplines (e.g. Bergtsen, 1980; Jeffcott et al., 1982; Lindner and Dingerkus, 1993; Philipsson et al., 1998). A detailed description of consequences of abnormal limb and foot structure and leg stances, and other instances of incorrectnesses of conformation as well, are presented by Stashak (1987). Some relationships are also reported by, for example, Magnusson (1985) and Dolvik and Klemetsdal (1994). According to studies, 7–11% of the variation in soundness of trotters and riding horses can be explained by conformation (Magnusson and Thafvelin, 1989; Holmström and Philipsson, 1993). These studies give a multiple correlation between the orthopaedic status and conformation of around 0.20–0.30. Saastamoinen et al. (1998b) found a genetic correlation of 0.46 between hardness of the legs and number of starts, also considered a measure of durability, in 5-year-old trotters. Trotters not started had a significantly lower mean score of orthopaedic health compared with those started (Magnusson, 1985). Trotters of middle size with long limbs and normal sized hooves but not tied-in at the knees, and not with curby, sickle hocks or straight fetlock angles, were found to have a better chance of remaining sound during training. Dolvik and Klemetsdal (1999) reported decreased start status for cow-hocked horses or horses with curby hocks. Magnusson (1985) found a multiple correlation of 0.33 between the conformation details and scores of orthopaedic health. There were also relationships between conformational details and type of injury. In saddle horses, high scores for medical status were associated, for example, with a long humerus and femur, and short metatarsus (Holmström and Philipsson, 1993). A long humerus, large angle of the elbow joint and small angles between the scapula and femur and the horizontal plane had positive effects on scores for orthopaedic status. Early conformation evaluation possibly can contribute to decrease the rate of wastage of young racehorses due to poor durability and ability to resist training stress. In sire selection, the largest weight in conformation evaluation should be put on conformational details scientifically shown to affect soundness of the horse. Further, systematic recording of serious diseases and conformational weaknesses is essential to improve the health of horses.

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Locomotor and Physiological Traits Related to Performance One of the challenges of sports science is to find variables which permit prediction performance potential in young horses. In each discipline, some physiological variables are related to competition performance. The relationships between locomotion and other physiological variables such as cardiovascular, respiratory and muscular ones have to be determined because a good prediction of performance would require an evaluation of the main functions involved in the exercise activity. A multivariate approach should be used to evaluate the exercise ability or to study the relationships to performance because this is the only way quantitatively to use all the horse characteristics obtained (Barrey et al., 1989; Valette et al., 1991; Rivero, 1996). Harness racing The racing ability of young trotters can be evaluated by measuring cardiac, metabolic and locomotor variables during an exercise test. From the energetic point of view, a group of good performers has a significantly higher aerobic capacity (VLa4) than a group of horses with poor performance. However, there is no significant difference for the cardiac capacity (V200) (Table 16.10). The cardiac capacity and the aerobic capacity are significantly correlated to the performance in a race, but there is only a weak linear relationship with the annual performance index (ITR): r = 0.28 and r = 0.17, respectively (Fig. 16.6). Stallions have significantly higher cardiac and aerobic capacity than mares and geldings. The exercise parameter values increase progressively with age and state of training. Other complementary information such as the locomotion patterns and muscle fibre types can be useful for improving predictions of racing capacity. It has been observed that good trotters show a short duration of the stance phase and a longer stance phase in the hindlimbs than in the forelimbs (Bayer, 1973). A locomotor test performed in trotters on a track confirmed that the best race performances were given by trotters which had higher maximal stride frequencies and long stride lengths (Barrey et al., 1995) (Fig. 16.7). The stride length was high both in good and middle level groups, but the main difference between those groups was the maximal stride frequency which was higher in

Table 16.10.

Effect of the performance level on the cardiac (V200) and aerobic capacity (VLa4).

Variable min−1)

V200 (m VLa4 (m min−1)

Good performers with ITR > 100

Poor performers with ITR ≤100

626.40 628.22

620.61 615.02

Significant at P < 0.01 for VLa4; non-significant for V200. ITR = annual performance index.

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Fig. 16.7. Correlations between the annual performance index (ITR) and the locomotor and physiological variables. These variables are used to predict the performance potential of a tested horse using discriminant analysis.

Fig. 16.6. Mean stride variables measured in the three classes of performance in race trotters (means followed by the same letter are not significantly different at P < 0.05).

the good performer group. These findings suggest that good Standardbred trotters are able to trot at a high speed using an optimal stride length and that they can accelerate by increasing stride frequency to finish the race. During the same test, the trot symmetry was found to be lower in the middle performance group than in the high performance group. This result might indicate that performance is limited by pathological or physiological gait asymmetry. Saastamoinen et al. (1998b) reported favourable genetic correlations (absolute values 0.33–0.53) between scores for gaits and various measures of performance in trotters (see Table 16.8).

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Thoroughbred racing In the Thoroughbred industry, horses usually race for the first time at the age of 2 years. Gait patterns are influenced by the age of the horse; however, so far, little is known about gait ontogeny in the horse. Leach and Cymbaluk (1986) analysed the relationships between conformation and gallop stride characteristics in foals aged 6–8 months. An increase in speed was obtained by a longer stride length in heavier foals and a higher stride frequency in taller foals. The ontogeny of the gaits has been investigated by longitudinal studies. The velocity-related changes in stride length of colts aged from 7 to 25 months showed that the speed increase was strongly correlated with the stride length at each age (D.H. Leach, N.F. Cymbaluk and A. Booth, personal communication, 1994). In older Throroughbreds, the maximum gallop velocity is also explained by stride length. The increase in this component is obtained by decreasing the overlap duration of the lead hindlimb and non-lead forelimb stance phase (Leach et al., 1987). A second suspension phase between the lead hindlimb and non-lead forelimb has been described at high speed in Thoroughbreds (Seder and Vickery, 1992). The overlap time of the diagonal limbs decreases linearly down to about 50 ms with increasing speed (Hellander et al., 1983; Deuel and Lawrence, 1984). Much kinematic data collected during races on various racing tracks were statistically analysed by Seder and Vickery (1992). Using a multivariate discriminant analysis, they concluded that only a set of temporal and linear stride variables could be sufficient to predict subsequent racing performance. Using the analysis of the gallop strides, they reported that they were able to predict correctly the racing ability of a horse five times better than the best traditional experts. In Thoroughbreds categorized as poor performers and tested on an inclined treadmill (10% slope) at a maximal velocity of 12 m s−1, the stride length and velocity at the maximum heart rate were the variables best correlated to run time (Rose et al., 1995). The heart rate (r = −0.56) and blood lactate (r = −0.68) measured during recovery after the exercise were correlated to the performance (timeform rating) in race (Evans et al., 1993). A higher percentage of fast myosin heavy chains favours greater muscular power, which is required for the propulsion phase in gallop racing. The gluteus medius muscle of the best performers is composed of a larger percentage of fast myosin heavy chains in Anglo-Arabians: good performers = 75.5% > poor performers = 69.6%. This result is consistent with previous findings in Thoroughbreds and Quarter Horses that were successful in racing (Barlow et al., 1984; Wood et al., 1988). Better performance in gallop racing has been observed in Thoroughbreds which had been 90 and 100% of fast twitch fibres in the semitendinosus muscle. For the same muscle in Thoroughbreds, the sprinters (race distance < 2500 m) had a higher percentage of fast twitch fibres than the stayers, which are suited to racing a longer distance (Snow and Valberg, 1994). Due to the significant relationship with performance and a low heritability value (0.13), it might be worthwhile improving

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this muscular trait for racing but a high pressure of selection should be applied.

Three-day eventing and endurance riding Analysis of gallop stride characteristics of horses in three-day eventing during the steeplechase of the 1988 Olympic Games in Seoul revealed some parameters and optimal values for successful performance: the stride frequency should be between 1.85 and 2.05 stride s−1, while the velocity should be between 13.0 and 14.3 m s−1 (Deuel and Park, 1993). The muscle fibre types of the gluteus medius muscle were studied using multivariate analysis in endurance horses competing at different levels. Horses on a higher endurance performance level had a higher percentage and relative area of type 1 and 2A fibres and lower percentages and relative areas of type 2X (= 2B in the horse) fibres than moderate performers (Rivero, 1996). The fibre types 1 and 2A are the first muscle fibres recruited during long and slow intensity exercise, as was demonstrated by the studies of glycogen depletion in endurance horses (Hodgson et al., 1983).

Show-jumping Kinematic studies of elite show-jumping horses were undertaken during the Olympic Games and World Equestrian Games. They allowed a description of some parameters to distinguish a good jumping technique. However, in order to know more about the relationship between the jumping technique and the jumping performance, a more heterogeneous and larger population of horses has to be studied to have a greater variability of jumping abilities. This could be done when a test procedure is available to collect the more interesting locomotor variables rapidly. During the 1988 Seoul Olympic Games, the kinematics of 29 horses jumping oxers was analysed (Deuel and Park, 1991). Fewer total penalties were associated with lower velocities during the jump strides, closer take-off hindlimb placements and closer landing forelimb placements. Another study on elite horses jumping a high vertical fence demonstrated that the push-off produced by the hindlimbs at take-off explained most of the energy required for clearing the fence (van den Bogert et al., 1994). The action of the forelimbs should be limited to place the body of the horse in a good orientation before the final push-off of the hindlimbs. A more vertical component of the initial velocity was observed in horses that successfully cleared a 4.5 m wide water jump (Clayton et al., 1995). The angle of the velocity relative to the horizontal was 15° in successful jumps as compared with 12° in unsuccessful jumps, and the vertical components of the velocity were about 0.5 m s−1 greater in the successfully completed jumps than in the unsuccessfully completed jumps. This initial velocity was generated by

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the impulse of the hindlimbs and determined the ballistic flight characteristics of the body. These kinematic findings agree with another study which showed that poor jumpers produced a high acceleration peak with the forelimbs and a low acceleration peak with the hindlimbs (Barrey and Galloux, 1997). This result means that the hindlimbs of a poor jumper produce a weaker force to push-off than those of good jumpers. This force determines the ballistic flight of the centre of gravity and also the characteristics of the body rotation over the obstacle during the airborne phase. The mean value of the stride frequency during the approach and its rapid change during the last stride was also related to the jump score of the horse. More penalties were recorded with horses that cantered at a low stride frequency (≤1.76 stride s−1) and suddenly reduced their stride frequency at take-off. Horses with a higher performance in show-jumping competition had a higher percentage of fast myosin heavy chains in the gluteus muscle: good performers = 74.1%; poor performers = 67.8%. The average performance index obtained in show-jumping was correlated more with the percentage of fast myosin heavy chains in the gluteus medius (0.47) than in the biceps femoris muscle (0.34) (Barrey et al., 1999a). No consistent relationship was found between jumping performance and other physiological variables such as heart rate, blood lactate or haematocrit (Barrey and Valette, 1993).

Dressage In dressage, the successful horse is expected easily to execute complex exercises, gait variations and gait transitions, always maintaining its equilibrium and suppleness. This discipline requires a high locomotion control by the rider which can be obtained progressively by suppling exercises and collecting the gaits. The ability of a horse to collect its gait appears to be one of the main limiting factors for dressage horses to compete successfully because it is impossible to execute the more complex exercises in competition correctly without attaining a good basic collection of the gaits. The collected gaits have been described extensively by kinematic studies (Holmström et al., 1994a; Clayton, 1994, 1995; Burns and Clayton, 1997). Other studies investigated the relationships between basic gait qualities required for dressage and the kinematic variables describing the trot. Some locomotor variables have been identified to predict the expressiveness of the trot (Back et al., 1994a; Holmström et al., 1994b). For example, a slow stride frequency including a long swing phase is required to have a good trot quality. The time elapsed between the hindlimb contact and the diagonal forelimb contact defines the diagonal advanced placement which should be positive and high at the trot. The horse should place its hindlimbs as far as possible under itself. The vertical displacement of the body during collected gaits is also an important factor for the dressage horse. The elasticity of the trot is obtained by storage of elastic strain energy in the fetlock, the hock, the stifle and the pelvis joints. To extend the trot, an inclined scapula (conformation) and the

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amplitude of the elbow joint appear to be important factors. The horses judged to have a good trot had a large flexion in the elbow and carpus joints at the beginning of the swing phase. A longitudinal study revealed that the duration of the trot swing phase, the maximal range of protraction–retraction of the limbs and the maximal flexion of the hock joint were well correlated between horses of 4–26 months of age (Back et al., 1994b). The relationship between the total score and the canter characteristics in Olympic dressage horses showed that the best horses were able to extend their gallop strides by increasing their stride length without changing their stride frequency (Deuel and Park, 1990). For three-day event horses at the Olympic games, extended canter stride length and velocity were positively related to points awarded by the judges. However, non-finishers of the event had higher extended canter stride lengths and velocities in the dressage phase than finishers (Deuel, 1995).

Use of Conformation, Gait and Physiological Traits in Breeding Due to the long generation interval in horse breeding, it is desirable to use evaluation schemes that supply information earlier in order to optimize genetic progress. For this purpose, conformation evaluation results, as well as applying various physiological traits, may be useful. For example, use of foal classification based on their conformation instead of that of adult mares can reduce the generation interval for conformation traits by 2 years (Preisinger et al., 1992). The efficiency of indirect selection for performance depends on the genetic variation of conformation traits and on the genetic correlations between conformation and performance. Due to the low or negative genetic correlations between dressage and jumping traits, it may be difficult to combine these traits beneficially in one population. Consequently, it is more relevant to select separately for different disciplines. In addition, because of the generally low genetic correlations between conformation and performance traits, it is not very beneficial to select for performance indirectly based on conformation only. If the main task is to produce performance horses, selection for performance should be done based on competition results, but inclusion of selected quantitative conformational measurements would improve the traditional judging of conformation as a means of predicting performance. Because conformation is highly heritable, it is an important tool in selecting for better soundness and locomotion, and thus, in practical selection, individuals with serious weaknesses and conformational defects should be culled. In addition, use of quantitative measurable conformation traits will be more advantageous than scored traits for these purposes. Further, only those conformational traits and details that have been proven to have the largest impact on performance and soundness of the horse should be included in the evaluation. Foal classification can also be integrated into a breeding programme as the first type of progeny test for conformation traits (Preisinger et al., 1991). Mare

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classification results can also be included in progeny tests of stallions later. In three-stage selection, conformation evaluation can be used at the first and second steps in classification of male foals and young stallions before test, respectively. Further, following the performance test at a station, a selection of stallions on the basis of conformation classification is possible (Preisinger et al., 1991). For all horses, the conformation evaluation results can be used as a part of an ‘information label’ to serve consumers, i.e. buyers, trainers, owners and breeders. Based on the positive, but low, genetic correlations between conformation and performance traits, it is possible to use the evaluation results as predictors of the later performance of a horse. Because body measures usually have strong genetic correlations (0.80–1.00) with each other (e.g. Saastamoinen et al., 1998b), it is possible to minimize the number of measures to be taken at stud book shows and include only those having the largest influence on performance and usefulness of the horse, e.g. rideability and gaits. Regarding a linear evaluation system, in which a large number of details are applied, the number of conformational details can also be reduced for the same reason, i.e. high correlations between them. The importance of applying various physiological and locomotion traits and standardized techniques for testing the exercise ability of a horse for selection and early prediction purposes will increase in the near future. These methods can contribute to the traditional conformation evaluations or even be used instead of them. One example of this is a standard field exercise test. The first genetic studies indicate that the procedure is promising (Barrey et al., 1999b). Moderate heritability of the cardiac and aerobic capacity allows these traits to be considered as early selection criteria in trotters. There are genetic factors also affecting the muscle fibre composition. Analysis of muscle fibre composition may, consequently, be a new tool in predicting performance capacity for racing and show-jumping, and can be applied together with other important traits. Gait analysis and other quantitative methods are applicable to evaluate the functional conformation traits in show-jumping and dressage. Because evaluation of conformation and gaits will have great importance in breeding selection of horses in the future, the main emphasis should be put on the traits which have the largest impact on performance, soundness and health of the horse. In this work, further development of various quantitative and objective evaluation methods will have the largest importance.

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Arnason, Th. (1994) Rapport om Genetiska Analyser av Kvalitetsbedömningar av Svenska Fyråriga Ridhästar 1973–1993. IHBC AB, Knubbo, Morgongåva, Sweden. Back, W., Barneveld, A., Bruin, G., Schamhardt, H.C. and Hartman, W. (1994a) Kinematic detection of superior gait quality in young trotting Warmbloods. Veterinary Quarterly 16, S91–S96. Back, W., Barneveld, A., Schamhardt, H.C., Bruin, G. and Hartman, W. (1994b) Longitudinal development of the kinematics of 4-, 10-, 18- and 26-month-old Dutch Warmblood horses. Equine Veterinary Journal (Suppl. 17), 3–6. Back, W., Schamhardt, H.C. and Barneveld, A. (1996) The influence of conformation on fore and hind limb kinematics of the trotting Dutch warmblood horse. Pferdeheilkunde 12, 647–650. Barlow, D.A., Lloyd, T.T., Hellhake, P. and Seder, J.A. (1984) Equine fibre types: a histological analysis of select thoroughbred yearlings. Journal of Equine Veterinary Science 4, 60–66. Barrey, E. (1999) Methods, applications and limitations of gait analysis in horses. Veterinary Journal 157, 7–22. Barrey, E. and Galloux, P. (1997) Analysis of the jumping technique by accelerometry. Equine Veterinary Journal. 23 (Suppl.), 45–49. Barrey, E. and Valette, J.P. (1993) Exercise related parameters of horses competing in show jumping events ranging from a regional to an international level. Annales de Zootechnie 42, 89–98. Barrey, E., Valette, J.P. and Wolter, R. (1989) Etude multifactorielle de l’aptitude à l’effort chez le cheval de selle. Annales de Zootechnie 38, 157–169. Barrey, E., Hermelin, M., Vaudelin, J.L., Poirel, D. and Valette, J.P. (1994) Utilisation of an accelerometric device in equine gait analysis. Equine Veterinary Journal (Suppl. 17), 7–12. Barrey, E., Auvinet, B. and Couroucé, A. (1995) Gait evaluation of race trotters using an accelerometric device. Equine Veterinary Journal. (Suppl. 18), 156–160. Barrey E., Valette J.P., Jouglin M., Blouin C. and Langlois B. (1999a) Heritability of percentage of fast myosin heavy chain in skeletal muscles and relationship with performance. Equine Veterinary Journal. 30 (Suppl.), 289–292. Barrey, E., Couroucé, A., Langlois, B., Blouin, C. and Auvinet, B. (1999b) Genetic components of exercise test parameters in French Trotters: first estimations. In: Lindner, A. (ed.), Proceedings of Conference on Equine Sport Medicine and Science. Cordoba, Spain, pp. 219–224. Bayer, A. (1973) Bewegungsanalysen an Trabrennpferden mit Hilfe der Ungulographie. Zentralblatt für Veterinärmedizin reihe A 20, 209–221. Bendroth, M. (1981) A survey of reasons for some trotters being non-starters as 2-, 3and 4-year olds. 32nd Annual Meeting of the European Association of Animal Production. Zagreb, Yugoslavia. Bergsten, G. (1980) The durability of the Swedish standardbred riding horse judged from a material of insured horses. 31st Annual Meeting of the European Association of Animal Production. München, Germany. Biedermann, G. and Schmucker, F. (1989) Körpermasse von Vollblutpferden und deren Beziehung zur Rennleistung. Züchtungskunde 61, 181–189. Bielen, E.C., Fagard, R.H. and Amery, A.K. (1991) Inheritance of acute cardiac changes during bicycle exercise: an echocardiographic study in twins. Medicine and Science in Sports and Exercise 23, 1254–1259.

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Genetic Improvement of the Horse 1 and T. Arnason Genetic 17 T. Improvement Arnason and L.D. Van of the Vleck Horse L.D. Van Vleck2 1IHBC, Knubbo, S-744 94 Morgongava, Sweden; 2Cornell University, Ithaca, NY and Agricultural Research Service, USDA, Lincoln, NE 68933-0166, USA

Introduction Breeding Goals General formulation of breeding objectives Racing horses Riding horses Other horse breeds Genetic Evaluations Genetic background and basic theory BLUP – the current standard method for obtaining EBV BLUP with the animal model – an effective tool for genetic evaluation of stallions and mares Applications of BLUP for genetic improvement of horses Use of non-additive genetic effects in horse breeding Selection – Genetic Response Factors determining genetic progress Observed genetic progress in several horse populations Effects of selection on genetic variation and long-term progress Selection in small populations Selection using genetic markers References

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Introduction From the time man domesticated horses some 5000–6000 years ago, horses have been subject to many forms of artificial selection that have resulted in gradual genetic changes. Differences found among breeds of horses are due to forces of artificial as well as natural selection, in addition to random change. Today there is growing interest in application of scientific animal breeding ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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theory to accomplish genetic improvement of valuable traits in many existing horse populations. This chapter will briefly introduce the tools available from animal breeding theory for the utilization of genetic variability within horse populations for genetic improvement. Successful application of modern knowledge of genetic improvement in animals, however, presumes that breeders have clearly defined breeding goals, that breeding populations are relatively large and, last but not least, that breeders are willing to accept scientific methods and to cooperate in a breeding programme. The same genetic principles, of course, are applicable for breeding of horses as for other farm animals. Some structural and demographic advantages and disadvantages specific to horse breeding compared with breeding of other livestock species should be highlighted:

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Thorough pedigree registration, often spanning many generations. Important traits are recorded on both males and females (often on a large proportion of the population). Low rate of reproduction. Long generation intervals. Wide overlapping of age groups among breeding candidates. Considerable non-random mating practised. Extremely large differences in economic value depending on assumed genetic merit.

Together, these factors support the application of the best available methods for accurate evaluation of breeding values for valuable traits including optimal use of all pedigree information. These factors are the main reasons for the early and widespread implementation of the animal model (AM) to obtain best linear unbiased predictions (BLUP) for genetic evaluation of horses in several European horse populations. The main aim of this chapter is to highlight some aspects of the basic theory behind the use of genetic evaluations, and the BLUP method in particular, for genetic improvement of horses. Several examples will be given for clarification. References will be given to basic sources and sometimes to suggest further reading. No attempt will be made to provide a complete review of literature of all the aspects of genetic improvement as related to horses.

Breeding Goals General formulation of breeding objectives The prerequisite for any rational breeding scheme with the goal of genetic improvement of quantitative traits is a clear definition of breeding goals. The breeding objectives are a statement of the relative values of genetic change in all of the desired traits that are included in a breeding plan. Breeding objectives traditionally are expressed in monetary terms as economic weights to be applied to each trait of commercial importance. The most common way

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of calculating these weights is an economically rational approach, such that the economic weights reflect the costs and returns in a production system without any consideration of the genetic parameters. An alternative means of developing breeding objectives is the desired gains approach, where the relative economic weights depend on the genetic parameters. Basically, the economically rational approach involves determination of the relative economic weight, αi, of the i-th of n different traits included in the overall objective (H). The overall objective, also termed aggregate genotype, is expressed as: H = α1A1 + α2A2 +... + αnAn, where Ai is the animal’s breeding value for the i-th trait. In the most simple form, the αi values are obtained by defining profit = return − cost, and then taking the partial derivatives of the profit function with respect to the n traits in the aggregate genotype (Hazel, 1943). This approach assumes that the vector of the relative weights is linear in H. Extensions and modifications of the economically rational approach to fit more complex economical models in other species are found widely in the literature (e.g. Amer et al., 1994). Until now, little scientific effort has been made to derive economic weights of traits which are included in the breeding objectives for horses. The use of ad hoc methods, at best based on empirical experience and some biological considerations, in combination with intuition, have predominated. There may be several reasons for this. A major reason may be the failure to devote enough effort and investment to resolve these issues by scientific means. Often the value of one unit of expression of a certain trait to the total value of the horse can be difficult to determine. The long time elapsing from time of mating until the traits are expressed in the progeny, and thus resulting in saleable products, add to the difficulties in determining a proper profit function. This time lapse pertains especially to riding horses, where important traits may not become expressed until 8–12 years after the mating took place. Another problem is that relative economic weights can be non-linear in the breeding objectives. The value of an increment of one unit in the genotype for a certain trait may be highly dependent on the genetic values of one or more of the other traits. For example, for a riding horse with outstanding jumping ability the additional value of genes affecting its dressage capability is presumably negligible or even negative, while for an inferior jumper a corresponding genotype for dressage traits may considerably increase its value. Clearly, much more research is needed for determining economic values of genetic improvement of individual traits and, more generally, for determining the breeding objectives in horse breeding schemes. Meanwhile, breeders should be provided with genetic evaluations for all the different traits that might be included in the breeding goal. Breeders would then have the opportunity to evaluate overall genetic worth using their own objectives. Breeders’ knowledge and predictions of marketing trends are usually good. The correct general direction of the breeding goal in a population is of course of greatest importance, while slight deviations of economic weights from the correct ones have a limited impact on selection efficiency (Vandepitte and Hazel, 1977).

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In situations where the relative economic values of genetic changes are difficult to determine, economic weights can be constructed that result in pre-chosen relative amounts of genetic change in each of the traits included in the aggregate genotype. This method is the desired gains approach. The weights for desired gains are influenced by the genetic parameters. Compared with the economical rational approach, traits exhibiting less genetic variation may receive higher economic weights in the desired gains approach. The total rate of genetic response in the linear function H will be less with the desired gains approach, but sometimes it is logical to expect that the traits that respond slowly to selection may increase in value relative to those changing more quickly. An example is the case in breeding of racing and riding horses where the traits interact in a non-linear manner in such a way that the more slowly responding traits may eventually critically suppress the horse’s total value. As a specific example, the relative value per unit of genetic improvement in the constitution of legs may increase compared with the more pure criterion of racing performance as the genetic level of racing capacity emerges, thus causing more strain on pasterns, tendons and hooves. According to economic rules for markets, breeders will automatically change the weights empirically. Brascamp (1984) gives an excellent description of methods of selection for restricted and desired genetic gains.

Racing horses At first glance, the breeding goal for racing horses may seem simple, i.e. the genetic ability to win races and thus earn prize money. However, the genetic components of racing performance may be quite complex and involve a complicated function of anatomical, physiological, neurological and endocrinological factors. Measures of racing performance generally are competition traits where the record of racing performance is always evaluated relative to that of competitors. Thus, for any single racing horse, the genetic level of the competitors acts as an environmental effect. Genetic improvement in the population as a whole also means that the level of competitors will have increased. Increased genetic value for racing ability within the population is also likely to alter the relative importance of various biological factors which affect racing performance. The history of breeding of Thoroughbred horses serves as an illustration of some of the problems encountered. The horse has an evolutionary history as a retreat animal which survived by escaping from its enemies at a full gallop (see Chapter 2). Speed at the gallop has certainly been an important fitness trait for horses. Selection for speed may therefore be looked upon as a continuation of a long history of natural selection. The Thoroughbred also has a long history of artificial selection for galloping speed, while being ridden by a jockey. Initially, the genetic ability for racing speed undoubtedly was an important factor for discriminating between winners and losers in Thoroughbred racing. Selection for winners in the major classic races efficiently yielded correlated

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improvement in racing time for the whole population (Cunningham, 1976). As the level of the population improved for racing speed, the rate of improvement in winning times of the classic races levelled off, gradually leading to an apparent plateau. The population seemingly also became more uniform, in terms of speed, at least on the untransformed natural scale of racing time. Slight differences in racing speed between horses raced against each other became less important relative to other factors determining racing success. The likelihood of winning against equally fast horses is dependent on the mental and physical ability of the horse to react successfully to the environment, such as competitors, signals of the jockey and variations in speed at different phases of the race. The capability of mobilizing an anaerobic muscular metabolism and a ‘fighting’ spirit have become essential for successful Thoroughbred racers. In breeding schemes for the modern Thoroughbred, little emphasis is put on speed as part of the declared breeding goal. Sufficient genetic value for speed is assumed to exist in the upper level of the population so that the keys to success in longer stakes and in major classic races are assumed to be other factors (Langlois, 1994). Robertson (1976) somewhat provocatively raised the question of whether the real goal in Irish and English Thoroughbred breeding was in fact high auction prices for yearlings. Variations in prices according to assumed differences in genetic merit of individual horses are tremendous. Many people would argue that this is an argument for rational use of available scientific methods for accurate and realistic genetic evaluations. However, the highly non-linear return function for racing performance may require skilful definition of the aggregate genotype and complex procedures for genetic evaluation of breeding animals. Some breeders and people in the racing industry may want to avoid rational evaluation of the breeding values as such evaluations might counteract the unrealistic expectations of gamblers on the value of slight presumed genetic differences. After all, the human desire to gamble is a prerequisite for the worldwide established marketing system for the Thoroughbred horse. In several European countries, trotters are genetically evaluated routinely for traits measuring racing performance by the BLUP method. Verbal formulations of the breeding goals usually include terms such as: racing speed, ability to win, temperament, conformation, precocity, endurance, health and regularity of gait. In reality, the aggregate genotype can be expressed simply in monetary terms as a strictly monotone increasing function of earnings over a given age span, as the cost of input is practically invariant for all horses.

Riding horses Breeding goals for riding horses are usually not as strictly related to performance in competition as for racing horses. The usage of riding horses can be divided into three categories: (i) leisure riding; (ii) competition at lower (amateur) levels; and (iii) competition at higher (Grand Prix) levels. The

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majority of riding horses are used in the first two categories, while the third category receives the most public attention. The traits representing Grand Prix level performance presumably dominate in the definition of official breeding goals. Such a definition is understandable because of: (i) the competition in the international market between breeders, (ii) the great difference in price between ‘high’ and ‘low’ level horses and (iii) the advertisement value related to Grand Prix champions. On other hand, most of the potential market is for the production of horses well suited for the average rider. Whether the same aggregate genotype is ideal for all classes (categories) should be considered by breeders. Studies to detect such genotype ×environment interactions in riding horses are scant. A primary consideration when defining breeding goals in riding horses is to decide whether the goal should be a composite genotype for both dressage and jumping traits, or the goal should be for success in only one of the events. Estimates of genetic correlations between dressage traits (gaits) and jumping traits that have been published have been somewhat contradictary. Some estimates have indicated rather low, but positive genetic correlations, while other estimates have shown clearly negative genetic relationships. Some evidence suggests that the negative genetic relationships are more prominent in analyses of competition results, while the positive correlations have been found mainly in analyses of field performances. Results showing positive correlations have been claimed to be based on data where the requirements for the level of performance in each event of the test have been low. On the other hand, results showing negative correlations may be criticized for possible confounding of genetic and environmental effects in the data. For example, progeny of a stallion which has proved to be an outstanding individual in one of the events are more likely to be trained for and competed in the same event, and thus would not be given the opportunity to express their ability in other events. When the breeding goal involves an aggregate genotype suited for lower level competitions and leisure riding, a tempting conclusion would be that both dressage and jumping traits could be included in the same objective for maximum success, with little or no cost, in terms of genetic progress, for either event. On the other hand, breeding schemes for higher levels such as Grand Prix horses would require specialized breeding populations where the breeding goals would include traits representing either jumping or dressage ability. The so-called 3-day events include dressage, jumping and a cross-country ride so that successful horses in 3-day events have to have all-round ability. Horses competing at higher levels in 3-day events frequently are the result of crossbreeding (often with Thoroughbreds), which seems to be a viable alternative to specialized breeding because the market for superior 3-day event horses is rather small in comparison with the market for specialized jumpers and dressage horses. Traditionally, body conformation traits reflecting type, function, soundness and grace have had a large place in the breeding goals for riding horses. More recently, health traits and longevity have received increased attention as

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a part of the breeding goals (Ricard and Fournet-Hanocq, 1997; Philipsson, et al., 1998).

Other horse breeds Most European horse breeds are able to perform the three fundamental gaits: walk, trot and gallop. Horses that are capable of performing the lateral four-beat movements called toelt, rack or slow gait, are called gaited horses. Gaited horses are quite common in many parts of the world. In North America, breeds such as the American Saddle horse and the Tennessee Walking horse are probably best known. In South America, several gaited horse breeds are represented, e.g. the Paso Fino horse. In the highlands of Ethiopia and in South Africa, many gaited horses are found. The Icelandic horse (sometimes called the Icelandic Toelter horse) is a good example of the so-called fivegaited horse, which can perform the two gaits, toelt and pace, in addition to the three fundamental gaits. The official breeding goal (H) of the Icelandic horse consists of a linear function of seven conformation traits and seven riding performance traits which are scored in special field performance tests. The estimated breeding values (EBVs) are obtained by a multiple-trait animal model BLUP procedure. In addition to a weighted EBV for the aggregate genotype consisting of the 14 traits, the breeders are provided with EBVs for each trait and also EBVs for two additional traits (wither height, and mane and tail hairiness). The EBVs are included in a database program that allows breeders to search for information on all registered animals in the population and to obtain predictions for the outcome of any possible mating within the breed based on the parental EBVs. The breeding goal for the Icelandic horse is to breed small but powerful fourand five-gaited horses which are well suited for participation in special gait competitions as well as for leisure riding by adults and teenagers. The Arabian horse has been divided into several subpopulations according to usage and, consequently, has several different breeding goals. In some countries, Arabians are used for races on a large scale. The common and successful participation of the Arabian horse in endurance competitions is well known. Nevertheless, a large portion of the breeding effort in the Arabian horse is directed to the imaginary perfection of the noble exhibition animal. Production of horses to be used as draught animals in agriculture and forestry may require breeding goals including pulling power, pulling technique and temperament. An appropriate breeding goal for hackney horses (drawing carriages) should include movement, temperament and elegance as a part of the aggregate genotype. The breeding goal for small horses (ponies) which are to be used by children for riding should have more emphasis put on temperament and suitability for their intended purpose as reliable companions for children and teenagers, with less emphasis on conformational details than seems to be a common practice in many countries. The general rule is that the breeding goal should include the traits which are of real functional importance for

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the breed, and should avoid wasting selection pressure on redundant traits. The breeding goals in many small horse populations of the world are often unique. Horses are bred for cutting performance and ‘cow sense’ for use on ranches and in rodeo shows. In Pakistan, some horses are even bred for dancing ability. The establishment of scientific breeding schemes for genetic improvement of such traits is a challenge that will continue in the future.

Genetic Evaluations Genetic background and basic theory Most traits included in breeding objectives for horses are influenced by many genes and many environmental factors and thus are termed quantitative traits. Animal breeding theory for such traits traditionally has assumed an infinitesimal model developed by R.A. Fisher more than 80 years ago. The model is illustrated by the following points:

• • •

The traits are controlled by the effects of an infinite number of genes. The effect of each gene is infinitely small and is equal for all genes. The environmental effects are randomly distributed and are independent of the genes and their effects.

The infinitesimal model obviously is only an approximation. More correctly, the traits are affected by (very) many genes, some of the genes are associated through linkage on the same chromosome (at least for several generations) and the effects of the genes on the traits vary in magnitude (major genes have been found to affect some quantitative traits in livestock). Nevertheless, the infinitesimal model has proved to be generally a good and robust approximation for modelling genetic variation and to use for genetic evaluation and for design of breeding plans. The main consequences of the infinitesimal model can be summarized:

• • • • •

Selection does not lead to any measurable changes in gene frequency within sufficiently large populations. Sums of gene effects (breeding values) are normally distributed within the population. The phenotypic values are sums of normally distributed breeding values and normally distributed environmental effects and, therefore, are also normally distributed. The covariance between breeding values of pairs of members of the population is proportional to the coefficient of relationship. The covariance of phenotypic values between pairs of members of the population is proportional to the coefficient of the relationship (i.e. phenotypic covariance between individuals is equal to the covariance between their breeding values). The phenotypic value (P) of any individual can be described as.

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P = Σgi + Σej + µ where: gi = average effect of gene i, which affects a particular quantitative trait, i = 1,...,n and n is the total number of genes affecting the trait (i.e, twice the number of loci). Usually n is large, and in fact with the infinitesimal model n = 64. ∞Σgi = A = the true breeding value (BV), ej = environmental effect j affecting the quantitative trait. The effects are expressed as deviations from population means. j = 1,...,m and m6∞4. Σej = E = randomly distributed environmental deviates and µ = a general form of the phenotypic population means (fixed effects in the statistical models). According to the infinitesimal model, the environmental deviates are assumed to be independent of the effects of the genes. Therefore, the variance of the phenotypic values in the population can be expressed as the sum of the additive genetic variance and the environmental variance: σ2P = σ2A + σ2E = h2 σ2P + (1 − h2) σ2P where heritability, h2 = σ2A/σ2P. The purpose of genetic evaluations is to estimate A, the breeding value of each individual animal. Breeding value is defined as the sum of average effects of the genes affecting a particular quantitative trait (or a function of several traits). Estimated breeding values always refer to a specific population. The breeding value of an individual i can be expressed as a function of the breeding values of its parents (S and D) plus a Mendelian sampling term, i.e. Ai = 1/2 AS + 1/2 AD + θi With the infinitesimal model, the Mendelian sampling terms follow a normal distribution with variance: σθ2 = 1/2 (1 − FS/2 - FD/2) σ2A = 1/2 (1 - FS/2 - FD/2) h2 σ2P where FS and FD are the inbreeding coefficients of the parents. The breeding values of the parents can be decomposed further into their Mendelian components plus the average of the breeding values of the grandparents. This decomposition can be repeated recursively through the pedigree all the way to an unselected base population. Thus, an individual’s breeding value is the weighted sum of the Mendelian components of all its ancestors traced to the base population (Kennedy et al., 1988; Woolliams and Thompson, 1994).

BLUP – the current standard method for obtaining EBV Unfortunately, true breeding values are never known. They can be estimated with varying degrees of accuracy depending on the amount of available information, heritability and genetic correlations among the traits. Rational methods for estimation of the breeding values with real data are based on a weighted

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sum of phenotypic values of related animals deviated from corresponding population means. The magnitude of the weights (regression coefficients) are inversely proportional to the amount and accuracy of the information. BLUP has become the standard method in animal breeding for combining such information in an optimal way. One important property of BLUP is that it yields an EBV which is the unbiased linear function of the phenotypic variables that has the highest possible correlation with the true unknown breeding value (Ai). The EBV of animal i can be expressed as: âi = 1/2 âS + 1/2 âD + φi This formula shows that the EBV of animal i is average of the parents’ EBV plus a deviation φi. The estimate of the Mendelian sampling term can be expressed as (ignoring possible inbreeding for simplification): φi = di/(di + λ) (yi − µi − 1/2 âS − 1/2 âD) where di = 1/2 if both parents are known (have EBV), 3/4 if only one parent is known and 1 if neither parent is known; λ = σ2E/σ2A = (1 − h2)/h2; yi = phenotypic value; and µi = phenotypic (sub-) population mean (correction for fixed effects with best linear unbiased estimates, BLUE). The decomposition of both the true and the estimated breeding values provides a key to understanding the nature of genetic improvement over successive generations. Selected animals become the parents of the next generation. When those parents are selected intensively on the basis of their EBV (âi), the level of the true breeding value of the selected individuals, which now become parents (1/2 AS + 1/2 AD), is raised compared with that of the parents of the previous generation. The Mendelian sampling term, θi, is not affected by selection in the parental generation, which means that its variance remains practically unchanged even after many generations. In the first round of selection counted from an unselected base population, the main source of information contributing to the EBV (âi) comes from φi since the average breeding value of the parent generation is 0. In a successful breeding scheme, the level of EBV will be raised for each generation due the increased values of parental EBV. The accumulated information from the whole pedigree will always be included in 1/2 âS + 1/2 âD. The additional information on the size and direction of the Mendelian deviation from the family mean, which is based on the adjusted record of the individual itself and, if it is a parent, also on the adjusted records of its descendants, is captured by the Mendelian sampling term, φi. With animal models, animals without identified parents build the base population. If the base animals cannot reasonably be assumed to belong to a single common population, but must be considered to be sampled from populations with different genetic means, a genetic grouping strategy must be applied to allow for the different expected genetic values of groups of base population animals (Westell et al., 1988). Animal genetic models used for genetic evaluation of horses with the BLUP method usually assume one common base population. If the breeding population is homogeneous and most

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pedigrees are well filled, tracing the majority of the younger animals to a common population of base animals is legitimate. If, on the other hand, the breeding population is a mixture from many base populations, there is importation of breeding stock from other countries or populations or there are many animals with incomplete pedigrees, an appropriate genetic grouping becomes essential in order to avoid serious errors in genetic evaluation.

BLUP with the animal model – an effective tool for genetic evaluation of stallions and mares Estimation of breeding values begins with definition of a linear model which should describe reality as well as possible in as simple a way as possible. The model should account for all major factors affecting variation in the data in a systematic way. In the simplest form, the model may be written for a single trait as: yij = µ + bi + aj + eij where the yij is the phenotypic observation on the j-th horse belonging to the i-th class of fixed (systematic) effects affecting the trait; µ represents the overall mean of the (base) population; bi represents the deviation from the population caused by the i-th class of fixed effects; and aj represents the breeding value of the j-th horse. The breeding values, aj, according to the infinitesimal model, are random normally distributed deviations from the genetic mean of the base population. Finally eij denotes the remainder of the model, which is assumed to include randomly distributed environmental effects pertaining to the j-th horse. With the BLUP method, the effects of fixed factors and the breeding values are estimated simultaneously by solving a large set of equations with equally as many unknown solutions. In this way, the estimated breeding values are adjusted for fixed effects and the estimates of fixed effects are correspondingly adjusted for differences in breeding values of the horses with records in the various fixed effect subclasses. The relative magnitude of the random effects (aj and eij) in the model depends on heritability of the trait. The elements of eij are assumed to be independent and distributed normally (if not, then a bias will be imposed into the estimated effects). In any practical case, the aj elements are not independent as horses are related to each other. The additive genetic relationships among all animals in the population are accounted for by use of the (numerator genetic) relationship matrix. Statistical models for practical applications of BLUP are usually far more complicated than that shown above. The model may include many fixed factors, many traits (multiple trait model), additional random factor(s) and also allow for repeated observations for some animals. The BLUP method assumes that the correct genetic parameters (i.e. variances, heritabilities, genetic correlations and environmental correlations) of the base population are known. In practice, this means that good estimates are needed.

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Studies have shown that the BLUP method is robust even for situations where the assumptions of the infinitesimal model do not apply. Simulations have proved that in situations where traits are controlled by few loci, the BLUP method with an animal model still provides a good approximation (e.g. Maki-Tanila and Kennedy, 1986). Most of the properties of the BLUP method as an optimal predictor of breeding values remain true even if the traits and the breeding values are not distributed normally in the population (Henderson, 1975). A comprehensive description of the BLUP procedure cannot be accomplished, even for the most simple models, without the help of matrix algebra. In fact, matrix algebra has become an essential tool for anyone seriously interested in quantitative genetics and in thorough understanding of the BLUP method. A technical description of the methodology is therefore outside the scope of this book. Interested readers are referred to Kennedy (1981), Van Vleck et al. (1989) and Mrode (1996) for comprehensible descriptions of BLUP techniques. The well proved genetic and statistical properties of BLUP as a selection criterion (Henderson, 1975; Kennedy et al., 1988) for quantitative traits have led to the use of BLUP for genetic evaluations in most livestock populations. The properties of BLUP for the animal model for obtaining EBVs are of great benefit in horse breeding and lead to the following practical consequences: 1. EBV based on full use of information from all relatives can be obtained for any member of the population. EBVs of potential progeny are easily obtained for any potential mating between any stallion and mare in the population by simply averaging the EBVs of the parents. This property can be used to assist breeders in planning of matings. 2. The EBVs are corrected for all fixed factors included in the model. The models used for genetic evaluation of horses may include effects of sex, age, year, birth-year, herd (stud) and others. 3. The EBVs of parents are corrected for EBVs of their mates. This property is particularly important in horse breeding, where the allocation of mares to breeding stallions is generally not random in terms of genetic merit. 4. The EBVs of later generations incorporate the genetic changes (progress) resulting from the effects of selection accumulated from the base population. That property means that animals from different generations can be fairly compared according to EBV. Genetic trends can be obtained easily by computing the mean EBVs for each birth-year. When year is included in the model, environmental trends, adjusted for genetic changes, can also be obtained from the corresponding solutions for year effects. A common characteristic of most horse populations is widely overlapping generations. The amount of information pertaining to the EBVs of individual animals is quite variable. When EBVs are published, estimates of their accuracy should be attached. A horse breeder often is faced with the decision of mating

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his mare with either an old or a young stallion. The EBVs from a correct model provide breeders with information for objective decisions. If the breeder is neutral concerning risk, the breeder will choose the stallion having a higher EBV irrespective of the amount of information included in the genetic evaluations of either stallion. Usually the older stallion will have an EBV with higher accuracy. If the stallions have equal EBVs, a risk-prone breeder would select the younger stallion which would have a larger variance of prediction for his EBV. In the same situation, a risk-adverse breeder would select the more accurately evaluated stallion. Unfortunately, there is often a conflict between the short-term risk considerations of the practical breeder and the risk aspects concerning the long-term breeding scheme for the whole population. This issue needs further investigation. Applications of BLUP for genetic improvement of horses The use of selection index (BLUP) principles for genetic evaluation of horses started in the mid-1970s with Langlois (1975) for French riding horses and with Minkema (1976) in The Netherlands for racing trotters. The BLUP method was first introduced to horse breeders with a simple example by Van Vleck and Hintz (1976). The first applications of the BLUP method with real data appear to have been in the Icelandic horse (Arnason, 1980) and in German trotters (Distl et al., 1982). The first BLUP applications in horses were based on sire models (progeny tests). The implementation of BLUP with the animal model in horse breeding was initiated by Arnason (1984) for the Icelandic horse. The application of animal models for estimating breeding values in horses has been reported for various breeds in at least ten European countries and for the Quarter Horse in the USA. Large-scale genetic evaluations are run routinely for trotters, riding horses and Icelandic horses in several countries. Breeders have access to the EBV through publications and computerized databases. A recent review of methodological developments in the genetic evaluation of performance traits in horses was given by Ricard (1998). A major problem has been to find good normally distributed measures of performance, which are appropriate for use in the framework of linear models. Various mathematical transformations of records have been used to approximate normal distributions. The alternative but computationally demanding non-parametric ranking methodology for genetic evaluation of competition traits developed by Ricard has potential for practical applications in horse breeding, due to the dramatic improvement in computer technology (Tavernier, 1991). The increase in international trade of sport horses and breeding stock has created a need for genetic evaluation of breeding horses across countries. Therefore, there is growing interest in the development of international genetic evaluations of horses. Inter-Nordic genetic evaluation of Nordic trotters and Icelandic horses (Arnason et al., 1994; Arnason and Sigurdsson, 1997) has already begun.

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Use of non-additive genetic effects in horse breeding Selection based on BLUP EBVs with the animal model is by far the most effective way of changing genetic merit of horse populations. The use of EBVs for planning matings and to predict potential genetic and phenotypic values of the candidate progeny are also of great value to the individual breeder. However, if non-additive genetic effects (dominance, epistasis) are important, then estimates of such effects would be useful for breeders as additional information for planning of matings. Both theory and experience show that traits closely connected to evolutionary fitness, or traits otherwise subject to long-term selection pressure, are more likely to show significant effects of dominance and epistasis. Traits connected with racing performance, especially in Thoroughbreds, are particularly likely to show such effects. No genetic analyses aimed at estimation of dominance effects or epistasis in performance traits of horses have yet been published. However, many practical horse breeders claim the existence of profound nicking effects, whereby mating of individuals from pairs of lines having certain relationship structures is supposed to be successful. Even if such effects as nicking have not been confirmed scientifically, that area is in need of more research. If reliable estimates of both the additive genetic effects (EBV) and of non-additive genetics effects for individual horses were available, they could be used in mate selection. Crossbreeding involving different breeds frequently is used for production of riding horses. Many outstanding show-jumpers and 3-day event horses have resulted from successful crosses. Crossbreeding, as generally applied in horse breeding, is an efficient way to utilize available specialized genetic resources to obtain economic heterosis through complementarity without creating permanent genetic improvement. In international competitions among riding horses at Grand Prix level, the traditional crossbred riding horses are generally losing ground in comparison with specialized synthetic horse breeds which are making rapid genetic progress. Progressive crossbreeding schemes in horses, involving selection for general or specific combining abilities, have not been implemented.

Selection – Genetic Response Factors determining genetic progress Successful selection in a population leads to gradual transmission of average breeding values. The changes can be mapped on the horizontal scale of a normal distribution in the desired direction according to the defined breeding goals as illustrated in Fig. 17.1. Genetic response is usually measured in units per year rather than units per generation, as the intention usually is to increase the mean as quickly as possible in the favoured direction. Genetic response depends on: intensity of selection (i); additive genetic variation in the aggregate genotype (σH), the correlation between the selection criterion and the

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Fig. 17.1. Effect of two different breeding schemes on accumulated genetic response over four generations. This difference in rate of response corresponds to what has been realized in horse populations implementing BLUP with animal model EBVs.

breeding goal (RIH); and the generation interval (L). In the simplest case, this relationship is expressed in the well-known formula (e.g. Falconer, 1989): ∆G = (iσHRIH)/L Because selection intensity, generation interval and accuracy of selection are not the same for males and females, the formula is modified to include two or four paths (Rendel and Robertson, 1950). Figure 17.1 illustrates the difference in genetic progress in two hypothetical horse populations. In one of the populations, the best 20% of the stallions according to phenotypic merit (h2 = 0.3) are used for breeding with no selection on the female side. In the other population, breeding animals are evaluated by the BLUP method with the animal model and the EBVs are used as a criterion for selection. On the stallion side,

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the best 2% are selected and, among the mares, the best 40% are selected. Generation intervals of 8.5 years on the male side and 11.5 years on the female side are assumed for both populations. The increased selection intensity and the increased accuracy of the selection from using BLUP is expected to result in three times faster genetic improvement in the latter selection scheme. The economic value of such an increase in progress over time would vary among horse breeds, but invariably would be equivalent to a considerable amount of money.

Observed genetic progress in several horse populations Racing trotters Intensive selection of stallions on the basis of phenotypic racing performance has been practised in many trotter populations for quite a long time. Recently, some trotting organizations have employed BLUP methodology to provide breeders with EBVs as selection criteria for racing performance. In Sweden, index values based on a multiple trait animal model have been available to the breeders of the Standardbred trotter since 1992. The following traits have been evaluated: number of races; percentage of races ranked first to third at the finish; earnings per race; total earnings; best racing time per km; and racing status, which is denoted as one if the horse did race and zero otherwise. All traits were based on accumulated racing results as 3- to 5-year-olds. The traits were transformed in order to approximate the normal distribution before being included in the BLUP analysis (Arnason, 1994a). About 40% of Swedish Standardbred trotters do not enter races as 3- to 5-year-olds and receive a start status of zero. Start status is genetically correlated with racing performance and is therefore a correlated pre-selection criterion for performance. Ignoring such a culling process can result in substantial selection bias (Klemetsdal, 1992). As a result, EBVs of stallions with relatively few raced offspring tend to be overestimated. A practical solution has been to generate canonical variables corresponding to imaginary performance records for non-racers based on the correlation structure and genetic relationship ties within the population (Ducrocq and Besbes, 1993). The implementation of an index based on BLUP with an animal model has increased greatly the selection intensity for both stallions and mares (Arnason, 1997). The annual rate of genetic response increased by 40% from 1988 to 1996 and corresponds to about 6% of one phenotypic standard deviation in racing performance. The trend in index values for the Swedish Standardbred trotters is shown in Fig. 17.2. For trotters, racing speed, winning ability and earnings are highly positively correlated traits. Selection for racing performance, where earnings are the main component, has resulted in substantial genetic as well as phenotypic improvement in racing time. Genetic improvement is estimated to account for about 60% of the phenotypic change. The phenotypic trends in racing time of male and female trotters in Sweden are shown in Figs 17.3 and 17.4.

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Fig. 17.2. Genetic progress in Swedish Standardbred trotters measured by trend in BLUP EBVs. Ten units in index correspond to one σH.

Fig. 17.3. Phenotypic trend in average racing time in s km−1 as 3- to 5-year-olds for Standardbred trotters in Sweden for both males and females.

Fig. 17.4. Phenotypic trend for slowest, average and fastest racing times in s km−1 as 3- to 5-year-olds for Standardbred male trotters in Sweden.

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Thoroughbred horses Gaffney and Cunningham (1988) used the BLUP procedure with a sire model to estimate genetic change in Timeform handicap ratings of Thoroughbreds in Great Britain. Timeform ratings express racing merit as weight in pounds which the compiler believes the horse should carry in an average free handicap race. Their results indicated genetic progress in Timeform ratings to be about 1% per year. This progress was not reflected in winning times of classic races, but the authors concluded that correlated genetic improvement in speed had been achieved in the Thoroughbred population as a whole. This conclusion assumes asymmetry in the distribution of racing speed. Arnason (1994b) showed how such scale effects in the distribution of racing speed might be caused by asymptotically diminishing marginal effects of gene substitutions in a genetic model with a finite number of loci. Such effects may be expected for traits where the best part of the population is approaching the physiological limit. The dilemma of lack of phenotypic progress in racing time in spite of intensive selection on racing performance traits has been discussed by Cunningham (1976), Gaffney and Cunningham (1988), Hill (1988), James (1990) and Cunningham (1998). It must be kept in mind that the selection has not been directly for racing time. The results need not be paradoxical, because the recent genetic gain may have been mainly for other traits more related to winning ability, as mentioned in the discussion about breeding objectives. A hypothetical illustration of the effects of scale on racing speed in km h−1 is shown in Fig. 17.5. The average value for racing speed is expressed by the graph of y = q0 (1 − e−pt), which is a mirror image of an exponential function, where y = km h−1 in racing speed, q0 is the asymptotic limit for racing speed over a certain distance (e.g. 62 km h−1), p is a positive constant that could be estimated (e.g. by least squares) from real data, and t indicates a time scale (e.g. generations). American Quarter Horse Racing performance in the American Quarter Horse was studied thoroughly in a series of papers by Buttram et al. (1988a,b,c). Quarter Horse races are run

Fig. 17.5. Hypothetical illustration of expected trend in racing speed over the classical distances for Thoroughbred horses. The graph of y = q0(1 − e−pt) is plotted for q0 = 62 km h−1 and the p constants as 0.18, 0.14 and 0.10 for the fastest, average and slowest horses in the population, respectively.

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over short distances, most commonly from 320 to 402 m. A BLUP procedure with an animal model was used for genetic evaluation of racing performance based on racing time records. Genetic trend in racing time in Quarter Horse racing was estimated from solutions for breeding values (Wilson et al., 1988). Annual progress of 0.004–0.009 s in the average EBV of parents was found, which corresponds to 0.06–1.8% of the phenotypic standard deviations in racing time for distances of 402 and 320 m, respectively. Icelandic horses Since 1950, Icelandic horse breeders have had their breeding stock evaluated in field performance tests, where several riding ability and conformation traits have been scored. In 1980, a research project was initiated with the goal of implementing BLUP procedures with an animal model for routine genetic evaluations based on the scoring results. A multiple trait animal model for Icelandic horses was introduced in 1983 (Arnason, 1984) and since then breeders have been provided annually with the EBVs. The latest analyses included over 100,000 horses, of which about 16,000 have been performance tested. Annual genetic changes in the official breeding goal (total score) are shown in Fig. 17.6. The rate of genetic progress has been accelerating over the last few years, probably to a large extent due to the fact that breeders are gaining confidence in use of the EBVs to assist with selection of breeding animals. Annual genetic improvement after 1990 was five times greater than progress made before 1980 and corresponds to 6% of the phenotypic standard deviation or 10% of the genetic standard deviation of total score (Sigurdsson, et al., 1997).

Fig. 17.6. Estimated genetic trend in total score for Icelandic horses. Ten units in index correspond to one σH.

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Effects of selection on genetic variation and long-term progress Selection across generations Additive genetic variation can be considered as the fuel for genetic response. A closer look shows what happens when this fuel is burned, i.e. breeding horses are selected on phenotypic values or functions of phenotypic values, e.g. EBVs. The additive genetic variance can be separated into variance within and between loci as shown by Bulmer (1971): σ2A = Σni=1 VAR(gi) + Σni≠j COV(gi, gj) Equilibrium genetic variance

Joint disequilibrium genetic variance

where VAR(gi) is the variance of genetic effects at the i-th locus and COV(gi, gj) is the covariance between genetic effects at the i-th and j-th loci. The summations are over the n loci affecting the additive genetic variation of the trait. For the infinitesimal model, all changes in the additive genetic variance can be shown to be caused by the disequilibrium part, i.e. the part due to covariance between genes (alleles) at different loci. Variance of gene effects within loci is unchanged in the population by generations of selection provided that the number of loci affecting the trait is large. Traditional directional selection, however, results in negative covariance between loci, i.e. a negative value for Σni≠j COV(gi, gj). Directional selection, therefore, leads to reduction in additive genetic variance. If selection ceases, the additive genetic variance will rebuild, because the negative value of Σni≠j COV(gi, gj) will be halved with each generation of random mating. Constant selection intensity for successive generations will lead to an equilibrium between reduction in genetic variance and the variation rebuilt due to recombination after several generations of constant selection intensity. This phenomenon (the Bulmer effect) can be compared with chemical reaction in both directions which eventually will reach equilibrium. When equilibrium is reached (steady state), the additive genetic variance will have been reduced in most breeding schemes by 10–30% depending on the selection intensity and accuracy of selection. Most economically important traits in breeding goals for horses are affected by many genes. Selection is therefore expected to be effective for changing the population mean in the desired direction for many generations, provided that deleterious effects of inbreeding can be avoided and that the effective population size is kept large enough. Sensible animal breeding is a fantastic process for sustainable utilization of biological resources. The level of fuel may become somewhat lower in the tank, but the process should never run out of petrol. Selection within generations The effect of selection on changes in genetic variance over generations was considered previously. The conclusion was that variance in quantitative traits

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amongst the annual crops of newborn foals is reduced somewhat over time with increased intensity of selection until equilibrium is reached and that thereafter genetic variance remains unchanged. What are the effects of selection within a generation? Within each generation, the genetic variance is obviously largest before any selection is practised, i.e. amongst the newborn foals. The variation in breeding values of selected animals is reduced in direct proportion to the selection intensity and to the correlation between the selection criterion and the true breeding value. The genetic variance among intensively selected stallions would therefore be far less than the genetic variance amongst newborn foals. Multi-stage selection in breeding schemes for horses was investigated by Hugason et al. (1987). As shown by VanRaden et al. (1984), selection on EBVs across the age structure of the population is close to optimum in terms of genetic progress. However, it is important to realize that the BLUP method will account properly for the selection bias only if all the traits on which selection is based are included in the analysis and the statistical model correctly adjusts for important fixed factors.

Selection in small populations Many horse populations are small and animal breeding principles that are optimal in (infinitely) large populations may not apply. In small populations, gene frequencies may fluctuate randomly from generation to generation as a result of the finite sampling of gametes. This phenomenon, called genetic drift, is quantified by the term effective population size, Ne (e.g. Falconer, 1989). In a closed population, the Ne is dependent on the number of parents in each generation, the variance of parental family sizes and selection. Hill (1979) gave a formula for computing Ne from the structure of random-mating populations with overlapping generations. Several modifications of Hill’s formula have attempted to account for selection as well (e.g. Wray et al., 1994). An alternative way of estimating Ne is to measure the rate of inbreeding over different generations (e.g. Falconer, 1989). Then Ne for generation t can be computed as: Ne = (1 − Ft−1)/2(Ft - Ft−1), where Ft is the mean coefficient of inbreeding for generation t. A relatively large Ne is advantageous in breeding plans in order to prevent loss of genetic variation and for accumulation of new mutations affecting valuable traits. In small closed populations, there is a risk that intensive selection on EBVs alone will lead to a small Ne. Consequently, strict use of unmodified EBVs for selection eventually may result in reduced long-term genetic improvement due to loss of genetic variation and inbreeding depression. Breeding plans for small horse breeds have to cope with a small Ne in attaining long-term progress. Selection criteria which involve EBVs modified such that the average relationship between selected animals is restricted have been proposed by Wray and Goddard (1994) and further developed by Meuwissen (1997). Such a selection criterion was tested in a simulation exercise (Arnason, 1996). The conclusion was that the method could be used to increase long-term

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response in Nordic trotters. Intense selection of males and variable family sizes may cause a surprisingly small Ne in horse populations of reasonably large actual sizes. The joint population of Nordic trotters in Norway and Sweden consists of 2000 brood-mares and 160 approved stallions. The Ne for this population has been estimated to be as small as about 30 animals (Klemetsdal, 1993; Anderson et al., 1998). In small horse populations, where there seems to be limited scope for genetic improvement, the first step to achieve improvement might be to target a market for the distinctive characteristics of the breed. One means of preserving this particular genetic resource would be to make the breed popular and to expand the breed on the basis of its special abilities, which should be strengthened with an effective breeding plan. Successful expansion of a breed sometimes follows from horsemanship associated with certain lifestyles. Interesting examples are the Western riding culture in relation to the American Quarter Horse and the global marketing of the Icelandic horse as an alternative to the traditional European riding style. Selection using genetic markers In the future, interest in the possible use of marker-assisted selection (MAS) for enhanced genetic improvement in horses is likely to increase. Genetic markers are DNA sequences that can be identified relatively easily in individual animals. As the gene map of the horse becomes more complete, the chance increases of finding genetic markers closely linked to major genes affecting some important quantitative traits included in the breeding goal. The only indication of a plausible marker for a quantitative trait in horses found in literature is the report of Anderson et al. (1987) on an association between different alleles at the serum esterase locus (Es) and racing status (all-or-none trait depending on whether the horse has started in a race or not) in Swedish Standardbred trotters. MAS is likely to be a valuable complement to selection on EBVs obtained by the BLUP method, rather than as a replacement for EBVs. The use of MAS in horse breeding schemes could be particularly useful for traits that are expressed late in the horse’s life because the genetic markers will become known in the foal. The effect of MAS would be to shorten the generation interval and thereby increase the rate of genetic progress. At present, evidence for the existence of quantitative traits loci (QTL) with large effects (major genes) in horses is scarce. Traits, however, such as the lateral gaits, pace and toelt, ‘cow sense’ and other unusual behavioural characteristics related to special movements affecting dressage and jumping characteristics in horses could quite probably be influenced by major genes.

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References Amer, P.R., Fox, G.C. and Smith, C. (1994) Economic weights from profit equations: appraising their accuracy in the long run. Animal Production 58, 11–18. Anderson, E., Nasholm, A., Gerber, E. and Arnason, T. (1998) Inbreeding and its impact on racing performance in Swedish and Norwegian coldblood trotters. 49th Annual Meeting of the European Association of Animal Production, 24–27 August 1998, Warsaw, Poland. Anderson, L., Arnason, T. and Sandberg, K. (1987) Biochemical polymorphism in relation to performance in horses. Theoretical and Applied Genetics 73, 419–427. Arnason, T. (1980) Genetic studies on Icelandic toelter horses (estimation of breeding values). 31st Annual Meeting of the European Association of Animal Production, 1–4 September 1980, Munich, Germany. Arnason, T. (1984) Genetic studies on conformation and performance of Icelandic toelter horses. IV. Best linear unbiased prediction of ten correlated traits by use of an ‘animal model’. Acta Agriculturae Scandinavica 34, 450–462. Arnason, T. (1994a) The importance of different traits in genetic improvement of trotters. In: Proceedings of the 5th World Congress on Genetics Applied to Livestock Production, Vol. 17. Guelph, Canada, 462–470. Arnason, T. (1994b) Simple (plausible) non-linear model for inheritance of racing speed and corresponding genetic changes from selection. 45th Annual Meeting of the European Association of Animal Production, 5–8 September 1994, Edinburgh, Scotland. Arnason, T. (1996) Selection criterion for increased long-term response in Nordictrotters. 47th Annual Meeting of the European Association of Animal Production, 25–29 August 1996, Lillehammer, Norway. Arnason, T. (1997) The selection intensity in Standardbred trotters in Sweden as measured by BLUP animal model index. 48th Annual Meeting of the European Association of Animal Production, 25–28 August 1997, Vienna, Austria. Arnason, T. and Sigurdsson, A. (1997) Genetic analysis of performance test traits in Icelandic toelter horses in Iceland and Sweden. 48th Annual Meeting of the European Association of Animal Production, 25–28 August 1997, Vienna, Austria. Arnason, T., Jensen, P., Klemetsdal, G., Ojala, M. and Philipsson, J. (1994) Experience from application of animal breeding theory in Nordic horse breeding. Livestock Production Science 40, 9–19. Brascamp, E.W. (1984) Selection indices with constraints. Animal Breeding Abstracts 52, 645–654. Bulmer, M.G. (1971) The effect of selection on genetic variability. American Naturalist 105, 201–211. Buttram, S.T., Willham, R.L., Wilson, D.E. and Heird, J.C. (1988a) Genetics of racing performance in the American Quarter Horse: I. Description of the data. Journal of Animal Science 66, 2791–2799. Buttram, S.T., Willham, R.L. and Wilson, D.E. (1988b) Genetics of racing performance in the American Quarter Horse: II. Adjustment factors and contemporary groups. Journal of Animal Science 66, 2800–2807. Buttram, S.T., Wilson, D.E. and Willham, R.L. (1988c) Genetics of racing performance in the American Quarter Horse: III. Estimation of variance components. Journal of Animal Science 66, 2808–2816.

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T. Arnason and L.D. Van Vleck Cunningham, E.P. (1976) Genetic studies in horse populations. Proceedings of the International Symposium on Genetics and Horse Breeding. Royal Dublin Society, Dublin, pp 2–8. Cunningham, E.P. (1998) The genetics of track performance in thoroughbreds. 49th Annual Meeting of the European Association of Animal Production, 24–27 August 1998, Warsaw, Poland. Distl, O., Katona, O. and Krausslich, H. (1982) Vergleich der Zuchtwertschatzmethoden BLUP und CC beim Traber. Zuchtungskunde 54, 157–164. Ducrocq, V. and Besbes, B. (1993) Solution of multiple trait animal models with missing data on some traits. Journal of Animal Breeding and Genetics 110, 81–92. Falconer, D.S. (1989) Introduction to Quantitative Genetics, 3rd edn. Longman Group UK, Harlow, Essex. Gaffney, B. and Cunningham, E.P. (1988) Estimation of genetic trend in racing performance of thoroughbred horses. Nature 332, 722–724. Hazel, L.N. (1943) The genetic basis for constructing selection indexes. Genetics 28, 476–490. Henderson, C.R. (1975) Best linear unbiased estimation and prediction under a selection model. Biometrics 31, 423–447. Hill, G.W. (1979) A note on the effective population size with overlapping generations. Genetics 92, 317–322. Hill, G.W. (1988) Why aren´t horses faster? Nature 332, 678. Hugason, K., Arnason, T. and Norell, L. (1987) Efficiency of three-stage selection of stallions. Journal of Animal Breeding and Genetics 104, 350–363. James, J.W. (1990) Selection limits in thoroughbred horses. In: Proceedings of the 4th World Congress on Genetics Applied to Livestock Production, Edinburgh, Vol. 16, pp. 221–224. Kennedy, B.W. (1981) Variance component estimation and prediction of breeding values. Canadian Journal of Genetics and Cytology 23, 565–578. Kennedy, B.W., Schaeffer, L.R. and Sorensen, D.A. (1988) Genetic properties of animal models. Journal of Dairy Science 71 (Suppl. 2), 17–26. Klemetsdal, G. (1992) Estimation of genetic trend in racehorse breeding. Acta Agriculturae Scandinavica 42, 226–231. Klemetsdal, G. (1993) Demographic parameters and inbreeding in the Norwegian trotter. Acta Agriculturae Scandinavica 43, 1–8. Langlois, B. (1975) Analyse statistique et génétique des gains des Pur Sang anglais de trois ans dans les courses plates francaises. Annales de Génétique et de Sélection Animale 7, 387–408. Langlois, B. (1994) An introduction to genetic analysis in the Thoroughbred. 45th Annual Meeting of the European Association of Animal Production, 5–8 September 1994, Edinburgh, Scotland. Maki-Tanila, A. and Kennedy, B.W. (1986) Mixed model methodology under genetic models with a small number of additive and non-additive loci. Proceedings of the 3rd World Congress on Genetics Applied to Livestock Production. Lincoln, Nebraska, USA, 12, 443–448. Meuwissen, T.H.E. (1997) Maximizing the response of selection with a predefined rate of inbreeding. Journal of Animal Science 75, 934–940. Minkema, D. (1976) Studies on the genetics of trotting performance in Dutch trotters: II. A method for the breeding value estimation of trotter stallions. Annales de Génétique et de Sélection Animale 8, 527–535.

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Mrode, R.A. (1996) Linear Models for the Prediction of Animal Breeding Values. CAB International, Wallingford, UK. Philipsson, J., Brendow, E., Dalin, G. and Wallin, L. (1998) Genetic aspects of diseases and lesions in horses. In: Proceedings of the 6th World Congress on Genetics Applied to Livestock Production, Armidale, NSW, Australia, Vol. 24, pp. 408–415. Rendel, J.M. and Robertson, A. (1950) Estimation of genetic gain in milk by selection in a closed herd of dairy cattle. Journal of Genetics 50, 1–8. Ricard, A. (1998) Developments in the genetic evaluation of performance traits in horses. In: Proceedings of the 6th World Congress on Genetics Applied to Livestock Production, Armidale, NSW, Australia, Vol. 24, pp. 388–395. Ricard, A. and Fournet-Hanocq, F. (1997) Analysis of factors affecting length of competitive life of jumping horses. Genetics Selection Evolution 29, 251–267. Robertson, A. (1976) What has genetics to contribute to horse-breeding? In: Proceedings of the International Symposium on Genetics and Horse Breeding, Royal Dublin Society, Dublin, pp. 64–70. Sigurdsson, A., Hugason, K. and Arnason, T. (1997) Breeding strategies and genetic progress in the Icelandic toelter population. 48th Annual Meeting of the European Association of Animal Production, 25–28 August 1997, Vienna, Austria. Tavernier, A. (1991) Genetic evaluation of horses based on ranks in competitions. Genetics Selection Evolution 23, 159–173. Vandepitte, W.M. and Hazel, L.N. (1977) The effect of errors in economic weights on accuracy of selection indexes. Annales de Génétique et de Sélection Animale 9, 87–103. VanRaden, P.M., Freeman, A.E. and Rothschild, M.F. (1984) Maximizing genetic gain under multiple stage selection. Journal of Dairy Science 67, 1761–1766. Van Vleck, L.D. and Hintz, R.L. (1976) Prediction of genetic value of stallions. In: Proceedings of the International Symposium on Genetics and Horse Breeding, Royal Dublin Society, Dublin, pp. 19–23. Van Vleck, L.D., Pollak, E.J. and Oltenacu, E.A.B. (1989) Genetics for the Animal Sciences. W.H. Freeman and Co., New York. Westell, R.A., Quaas, R.L. and Van Vleck, L.D. (1988) Genetic groups in an animal model. Journal of Dairy Science 71, 1310–1318. Wilson, D.E., Willham, R.L., Buttram, S.T., Hoekstra, J.A. and Luecke, G.R. (1988) Genetics of racing performance in the American Quarter Horse: IV. Evaluation using a reduced animal model with repeated records. Journal of Animal Science 66, 2817–2825. Woolliams, J.A. and Thompson, R. (1994) A theory of genetic contributions. In: Proceedings of the 5th World Congress on Genetics Applied to Livestock Production, Guelph, Canada, Vol. 19, pp. 127–134. Wray, N.R. and Goddard, M.E. (1994) Increasing long-term response to selection. Genetics Selection Evolution 26, 431–451. Wray, N.R., Woolliams, J.A. and Thompson, R. (1994) Prediction of rates of inbreeding in population undergoing index selection. Theoretical and Applied Genetics 87, 878–892.

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Standardized Genetic Nomenclature for the Horse C.H.S. Dolling Standardized 18 C.H.S. Genetic Dolling Nomenclature COGNOSAG, P.O. Box 74, McLaren Vale, South Australia 5171, Australia Introduction Development of Nomenclature The first guidelines for sheep and goats The 1991 guidelines for ruminants The 1993 guidelines for ruminants The 1998 guidelines The Guidelines for Genetic Nomenclature for the Horse Locus Alleles Genotype terminology Phenotype terminology Additional Comments for Blood and Milk Polymorphisms and for Mapped Loci Acknowledgements References

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Introduction The Committee on Genetic Nomenclature of Sheep and Goats (COGNOSAG) was founded with the express purpose of drawing up guidelines for the nomenclature of loci and alleles in sheep and goats. It was launched at Palmerston North, New Zealand, in 1984, and is an association registered in France under the Loi de 1901 as the Comité de Nomenclature Génétique des Ovins et Caprins (COGOVICA). The need for guidelines became manifest during the National Congress on Breeding Coloured Sheep and Using Coloured Wool in Adelaide, South Australia, in 1979, at which there were almost as many systems of nomenclature for coat colour and pattern as there were speakers from half a dozen countries. ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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Development of Nomenclature The first guidelines for sheep and goats A project for standardizing genetic nomenclature in sheep was developed by Lauvergne (1984) and presented to the COGNOSAG Workshop of 1986. At the same workshop, Searle (1988) reviewed the mouse nomenclature rules and gene nomenclature in the human and in other mammals. The rules proposed for sheep by the workshop were a compromise between nomenclature regulations for mice and those for humans. The rules permitted the use of mainly human rules for loci defined by biochemical variants and tissue or blood groups, and retained useful rules from the mouse nomenclature for the loci where dominance and recessivity could be distinguished in the various genotypes (Lauvergne, 1988). The 1986 Workshop guidelines for sheep and goats were reviewed at the COGNOSAG Workshop of 1987 (COGNOSAG, 1989).

The 1991 guidelines for ruminants The guidelines from the 1987 Workshop were revised during the 1988 and 1989 Workshops. A further development was the revision at the 1991 Workshop to accommodate all ruminants and to facilitate the development of a comparative genome nomenclature. This development had been stimulated by the interest expressed by workers with cattle, at the 1990 Workshop, for the guidelines to be suitable for use with cattle as well as with sheep and goats. The ruminant guidelines were published by COGNOSAG (1991).

The 1993 guidelines for ruminants Revised guidelines for gene nomenclature in ruminants were prepared by the COGNOSAG ad hoc committee at the 1993 Workshop (COGNOSAG ad hoc committee, 1995). The core of these revised guidelines consists of the 1991 proposals of COGNOSAG (1991). Their rewording was undertaken to reduce their length and to increase their clarity. The earlier recommendations to limit the length of symbols of loci and alleles to a maximum of five and four characters, respectively, were relaxed. The designation of top dominant and codominant alleles by an upper case initial letter was undertaken to assist in the recognition of alleles with visible effects. Thus, the changes made are intended to render the guidelines more permissive and user-friendly, while retaining consistency with the human and mouse systems of nomenclature. The new recommendations include the use of species prefixes, e.g. ECA for the horse, BTA or BBO for cattle, OAR or OOV for sheep, and the adoption of the nomenclature for keratins and keratin-associated proteins proposed by Powell and Rogers (1993). An additional proposal for provisionally assigning

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symbols and for listing newly reported DNA segments and proteins that have no known homologues, official names or symbols was also outlined. The 1998 guidelines Some minor changes in wording were made to the 1993 guidelines at the 1997 and 1998 Workshops (COGNOSAG, 1999). At these two workshops, genetic nomenclature for the horse was discussed. The 1998 guidelines form the basis of this chapter. As far as possible, COGNOSAG respects the names of loci and alleles proposed by authors, and COGNOSAG will propose new names only in the light of new knowledge or to maintain consistency with the existing nomenclature. The use of names and symbols in italics for loci and their alleles is preferred. However, if it is not possible to comply with this, then those names and symbols should be underlined. Authors are asked to be consistent in the underlining or use of italics for the names and symbols within a document and/or file. Locus and allele symbols need not be in italics or underlined in databases, but should be in italics in hard copy from these databases.

The Guidelines for Genetic Nomenclature for the Horse Locus Locus name Choice of name

•

• • • •

The name in English should be as brief as possible but not consist of a single letter, and should convey as accurately as possible the character affected or the function by which the locus is recognized. The name may indicate, for example, a morphological character, a disease character or a biochemical property. As far as possible, the locus name should reflect interspecies homology. All Greek symbols should be spelt out in roman letters and placed after the name, e.g. b Haemoglobin becomes Haemoglobin Beta. If a newly described locus has an effect similar to that of a locus which has already been named, it may be named according to the breed, geographic location or population of origin. Should a new locus be identified later as being the same as a locus already named, the name invoking breed, geographic location or population of origin should be abandoned.

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Printing the name

• • •

The locus name should be in roman letters or a combination of roman letters and arabic numerals. Wherever possible, the locus name should be printed in italics; otherwise it should be underlined. The initial letter of the locus name should be an upper case roman letter. Both nouns and adjectives in locus names should begin with upper case roman letters, e.g. Haemoglobin Beta; Haemoglobin Beta.

Locus symbol Choice of symbol

•

• • • • • • • •

For newly reported loci, unmapped DNA segments and proteins which have no known homologues or official names or symbols, special care should be exercised in selecting an appropriate symbol to avoid duplication and confusion with existing nomenclature. Every effort should be made to ensure that the symbols selected conform with those in current use for homologous loci. The locus symbol should consist of as few roman letters as possible, or a combination of roman letters and arabic numerals. The initial character should always be an upper case roman letter which, if possible, should be the initial letter of the name of the locus. For loci other than those for coat colour and visible traits, upper case roman letters only, or upper case letters combined with arabic numerals, should be used. If the locus name is of two or more words and the initial letters are used in the locus symbol, then these letters should be in upper case. All characters in a locus symbol should be written on the same line; no superscripts or subscripts should be used, nor should roman numerals or Greek letters. Where appropriate, the symbol should indicate the biochemical property or designate a particular nucleotide segment. The rules of mammalian interspecific homology already used in the choice of the name of the locus should be applied to the choice of the symbol. The designation of prefixes denoting mammalian species of origin, when being used to distinguish between the species homologues of a locus (e.g. ECA for the horse and HSA for humans), should follow the recommendations of the Human Genome Nomenclature Committee.

Printing the symbol Wherever possible, the locus symbol should be in italics; otherwise it should be underlined, e.g. the symbol of the Agouti locus: A or A.

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Alleles Allele name Choice of name

• • •

The name should be as brief as possible, but should convey the variation associated with the allele. If not given names, alleles should be given symbols as described in the ‘Allele symbol’ section below. If a newly described allele is similar to one which is already named, it should be named according to the breed, geographic location or population of origin. The names of new alleles at a recognized locus should conform to the nomenclature established for that locus. Should a new allele be identified later as being the same as an allele already named, the name invoking breed, geographic location or population of origin should be abandoned.

Printing the name Wherever possible, the allele name should be in italics; otherwise it should be underlined. A lower case initial letter for the allele name is preferred. This does not apply when a symbol is used instead of an allele name; for example, an allele at the Haemoglobin Beta locus: B or B. Allele symbol Choice of symbol

• • • • •

The allele symbol should be as brief as possible, consisting of roman letters and/or arabic numerals. As far as possible, the allele symbol should be an abbreviation of the allele name, and should start with the same letter. In the loci detected by biochemical, serological or nucleotide methods, the allele name and symbol may be identical. Greek letters and roman numerals should not be used. The symbol + can be used alone for identification of the standard allele (‘wild-type’) for alleles having visible effects. Neither + nor − symbols should be used in alleles detected by biochemical, serological or nucleotide methods. Null alleles should be designated by the number zero. The initial letter of the symbol of the top dominant allele should be an upper case letter. When there are codominant alleles only, they should each have an upper case initial letter. The initial letter of all other alleles should be lower case.

Printing the symbol

•

The allele symbol should always be written with the locus symbol. It may be written as a superscript following the locus symbol or it may be written following an asterisk on the same line as the locus symbol. The allele symbol should be printed immediately adjacent to the locus symbol, i.e. with no gaps.

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Wherever possible, the allele symbol should be in italics; otherwise it should be underlined.

Genotype terminology

• • • • •

The genotype of an individual should be shown by printing the relevant locus and allele symbols for the two homologous chromosomes concerned, separated by a solidus. Unlinked loci should be separated by semicolons. Linked or syntenic loci should be separated by a space and listed in alphabetical order when gene order and/or phase are not known. For X-linked loci, the hemizygous case should be designated by /Y following the locus and allele symbols. Y-linked loci should be designated by /X following the locus and allele symbols.

Phenotype terminology The phenotype symbol should be in the same characters as are the locus and allele symbols. The difference is that the characters should not be in italics, should not be underlined, and should be written with a space between locus characters and allele characters instead of with an asterisk. Square brackets [ ] may also be used.

Additional Comments for Blood and Milk Polymorphisms and for Mapped Loci The group of COGNOSAG members working on blood and milk polymorphisms has given detailed consideration to the nomenclature for blood and milk polymorphisms in cattle, sheep and goats. To comply with the decision made at the 21st International Conference on Animal Genetics, Michigan, 1990, to change existing gene nomenclature closer to that used for man, additional proposals were put forward for cattle, sheep and goats (Larsen et al., 1992). These were that the locus symbols would be written in upper case letters but that lower case letters would be retained to designate recessive alleles. Lower case letters were also retained for sheep and goat blood group factors. The guidelines for gene nomenclature for mapped loci and other genetic systems are as described in the previous section, but, in addition, COGNOSAG recommends that, if an un-named, newly mapped locus is identified, contact be made with Dr Sue Povey, MRC Human Biochemical Genetics Unit, University College London, Wolfson House, 4 Stephenson Way, London NW1 2HE, UK. Phone +44–207–679 7410; Fax +44–207–387 3496; email [email protected], before the locus is named to ensure that the

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symbol proposed for the horse has not already been adopted for the human. Researchers may contact the cattle genome databases – BovMAP of INRA, France; or BOVGBASE of Texas A and M, USA, for assistance in avoiding symbols already adopted for cattle. Listings of the mapped loci in cattle have been prepared in the following: BOVGBASE, of Texas A & M, USA http://bos.cvm.tamu.edu/bovgbase.html BovMAP, of INRA, France http://locus.jouy.inra.fr/cgi-bin/bovmap/Bovmap/main.pl Cattle Genome Database, CSIRO, Australia http://spinal.tag.csiro.au/ Animal Genome Database, Japan http://ws4.niai.affrc.go.jp/jgbase.html OMIA – Online Mendelian Inheritance in Animals, Australia http://www.angis.org.au/Databases/BIRX/omia/ MARC – Meat Animal Research Center, Nebraska, USA http://sol.marc.usda.gov/marc/html/gene1.html

Acknowledgements Grateful acknowledgement is made to my colleagues who have attended the COGNOSAG Workshops over the years. It is a pleasure to acknowledge the assistance of the following members of COGNOSAG: Tom Broad (New Zealand), Frank Nicholas (New South Wales, Australia), and Phil Sponenberg (USA) for helpful comments, and Verle Wood (South Australia) for the preparation of the typescript.

References COGNOSAG (1989) Gene nomenclature in sheep and goats 1987. In: Lauvergne, J.J. (ed.), Standardized Genetic Nomenclature for Sheep and Goats 1987. Loci for Visible Traits Other than Colour and Blood and Milk Polymorphisms. Proceedings of the COGNOSAG Workshop, 1987. TEC and DOC, LAVOISIER Paris, pp. 17–21. COGNOSAG (Andresen, E., Broad, T., di Stasio, L., Dolling, C.H.S., Hill, D., Huston, K., Larsen B., Lauvergne, J.J., Levéziel, H., Malher, X., Millar, P., Rae, A.L., Renieri, C. and Tucker, E.M.) (1991) Guidelines for gene nomenclature in ruminants 1991. Genetics, Selection, Evolution 23, 461–466. COGNOSAG ad hoc committee (1995) Revised guidelines for gene nomenclature in ruminants 1993. Genetics, Selection, Evolution 27, 89–93. COGNOSAG (Broad, T.E., Dolling, C.H.S., Lauvergne, J.J. and Millar, P.) (1999) Revised COGNOSAG guidelines for gene nomenclature in ruminants 1998. Genetics, Selection, Evolution 31, 263–268. Larsen, B., di Stasio, L. and Tucker, E.M. (1992) List of alleles for blood and milk polymorphisms in cattle, sheep and goats. Animal Genetics 23, 188–192.

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C.H.S. Dolling Lauvergne, J.J. (1984) A Project for Standardizing Genetic Nomenclature in Sheep. Bulletin technique du Département de Génétique animale No 38, INRA Département de Génétique animale, CNRZ, Jouy-en-Josas, France, 59 pp. Lauvergne, J.J. (1988) The project of genic nomenclature for the COGNOSAG. In: Lauvergne, J.J. (ed.) Proceedings of the COGNOSAG Workshop, 1986. TEC and DOC, LAVOISIER, Paris, pp. 21–25. Powell, B.C. and Rogers, G.E. (1993) Differentiation in hard keratin tissues, hair and related structures. In: Leigh, I., Watt, F. and Lane, E.B. (eds), Keratinocyte Handbook. Cambridge University Press, Cambridge, UK. Searle, A.G. (1988) The genetic nomenclatures of the mammals. In: Lauvergne, J.J. (ed.). Proceedings of the COGNOSAG Workshop 1986. TEC and DOC, LAVOISIER, Paris, pp. 15–20.

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Genetic Aspects of Domestication, Breeds and Their Origins 1 and A. Ruvinsky2 A.T. Bowling 2Genetic A.T. Aspects and Bowling ofA.Domestication Ruvinsky 1Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, CA 95616–8744, USA; 2Animal Science, SRSNR, University of New England, Armidale, NSW 2351, Australia

Introduction Origin of the Domestic Horse The wild ancestors of the horse Reasons, pre-condition and the initial steps of horse domestication History of horse domestication Genetic Studies of Domestic Horses and Przewalski’s Horse Chromosomes Nuclear genes detected by immunogenetic and biochemical techniques Molecular polymorphisms – mtDNA and microsatellites Worldwide Distribution of the Domestic Horse Stud books define the gene pool of a breed and the rules for managing it Examples of horse breeds Genetic similarity studies of breeds provide information to assess genetic relatedness Addressing questions of introgression between domestic breeds Future Prospects for Domestic Horses Acknowledgements References

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Introduction In human history, no other domestic animal has played such a direct role in accelerating social processes and political developments as the horse; it has been central to the rise and fall of empires, the conquest of entire continents. ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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No other domestic species has been used so widely as a means of transport in war and peace, for communications and agricultural progress; only the dog has been used more widely in sport and recreation. The 20th century has seen an end to many practical uses for which horses have traditionally been important throughout the 6000 years since their domestication. However, the recent trend shows that humans choose to maintain their association with horses, even in an era of modern cars, tractors and planes, if for no other reason than appreciation of the horse’s beauty and romantic traditions. Mutual trust and strong affiliation between humans and horses is the basis for stories, legends and myths. Research increasingly shows that the human–animal bond plays an important role in maintaining physical and mental health in the human half of the partnership. The highly developed ability of the wild horse to run fast and for a long distance was possibly the major attraction for domestication. This adaptation was vitally important for survival of the horse in natural conditions and possibly reached morphological and physiological limits. It seems unlikely that this trait improved significantly during domestication of the horse. However, other traits, which were focuses of artificial selection, changed dramatically (Fig. 2.1). Tremendous progress in mammalian genetics during the last decade did not by-pass the horse. This knowledge is very important for a number of reasons including a better understanding of the history of horse domestication, which still is full of gaps and questions. Preservation of existing breeds and their further development, as well as creation of new horse breeds, is a continuing focus of activity. Hopefully this book will facilitate further investigations which will enrich our understanding of horse genetics and domestication.

Origin of the Domestic Horse The wild ancestors of the horse While horses present a great spectrum of morphological differences, including size, shape, gait and colours, all breeds of horses are contained within the single species Equus caballus (L.). All breeds of horses have the same karyotype; crosses between breeds produce viable and fertile offspring. The immediate progenitor species for the domestic horse is not clearly defined but, by most accounts, the first domestic horses probably derived from wild horses in Eurasia (Fig. 2.2). At present, the earliest evidence for domestic horses is at an excavated settlement in southern Ukraine at Dereivka, radiocarbon dated to 4200–3800 BC (Anthony et al., 1991; The Institute for Ancient Equestrian Studies, 1997; http://www.hartwick.edu/anthropology/iaes.html). There were at least two subspecies of wild horse in historic times, a western subspecies Equus ferus ferus and the eastern Equus ferus przewalskii. Separate domestication events may have occurred in central Asia and Western Europe, but there is no direct evidence to substantiate or reject that possibility at present.

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A

B

Fig. 2.1. (A) Tarpan-like horse, reconstructed in Germany by the Heck brothers, to resemble the tarpan. Selected mares from Polish Koniks, Icelandic ponies, Swedish Gotlands and Polish primitive horses (from the preserve in Bialowieza) were mated to Przewalski’s horse. Heck assumed that the wild Przewalski horse would serve as a catalyst to draw out the latent tarpan characteristics dormant in these more modern breeds (http://www.ansi.okstate.edu/breeds/horses/). (Photograph by ©Sorrel, Germany; permission kindly granted by Ms Gaby Kärcher). (B) Arabian horse (photograph by Michael Bowling, permission kindly granted).

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Dogs, sheep, goats, pigs and cattle were domesticated earlier, presumably to ensure food sources. The horse was likely to have been domesticated primarily for purposes other than food, namely for transportation or for draught power, although these uses do not preclude use as a food source (meat or milk). Still, domestication and selection of the horse differs from that of other species, in that the chief functions for which the horse was useful to humans reinforced the trends of natural selection. Cows producing 8000 kg of milk per lactation would never appear in natural conditions; nor would sheep producing 7 kg of superfine wool per year. Horses in the wild, however, would be selected for speed, strength and endurance – the very traits that would make them useful under domestication. Equus f. ferus (http://www.ansi.okstate.edu/breeds/horses/tarpan/index. htm), or tarpan, persisted in the southern regions of Eastern Europe until the 19th century (Fig. 2.2). The last tarpan in the wild was killed in December 1879, 35 km from Askania-Nova in the Herson region, Ukraine. The last tarpan captured in the same area lived in the Moscow Zoo until the late 1880s. One stallion, which may not have been a pure tarpan, lived on a farm in the Poltava region, Ukraine, until 1918 (Bannikov and Flint, 1989). The Russian naturalist Gmelin in 1769 provided the first good description of the tarpan in nature (from a site near Voronezsh ~400 km southeast of

Fig. 2.2.

Area of horse domestication and likely directions of migration.

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Moscow). The horses that Gmelin saw were ‘hardly as large as the smallest Russian’ and had extraordinary thick heads, pointed ears and short upstanding manes. These horses were mouse-coloured; according to other descriptions, some animals were ash-grey and even white. The belly was ash-grey and the points were black. Their hair was very long and thick and like a pelt. The tail was more or less covered with hair, but always shorter than in domestic horses. The tarpan ran with ‘the utmost speed, and at least twice as fast as a good tame horse’ (cited from Zeuner, 1963, pp. 311–313; Groves, 1994). It seems doubtful that the speed estimation was correct, but it is likely that the tarpan, in spite of its small size (~130–140 cm), was very fast indeed. It is nearly universally accepted that the tarpan was the wild ancestor of the modern horse – unfortunately, direct karyological and molecular comparisons are not possible due to the extinction of this species. Equus f. przewalskii, also known by its Mongolian name takh, is the eastern subspecies of the wild horse (http://www.ansi.okstate.edu/breeds/ horses/przew/index.htm). Its name commemorates the Russian naturalist and explorer of Asia, Colonel N.M. Przewalski, who first encountered wild horses in northwest China near the Mongolian border in 1879. A detailed description of external characters of Przewalski’s horse can be found elsewhere (Groves, 1994; see Chapter 1). This is a robust animal with a thick short neck and a heavy head. Males are about 138–146 cm at the withers, females generally 5 cm smaller. The body colour is pale grey–yellow or bright yellowish red–brown. A dark dorsal stripe and dark points are typical, along with a more or less pale belly and light muzzle. Most authors exclude E. f. przewalskii from direct ancestry of the domestic horse mainly due to differences in karyotype. Przewalski’s horse has 66 chromosomes while the domestic horse Equus caballus has 64 (see Chapter 9 for details). It is not ruled out that the takh could have been the subject of independent domestication in Mongolia and northwestern China and later have been replaced by domestic horses of western origin.

Reasons, pre-condition and the initial steps of horse domestication High intelligence of the wild progenitors and plasticity of their behaviour were among key characters essential for successful adaptation to domestication. Indeed, the animals that successfully underwent the domestication process have been ‘pre-adapted’ by their previous evolution. Candidates must have had abilities that would not fully overlap with those of other domestic animals. The tarpan seems to fit these demands (Budiansky, 1997). It was a large animal able to run for a long time at high speed. A very specialized caecal digestive system allowed it to run immediately after eating. This specialized digestive system enabled horses to survive on a diet that is not sufficient for ruminants (cattle, sheep and goats) and thus reduced food competition with previously domesticated mammals.

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Shifts in genetically determined behaviour may cause changes in the neuroendocrine system and in levels of hormones. These changes in hierarchical systems regulating gene expression during development may affect basic morphogenetic processes (Belyaev, 1979; Trut, 1997), including a shift toward neoteny. It is obvious that a significant change in behaviour was a precondition for success in managing at least the first several generations of nearly wild horses. It can be presumed that taming and feeding of young foals may somewhat lessen the problem when they reached maturity, but strong selection must have operated for individual horses able to cooperate with people and demonstrate submissive behaviour. Interestingly a comparison of concentration of α-tocopherol in plasma showed that in Przewalski’s horse this parameter was substantially higher than in the domestic horse (Dierenfeld et al., 1997). We do not know much about different types of behaviour in the tarpan population, but experience with Przewalski’s horses living for a number of generations close to humans (e.g. in zoos) shows that while some Przewalski’s horses can be petted and brushed, they resist restraint and generally cannot be haltered and led. For the most part, they must be substantially restrained even to obtain a blood sample. Unlike feral horses, they cannot be easily ‘tamed’. There are pictures of Przewalski’s horses saddled and with riders on them, but this does not necessarily mean that these horses were useful riding horses. Attempts to tame zebras, another representative of wild equids, have been numerous and unsuccessful. Budiansky (1997) considers this fact as an indication of behavioural pre-adaption of the tarpan to domestication. Nevertheless, genetic and phenotypic variability for ‘domestic type’ behaviour might exist in the ancestor tarpan populations and might provide the necessary basis for successful behavioural selection and domestication. As experiments with fox domestication show, radical transformation of behaviour may occur in less than a dozen generations (Belyaev, 1979). It may mean that the initial but crucial steps could be accomplished within a limited period. A diastema (a gap between the front incisors and rear grinders) provided an opportunity for the innovation of bridle and bit. Without them, managing a horse would be much more difficult.

History of horse domestication Clearly, the domestic horse could reach the Middle East only by two ways (Fig. 2.2): over the Caucasus, or through the steppes and semideserts to the east from the Caspian sea. Use of horses probably started spreading between 3500 and 3000 BC and reached the Middle East in the middle of the third millennium BC. The earliest known clay figurine of a domestic horse (~2300 BC) was found recently at Tell Es-Sweyhat, about 300 km northeast of Damascus (Bower, 1993). The horse reached Egypt close to the end of the Hyksos dynasty (~1580 BC) when it was considered a rare animal.

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The horse appeared in southeastern Europe (Greece) at about the same time, soon after 1900 BC (Bökönyi, 1984). Much later, in the time of Philip of Macedon, the father of Alexander the Great (430–354 BC), horses still were imported extensively into Greece from the kingdom of Scythia, the area of modern Romania, Ukraine and south Russia (Clutton-Brock, 1987). The Greek mythological figure of the man–horse Centaur shows the spread of horseriding culture in ancient Greece and particularly in its northern neighbour Scythia, where these skills were highly developed and significantly affected contemporary arts. The horses on Scythian artefacts are characteristically large and Arabian type in their conformation (Clutton-Brock, 1987). From this time, expansion of the horse into the major western civilizations spread rapidly, with the recognition of its military usefulness. The domestic horse penetrated eastward at least by the early first millennium BC. A Scythian horse wearing a ritual mask was found in the frozen tomb at Pazyryk in the Altai mountains, dated to the 5th century BC (Zeuner, 1963; Rudenko, 1970). There are indications that horse riding appeared in the Altay-Sayan region as early as 1500 BC (Bökönyi, 1984). In this case, Sarmatians and other seminomadic people who lived eastwards from the Ural mountains and Caspian Sea might have possessed domestic horses. It is well known that the steppes of modern Kazakhstan, southern Siberia and Mongolia were the places where the ‘horse culture’ reached its highest development. For the nomadic tribes that lived there, it was a way of life compatible with vast territories and natural conditions. These tribes rode horses, milked them, fermented ‘kumyss’ (an important part of their diet), ate horse meat and used their hides. There is no historic evidence that the domestic horse was used in China before the second or third centuries BC. In 102 BC, Emperor Wudi sent a huge military expedition to Middle Asia (‘Fergana’) to obtain ‘heavenly horses’ (Zeuner, 1963; Olsen, 1988). However, there are indirect indications that Scythians had contact with China long before this time. Silk and a bronze mirror found in the Scythian Pazyryk tomb in the Altai mountains confirm contacts between China and Scythia in about 500–400 BC. These data may indicate that the domesticated horse reached China through the Altai region as early as 500–400 BC. It seems likely that the domestic horse reached Mongolia by a similar but more northerly route. World history would be quite different without the domestic horse. Interestingly, wheeled vehicles were in use on the Russian steppes by 3650 BC (Haywood, 1997), but military chariots were introduced only in the 16th century BC in Egypt. Egyptian cavalry and horse-drawn chariots were first mentioned in the biblical description of Exodus, when the whole army was swept away by the waves of the Red Sea. The domestic horse became an increasingly powerful component of Eurasian civilizations from the middle of the second millennium BC (The International Museum of the Horse 1998. Lexington, Kentucky http://www.imh.org/imh/imhmain.html). Horse transport enabled humans to increase the speed at which they could move across land, in a way that none of the animals domesticated earlier

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had provided. That inherited differences occur in gaits (footfall patterns) of horses and in no other domestic animal is probably also an indication of the significance of ridability to its domestication. From the early centre (or centres) of domestication, horses spread in all directions (Bökönyi, 1984). People migrating into new territories brought their domestic animals, including horses, with them. Since the horse was a major means of transportation, horses were also spread along major trade routes, and probably gained new homes as captured booty from skirmishes with invaders.

Genetic Studies of Domestic Horses and Przewalski’s Horse Morphology and historical information classically have been used to describe the relationships of breeds. More recently, studies of objectively determined genetic markers have been used to understand the fundamental relationships of breeds to one another and to other equids, particularly Przewalski’s horse, the most closely related species. An inherent problem in genetic comparison conclusions concerning domestic and wild horses is that a Mongolian domestic mare is included in the pedigree lineage of many extant Przewalski’s horses (Kus, 1997). Although the other 12 surviving species founders for Przewalski’s horse are not considered to represent domestic horse hybrids, this point is not known with certainty.

Chromosomes The genus Equus is characterized by rapid karyotype evolution (see Chapter 9). Rapid evolution of satellite DNA in equids (Wijers et al., 1993) may be a factor in their rapid chromosome evolution (Wichman et al., 1991; Bradley and Wichman, 1994). The karyotype of the domestic horse is composed of 64 chromosomes (13 metacentric autosomal pairs, 18 acrocentric autosomal pairs and a pair of sex chromosomes). That of Przewalski’s horse is very similar, but the diploid number is 66 (12 metacentric autosomal pairs, 20 acrocentric autosomal pairs and a pair of sex chromosomes). The difference appears to involve chromosomal fission or fusion (depending on the direction of evolutionary change) of ECA5 (E. caballus chromosome 5). The karyotype difference between Przewalski’s and domestic horses may have alternative evolutionary scenarios. According to the first one, the tarpan had 64 chromosomes at the time of domestication, which explains why domestic horses have 64 chromosomes. The possible difference between the tarpan and the takh may be a result of fixation of the Robertsonian translocation in the tarpan if the ancestor of both subspecies had 66 chromosomes. The second proposal is based on the assumption that the Robertsonian translocation occurred in the very early stages of domestication, essentially in one herd of tarpan that originally had 66 chromosomes. Due to random events, only homozygotes for this translocation survived. All domestic horses would

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originate from this single herd. However, if the common ancestor of both wild subspecies had 64 chromosomes then a fission event of metacentric or submetacentric chromosomes might increase the number of diploid chromosomes from 64 to 66 in Przewalski’s horse (Ishida et al., 1995). Hopefully, future investigations will provide information to rule out one of these proposals. All Przewalski’s horses that have been studied have the same diploid karyotype of 66 chromosomes. All horse breeds so far studied have a diploid set of 64 chromosomes, except for the Caspian pony, a rare group of horses found in the northern part of Iran around the Caspian Sea, which shows polymorphism in the diploid number of chromosomes (Hatami-Monazah and Pandit, 1979). Out of 17 studied animals, 11 had 64 chromosomes while six had 65 chromosomes, demonstrating heterozygosity for centromeric fission in one of a pair of metacentric chromosomes. None had 66 chromosomes. The G-band pattern of these chromosomes indicated close resemblance to both E. caballus and E. f. przewalskii. The fertility of mares in the group was low, about 40%, as expected for heterozygosity for a Robertsonian translocation. The stallions had a low sperm count and poor sperm motility. Possible explanations for this chromosomal polymorphism in Caspians could be natural hybridization of domestic and Przewalski’s horses in the past or it could have been produced by a spontaneous, independent event involving the same chromosomes as the karyotype difference between Przewalski’s and the domestic horse.

Nuclear genes detected by immunogenetic and biochemical techniques Starting in the late 1960s and continuing to the early 1980s, genetic studies of horses were based on immunogenetic and biochemical markers developed for parentage verification of domestic horses registered by breed societies (see Chapter 5). Development of these tests involved extensive research with a few major breeds (e.g. Thoroughbred, Arabian, Quarter Horse, American Standardbred and Swedish Trotter) but subsequently the tests proved highly effective for examining parentage questions in any breed – and for looking at genetic similarities among breeds and with Przewalski’s horse. Investigators with access to genetic material from Przewalski’s horse applied these gene assays to that species (Braend, 1979; Kaminski, 1979; Ryder et al., 1979; Scott, 1979; Putt and Whitehouse, 1983; Bowling and Ryder, 1987; Bowling and Dileanis, 1990; Patterson et al., 1990; Fincham et al., 1992). These studies generally confirmed a significant level of similarity among breeds and between E. caballus and E. f. przewalskii compared with other Equidae species, and, for several loci, genetic variants were present in some Przewalski’s horses that were not found in domestic horses. The first highly effective parentage test developed for horses, still used by many breed registries throughout the world, consisted of a battery of tests detecting blood group and blood protein polymorphisms developed by collaborating laboratories belonging to the International Society for Animal Genetics

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(ISAG) (described in Bowling, 1985; Bowling and Clark, 1985; and see Chapter 5). Several comparative studies of breeds and Przewalski’s horses have been conducted based on the parentage test battery. Bowling and Ryder (1987) used 18 polymorphic loci to investigate five breeds of domestic horses and Przewalski’s horse. The genetic distance between the breeds of domestic horses and Przewalski’s horse was estimated as 0.3 or slightly greater, while the domestic breeds clustered rather closely at distances of about 0.1 or lower. Interestingly the average heterozygosity for Przewalski’s horse did not differ significantly from the values calculated for several domestic breeds. Dubrovskaya et al. (1992), using a smaller set of polymorphic systems and a different group of domestic breeds, came to a similar estimation of genetic distances. Surprisingly, the genetic distance between Przewalski’s horse and the Shetland pony was rather small, possibly reflecting not only the real relationships between these two populations, but also results of genetic drift. The cluster analysis grouped several phenotypically quite different indigenous breeds from the eastern part of the former USSR, such as the Akhal-Teke and Yakut horses. This unexpected conclusion was confirmed recently in the study by Tikhonov et al. (1998) based on a much larger set of breeds and on a more powerful set of polymorphic loci than used by Dubrovskaya et al. (1992) (comparable with the parentage panel described by Bowling and Clark, 1985). In the Tikhonov study, indigenous breeds with such distinctly different phenotypes as Akhal-Teke and Yakut show high levels of relatedness. Rogers coefficient of similarity between these two breeds was equal to 0.681. Even more surprising is that the similarity of Arab and Yakut horse was about 0.725. However, these data do not contradict the discussion concerning the spreading of horses from a horse domestication centre in Scythia. Tikhonov and colleagues expressed the view that Yakut and several autochthonous breeds of Middle Asia had significant association with the ancient horse that moved eastwards from the centre of horse domestication. Przewalski’s horse consistently takes an outgroup position in the dendrograms of domestic horses based on nuclear genes (Bowling and Ryder, 1987; Dubrovskaya et al., 1992; Tikhonov et al., 1998). This is an additional argument apart from morphology, karyology and behaviour that Przewalski’s horse did not contribute to the gene pool of the domestic horse.

Molecular polymorphisms – mtDNA and microsatellites Polymorphisms in DNA sequence have also been used to decipher the recent evolution of horses. The first studies involved mitochondrial DNA (mtDNA) (George and Ryder, 1986). Comparisons of Przewalski’s horse and domestic horse mtDNA cleavage maps (based on shared restriction enzyme recognition sites) yielded estimates of divergence from 0.27 to 0.41%. Using an average estimation of divergence rate for mtDNA of 2% per million years, it can be assumed that the common ancestor for Przewalski’s and domestic horses lived

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about 100,000 years ago. Ishida et al. (1995) confirmed data presented by George and Ryder and provided new information concerning evolution of equids, particularly for domestic and Przewalski’s horses. From the sequence of the variable region of the horse mtDNA (D-loop segment), they concluded that ‘the lineage of the Przewalski’s wild horse is not located at the deepest branching among the E. caballus sequences’ in the neighbour-joining trees they constructed. The observed topology of the trees clearly contradicts the origin of the domestic horse from Przewalski’s horse. However, it is difficult to reconcile the topology of the phylogenetic trees constructed by Ishida et al. (1995) with comparative morphological, karyological and behavioural data and with trees constructed by others. On the dendrograms of Bowling and Ryder (1987), Tikhonov et al. (1998) and Dubrovskaya et al. (1992), Przewalski’s horse shows early separation between wild and domestic horses, while that of Ishida et al. includes Przewalski’s horse within the branches of different breeds of domestic horses. Oakenfull and Ryder (1998) determined the mtDNA D-loop sequence in surviving Przewalski’s horse pedigrees using representatives of the four extant matrilineal mitochondrial sources. Only two sequences were found, one of which corresponded to that of Ishida et al. (1995). The other sequence differed from the first, but both were certainly more similar to the published sequences of three Thoroughbreds and a Mongolian horse than to other equids, corresponding to the close relatedness found from nuclear gene studies for domestic and wild horses. There is no other information suggesting that the mtDNA sources in surviving Przewalski’s pedigrees are derived from domestic horses. However, Przewalski’s horse and domestic horse hybrids are viable and fertile. The contradiction between the mtDNA and nuclear DNA studies may be an indication that in addition to the known introgression event that occurred within a captive breeding programme, introgression also occurred in the wild. Dinucleotide tandem repeats in horse DNA (microsatellites) in non-coding regions of undefined genes have been described (e.g. Ellegren et al., 1992; Marklund et al., 1994; and see Chapter 6). Microsatellites can be assayed subsequent to polymerase chain reaction (PCR) fragment amplification using semi-automated procedures for efficiency of sample testing. Frequency data for microsatellite fragment sizes (allelic markers) of Przewalski’s horse and domestic horses were reported by Breen et al. (1994). While there was considerable overlap among variants observed in both species, some alleles appeared to be present in the wild species, but not in domestic horses.

Worldwide Distribution of the Domestic Horse The current estimated global population of domestic horses is 62 million (FAOSTAT Database 1998), consisting of approximately 500 ‘breeds, types and varieties’ (Mason, 1996). Classically, a breed is a distinctive set of animals associated with a restricted geographical area in which it was developed to meet human needs under particular local conditions (an autochthonous

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breed). Increasingly often, horse breeds are defined by societies or registries that record pedigrees and maintain a stud book for a selected subset of horses based on geographical origin, phenotype or function. Horses registered with a breed society constitute about 10% of the global population (Bixby et al., 1994). Horses not associated with breed societies include feral horses and the majority of those working in transport and agriculture. Autochthonous breeds not constrained by stud book regulations potentially constitute a significant genetic resource, often overlooked. Attempts have been made to organize horse breeds into major subdivisions, for example based on physical size and proportions (pony, light horse or draught horse), or on temperament (coldblood, warmblood or hotblood), reflecting perceived evolutionary relationships. However, no such system provides a robust, intellectually coherent classification scheme in which each contemporary breed would have an unequivocal assignment. Notwithstanding the difficulties in arriving at a comprehensive classification scheme, from time to time the above terms are useful to describe certain subsets of horses. For example, ‘European warmblood’ has become the accepted descriptor for a group of horse breeds, not so much defined by temperament as by related origins, physical appearance and use.

Stud books define the gene pool of a breed and the rules for managing it Horse breeds defined by stud books represent a diverse collection of options for assembling and managing a gene pool. In most countries, there is no overriding authority that oversees compliance with a single stud book model to define and maintain a breed, such as exists for purebred dogs. The rapidly accumulating and widespread evidence concerning the poor genetic health of many dog breeds provides a dramatic example of the importance of considering genetic implications of stud book policies, both at the stud book inception and during the maintenance phases. Horse breeds may appear to be less phenotypically distinctive than dog breeds, perhaps a reflection of the historical relatedness of many breeds and more recent crossbreeding schemes. For horses registered with a breed society that allows crossbreeding, the potential for generating heterozygous genotypes may contribute to overall breed health, but compromise the predictable genetic breeding uniformity of individuals. Some horse stud books are under the authority of a government agency (e.g. Spanish Purebred horse (Andalusian)), but most stud books are maintained by and subject to the legal obligations of a private corporation. The keepers of the stud book (generally, the elected or appointed Directors of the Breed Society) determine the registration rules. The rules may be changed by the current authorities when deemed essential for breed viability. In the last few decades, authorities have had to make decisions about whether to allow assisted reproductive technologies (ART) (such as artificial insemination, transported cooled semen, frozen semen and embryo transfer) which have potential to affect the genetic future of the breed. While many breeds have endorsed

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these technologies, a notable exception is the Thoroughbred, for which stud books worldwide have agreed only to register animals conceived by ‘natural cover’ and delivered by their own (genetic) mother. Stud book breeds suit a variety of purposes. The founder populations usually have been selected particularly on appearance (‘type’) and utility. Selection may also be based on geographical origin (Arabian, Connemara, Icelandic, Kiger), colour (Appaloosa, Paint), gait (Tennessee Walking Horse, Paso Fino), racing speed (Thoroughbred, American Standardbred) or relationship to a single stallion (Morgan, Shagya). Crossbreeding initially may be allowed (and recorded in pedigrees) for several generations to build up a viable breeding nucleus. Subsequently, many stud book authorities close registration to any but the offspring of horses already registered with the society. In the case of a closed stud book (e.g. Arabian or Thoroughbred), the breed today is based on a selected set of founders and there are no (or extremely few) options to increase diversity in the gene pool. One possibility to expand the gene pool is reciprocity with stud books in other countries. Even if breed stud books in different countries are based on the same founders, genetic differences due to the mutation process, recombination and genetic drift probably will develop provided the populations remain separated for a sufficient length of time. Breed societies that have elected to keep their stud books open usually restrict crossbreeding to horses registered with specified stud book authorities. The breeds allowed for outcrossing most commonly include Thoroughbred and to a lesser extent Arabian, such that modern breeds with open stud books may be losing their distinctiveness (Bixby et al., 1994). Crossbred horses in open stud books may be placed in a separate section (e.g. Thoroughbred crosses in the Appendix section of the Quarter Horse stud book) but may advance to regular registry status after meeting performance or breeding standards. Due to the pedigree restrictions of most breed societies, there is little opportunity for proven useful gene combinations from unregistered horses (e.g. feral horses) to be added to their gene pools. However, new horse breed societies continue to emerge (e.g. National Show Horse, Azteca, Tiger), permitting breeders to take advantage of gene sources and combinations not otherwise accommodated by extant breeds. This continuing development of breeds is not nearly as common among other domestic animal species but, for horses, there appears to be sufficient interest and economic incentive to do so. For the most part, the offspring of a pair of registered (stud book recorded) parents of the same breed will also be eligible for registration in the same stud book, but some breed societies have defined restrictions. For European warmbloods, foals may be recorded in a ‘foal book’, but not attain full breeding registry status until they pass a series of requirements, including physical inspection, performance and breeding tests. Particularly for colour breeds, foals that do not have the defining colour may be assigned to a separate section and may not be allowed to participate in breed shows, but their offspring are fully eligible for regular stud book status if they meet the colour and pedigree requirements. Even ‘non-colour’ breeds may have ‘colour’ requirements. For example, until recently, Morgan foals could not be

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registered if they were of cremello or perlino colour (i.e. ~25% of foals arising from the inter se breeding of palomino or buckskin parents) or had ‘excessive’ white markings (genetics not defined). Those registration restrictions were lifted in 1996 for Morgans, but similar ones are in place for Quarter Horses. Breeds defined by stud books are young compared with the length of time that horses have been domesticated. Breeds with the longest pedigree records include Lipizzaners (since the early 1700s) and Thoroughbreds (since the late 1700s) (both breeds from acknowledged crossbred sources). Arabians are considered to have been a recognized breed for longer than either Lipizzaners or Thoroughbreds, theoretically without any crossbreeding, but for most Arab horses the bulk of the recorded pedigree consists of lines that trace to desert sources representing only 100 years or less of written records, and no line can be carried back before 1800. Examples of horse breeds Examples of modern horse breed diversity are provided here by riding horse, draught horse, pony and mixed-use breeds. Breed information, chosen to illustrate the kinds of information available about the genetic history of breeds, was obtained from comprehensive material provided by breed registries, Edwards (1980) and Hendricks (1995). The breeds selected provide background for the genetic distance comparisons that follow later in this chapter. Arabian (AR) The Arabian as a breed has no origin in recorded history. Desert nomads in the Arabian peninsula developed a moderately sized horse noted for such beauty, stamina and conformational strength that for centuries horse breeders went to the Arabian desert to import horses for improvement of local stock. The Thoroughbred (TB) and continental cavalry breeds are the noteworthy result of this process. Historically, a few breeders in Europe and Egypt imported ARs not only for crossbreeding, but also to establish AR breeding programmes. Most modern AR pedigrees trace to desert imports from about the mid-1800s to the early 1900s. AR stud books are maintained throughout the world. The international gene pool of ARs is essentially closed, but it is not homogeneous. Stud books could still be acquiring new genetic material from other countries. ARs have been particularly successful in events requiring endurance, but are primarily bred for competitive show ring events in which excellence is judged subjectively. In traditional classification schemes for horse breeds, ARs are typically presented along with TBs as a standard example of the hotblood or light horse. The breed data for this chapter are derived from horses registered with The Arabian Horse Registry of America. Iberian (IB) Horses of the Andalusian (Purebred Spanish horse) and Lusitano breeds are riding horses that have been developed in Spain and Portugal, respectively,

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from historically similar autochthonous Iberian peninsula breeding elements. Differences seen today can probably be attributed to relatively recent differences in selection emphasis. For example, most Andalusians are grey, with occasional examples of other colours except chestnut (while chestnuts occur, they are not considered acceptable as breeding stock). Any solid colour is acceptable in Lusitanos, including chestnut. While both breeds are used as riding horses, the Lusitano is an integral part of the Portuguese bull-fighting tradition and is selected primarily for excellence in this sport, while the Purebred Spanish horse is selected for beauty and sound, functional conformation. Iberian horses historically have been used as a source of strength and elegance for breeding stock improvement at European state studs and are highly valued for those same traits today. The stud book of the Purebred Spanish horse is maintained for horses worldwide by the Cria Caballar, a branch of the Spanish military. Lusitanos have a more restricted worldwide distribution. In the USA, horses of each breed and their combinations are registered by The International Andalusian and Lusitano Horse Association and the breed data for this chapter are derived from those horses. Lipizzaner (LI) This breed was developed in central Europe, in an area associated variously with Italy, Austria or Yugoslavia, depending on national border changes established by peace treaty agreements. Lipizzaners are distributed around the world, although the number of horses overall is not large. The American Livestock Breeds Conservancy (ALBC) classifies LIs as rare. The predominant colour for LIs is popularly referred to as ‘white’, although that colour would be recognized by geneticists as being produced by the gene for grey colour whose epistatic action (see Chapter 3) causes the progressive and gradual lightening of the dark hair of the foal coat. LIs are riding horses characterized by great strength. Today, LIs are best known for their association with the Spanish Riding School in Vienna, the oldest riding school in the world, and for their excellence in classical riding disciplines (dressage). Archduke Charles, son of Emperor Ferdinand I, established a stud farm at Lipizza in 1580 that exchanged horses with the court stud of his brother at Kladrub and with other state studs. The LI has one of the oldest stud books, dating to 1701. The genetic elements of LIs, in addition to autochthonous horses, include IB, AR, Danish, Neopolitan and Kladrub sources. Breeding traditions emphasize paternal and maternal line founder elements and attempt to preserve all the extant pedigree sources. The breed data for this chapter are derived from horses registered with United States Lipizzan Registry. Miniature (MI) The ideal for this breed is an animal that is a miniature version of a standard horse but measuring no more than 34 inches at the withers (base of the mane). While animals are seen that simulate this ideal, most tend to reflect their pony origins. Miniatures are too small for riding but they can be used for light driving. They are particularly valued as companions and for show ring

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competition. In addition to small size, these horses are known for their great variety of colours and patterns – indeed it is reasonable to propose that all the colours and patterns distributed among various horse breeds can be found in MIs. Their earliest origins are unrecorded and subject to various interpretations, as with other breeds pre-dating stud book records. There is no compelling evidence of a recent relationship with any horse-sized breed, despite physical resemblance of some animals, for example to ARs or TBs. Certainly some MIs are of relatively recent descent from Shetland ponies meeting MI size requirements. Another source is from the breeding programme of the Falabella family in Argentina. Pedigrees of MIs may be relatively short, since the breed association is relatively new, and horses meeting size requirements may be accepted for registration without a pedigree. The breed data for this chapter are derived from horses registered with The American Miniature Horse Association. Morgan Horse (MH) All horses of this breed trace to one stallion, a horse owned in the late 1700s by a Vermont school teacher named Justin Morgan. The breeding origins of this stallion are not documented reliably. Mixtures of TB, AR, Dutch Draft and Welsh Cob breeding have all been suggested, but nothing can be proved. He was bred to the variety of mares available locally and his descendants demonstrated tractability and versatility, from weight-pulling, to saddle and harness racing, and use as a utility saddle horse – all by the same horse. A registry was established in 1909 incorporating in many cases pedigrees with elements tracing back 100 years. In the early decades of the registry, crossbreeding with such breeds as AR, Standardbred and American Saddlebred, followed by back-crosses to MHs, was allowed. At present, the registry is closed. The gene pool of this breed is expected to be quite diverse, despite descent from a single sire. Reasons for this include the variety of mare lines used in the establishment of the breed, the more recent use of crossbreeding, and selection practices based on somewhat subjective characters such as physical type which have a low heritability. MHs provide an example of a horse breed that logically should be classified as a warmblood, since it has elements of hotblood and coldblood breeds. However, the term ‘warmblood’ is applied now to mixtures of European breeds with TBs and it is likely that there would be some objections to the inclusion of MHs in the warmblood category. The breed data for this chapter are derived from horses registered with the American Morgan Horse Association. Norwegian Fjord (NF) Horses of this breed are used as small draught horses and as riding and driving horses. They are hardy horses of ancient origin and today are of a distinctive colour (dun), although the uniformity of pattern was imposed relatively recently (selective breeding programmes dating from the late 19th century). The predominant colours – red, brown and grey dun – are produced by the interactions of the basic coat colour genes with the Dun gene. To maintain

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hardiness and diversity of the gene pool, registration is denied for horses conceived from breeding between first generation relatives. The breed data for this chapter are derived from horses registered in the USA with the Norwegian Fjord Horse Registry. Paso Fino (PF) This gaited (lateral rather than diagonal footfall pattern) riding breed is being developed in the USA in a relatively recent effort from Caribbean and South American sources. The horses descend from Barbs, Iberian horses and Jennets brought to the New World by Spanish explorers and colonists as long as 500 years ago. The PFs have a natural lateral gait, a characteristic of most of the world’s riding horses prior to the 17th century, that contrasts sharply with the square trotting gait seen in most horses today. This gait is extremely comfortable for the rider and is relatively non-tiring for horses to perform. In the USA, these horses are used primarily for pleasure riding and showing. The breed data for this chapter are derived from horses registered with the Paso Fino Horse Association. Percheron (PN) This draught horse was developed in northern France, from crosses of oriental stallions with local mares of undocumented, but ancient origins. These horses, along with other European draught horses, were widely exported in the midto late 19th century to provide the breeding basis for agricultural horsepower, particularly for the USA. While breed numbers declined dramatically with the advent of agricultural mechanization in the early years of the 20th century, in the last few decades the number of foals registered has been increasing so that this breed is no longer considered rare. The horses are still popular for agricultural uses, for show ring competition and for crossbreeding with TBs to produce competition sport horses. The horses today are black or grey, with minimal white markings. The breed data for this chapter are derived from horses registered with the Percheron Horse Association in the USA. Thoroughbred (TB) The recorded origins of this breed trace to the use in the early 1700s of imported ‘eastern’ (‘Arabian’, ‘Barb’ or ‘Turk’) stallions with English mares to beget horses for racing. Weatherby’s General Stud Book (GSB), Vol. 1, appeared in 1808. Registered TBs must trace in all lines to horses registered in the GSB or in similar TB stud books maintained outside Great Britain. Thus, this breed, originally a crossbred, has had a closed stud book for nearly 200 years. Individual TBs may excel at jumping, cross-country racing and dressage, but for the most part these horses have been selectively bred only for galloping speed. In traditional classification schemes for horse breeds, TBs typically are presented as a standard example of the hotblood or light horse. The breed data for this chapter are derived from horses registered with The Jockey Club in the USA.

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Trakehner (TK) This breed was developed in the 18th century as a cavalry horse from Prussian local horses of various types including heavy horses, augmented with selected TBs and ARs. The TK is one of the breeds traditionally referred to as a warmblood because of its origins from mixed breed types, including TB with continental European autochthonous breeds. The breed was almost destroyed during World War II, but, through the efforts of dedicated breeders and German federal government support, it has been revived as a vigorous and highly regarded breed, particularly valued for its abilities in sport horse competitions, including jumping, dressage and cross-country events. The breed is distributed worldwide. It is also used as a breeding element in other European warmblood breeding programmes. The breed data for this chapter are derived from horses registered with the American Trakehner Association in the USA. Genetic similarity studies of breeds provide information to assess genetic relatedness The early breed comparison studies were based on easily identified allelic differences in protein-coding genes. With the rapid development of techniques for identification of DNA sequence variation, assays of highly polymorphic microsatellites as markers of non-coding genetic sequence have become available. A microsatellite-based alternative parentage test that can be applied to blood or other biological samples, including hair roots, teeth and bone, has been developed and provides a standard, readily assayed battery of molecular markers for descriptive breed data (Bowling et al., 1997). A new generation of breed comparisons can now be carried out. The more comprehensive the genome coverage, the more likely it is that genetic similarity measures will reflect the relatedness of breeds and allow us to provide reasonable assessments of the early stages of breed developments. A population analysis of allelic frequency data for ten breeds is presented. The analysis is based on tests of over 50,000 horses for 22 loci of blood group and protein polymorphisms and 16 loci of microsatellites, includes loci representing 18 autosomes, the X chromosome and six loci whose autosomal assignment at present is not known (Table 2.1) (for a discussion of the loci, see Chapters 5 and 10). For each locus, estimates of the total gene diversity (HT)*, the coefficient of gene differentiation (GST)* and average gene diversity (HS)* are provided for domestic horses (Table 2.1). The high gene diversity (HT) values overall reflect the selection of moderately to highly polymorphic loci to provide power for parentage testing. The measures HS and GST provide an estimate for the partitioning of the variation within and between breeds. The * HT is the expected heterozygosity of an individual produced by random mating within the total population of horses, disregarding breed; HS is the expected heterozygosity of an individual produced by a random mating with a subpopulation (breed); GST relates HT to HS. It is a measure of loss of diversity within subpopulations compared with the total population of horses.

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Table 2.1. Loci used for analysis of breed differences and data for construction of dendrogram. Estimates of the total gene diversity (HT), the coefficient of gene differentiation (GST) and average gene diversity across breeds (HS) are provided for each locus. Locusa Blood groups

RBC proteins

Serum proteins

Microsatellites

Chrb

20 U U 2 U [8] 24 Average U AP U CA U CAT 13 HBA 2 PGD 5 PGM 10 GPI Average 3 ALB 7 C3 3 ES 3 GC 31 PLG 16 TF 24 PI 10 XK Average 2 ASB17 30 VHL20 21 HTG10 4 HTG7 9 HTG4 [8] AHT5 24 AHT4 4 HMS6 9 HMS3 1 HMS7 15 HMS1 X LEX3 4 LEX33 15 ASB2 [28] CA425 15 HTG6 Average Average (38 loci) EAA EAC EAD EAK EAP EAQ EAU

Alleles

HT

GST

HS

12.4 2.4 25.4 2.4 3.4 6.4 2.4 7.4 2.4 6.4 3.4 5.4 3.4 3.4 5.4 3.9 3.4 5.4 12.4 2.4 2.4 15.4 25.4 4.4 8.5 22.4 10.4 12.4 5.4 8.4 11.4 11.4 8.4 11.4 10.4 8.4 14.4 12.4 14.4 11.4 11.4 11.8 7.9

0.740 0.467 0.889 0.115 0.512 0.667 0.429 0.546 0.076 0.137 0.357 0.530 0.293 0.202 0.094 0.241 0.451 0.450 0.504 0.161 0.327 0.766 0.876 0.173 0.463 0.871 0.830 0.845 0.606 0.687 0.809 0.809 0.759 0.822 0.803 0.664 0.859 0.834 0.847 0.670 0.622 0.771 0.567

0.139 0.099 0.079 0.115 0.036 0.152 0.156 0.111 0.018 0.071 0.161 0.119 0.106 0.034 0.129 0.091 0.090 0.061 0.124 0.060 0.116 0.097 0.089 0.097 0.092 0.080 0.094 0.067 0.124 0.092 0.083 0.076 0.074 0.078 0.093 0.040 0.076 0.089 0.111 0.051 0.181 0.088 0.094

0.638 0.421 0.819 0.102 0.494 0.566 0.362 0.486 0.074 0.127 0.300 0.467 0.262 0.195 0.082 0.215 0.411 0.423 0.442 0.151 0.289 0.691 0.798 0.156 0.420 0.801 0.752 0.788 0.531 0.624 0.741 0.747 0.703 0.758 0.728 0.637 0.794 0.759 0.753 0.636 0.510 0.704 0.514

aLoci

identified here by abbreviations are explained and referenced in Chapters 5 and 10. chromosome; U: unassigned to a chromosome; brackets designate provisional assignment. More information on chromosome assignments is provided in Chapter 10.

bChr:

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higher values for HS (within-breed variation) and the lower values for GST (between-breed variation) show that most of the genetic diversity found is shared among breeds. Notice that, at least among the loci chosen here, microsatellites are slightly less effective in distinguishing between breeds (lower GST values) than the gene loci of the conventional test (blood groups and protein polymorphisms). The ten breeds of horses (described in previous paragraphs) have been chosen to provide a sampling of physical types and uses, breed origins and pedigree structures. Estimated average heterozygosity values for each breed and for Przewalski’s horse are provided in Table 2.2. The breeds with the oldest pedigree records (TB and LI) have the lowest heterozygosity values, and the newer breeds (based on the age of the stud books) (PF and MI) have the highest values. Average heterozygosity for Przewalski’s horse, despite the very small number of founders (13) recorded in the stud book (Kus, 1997), has values comparable with some of the domestic horse breeds. Genetic distances (D) among these breeds and Przewalski’s horse, based on 38 loci, were calculated using DISPAN (Ota, 1993) (Table 2.3). As in other genetic distance studies, Przewalski’s horse provides the most dissimilar of the paired comparisons for any breed, shown here with a value of at least 0.308. Between domestic breeds, no distance measure exceeds 0.214 ± 0.05 (TB versus NF), which seems appropriate considering the lack of a documented or even anecdotal connection between these breeds. Also, the very close distance value between TK and TB (0.041 ± 0.01) is expected considering the contemporary use of TBs in TK breeding programmes (but not vice versa). As a means of visualizing these distance data, a dendrogram was constructed based on a neighbour-joining algorithm (NEIGHBOR) using PHYLIP (Felsenstein, 1993) (Fig. 2.3). The arrangement agrees reasonably well with anecdotal and published information available for these breeds and with Table 2.2. Estimated average heterozygosity at 38 loci (listed in Table 2.1) for ten breeds of horses and Przewalski’s horse, arranged from lowest to highest values. Breed or taxon TB LI PZ AR IB TK NF PN MH PF MI

Estimated average heterozygosity (±SD) 0.461 (0.047) 0.473 (0.042) 0.474 (0.044) 0.478 (0.045) 0.491 (0.046) 0.511 (0.043) 0.531 (0.039) 0.535 (0.041) 0.537 (0.042) 0.551 (0.045) 0.579 (0.038)

AR, Arabian; IB, Iberian; LI, Lipizzaner; MI, Miniature Horse; MH, Morgan; NF, Norwegian Fjord; PF, Paso Fino; PN, Percheron; PZ, Przewalski’s horse; TB, Thoroughbred; TK, Trakehner.

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0.124±0.02 0.214±0.05 0.152±0.04 0.175±0.04 0.194±0.05 0.115±0.03 0.168±0.03 0.092±0.02 0.354±0.08

0.109±0.02 0.057±0.01 0.114±0.03 0.069±0.01 0.078±0.02 0.113±0.02 0.079±0.02 0.107±0.03 0.091±0.02 0.345±0.08

Breed abbreviations as in Table 2.2.

NF PF TB TK AR LI PN IB MI PZ

NF

0.129±0.03 0.094±0.02 0.099±0.02 0.126±0.02 0.092±0.02 0.092±0.02 0.083±0.02 0.323±0.08

PF

0.041±0.01 0.105±0.02 0.202±0.05 0.194±0.04 0.170±0.04 0.182±0.04 0.382±0.09

TB

0.065±0.02 0.155±0.03 0.150±0.03 0.137±0.03 0.141±0.03 0.382±0.09

TK

0.139±0.03 0.156±0.03 0.109±0.03 0.151±0.03 0.394±0.09

AR

0.132±0.03 0.199±0.04 0.136±0.03 0.394±0.09

LI

0.139±0.03 0.089±0.02 0.344±0.08

PN

0.137±0.03 0.389±0.09

IB

Standard genetic distances (±SD) between ten breeds and Przewalski’s horse based on 38 polymorphic loci (listed in Table 2.1).

MH

Table 2.3.

0.308±0.07

MI

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Fig. 2.3. Thirty-eight loci of genetic markers (blood groups and proteins as well as microsatellites) were used to construct this dendrogram for ten breeds and Przewalski’s horse based on a neighbour-joining algorithm using NEIGHBOR in PHYLIP (Felsenstein, 1993) and bootstrap resampling of 100 data sets. Bootstrap values are provided at branch points.

previously published dendrograms using fewer loci and less extensive genome coverage. As in previously published dendrograms based on nuclear genes, Przewalski’s horses remain as an outgroup to the domestic horse breeds. MI and NF horses form a cluster, set off from the other breeds. The remaining breeds form a somewhat loosely defined cluster, with the closest relationship being between TBs, TKs and ARs, and the others branching off from this set. This arrangement probably reflects the similarity in early origins of most breeds of domestic horses, with the evident phenotypic distinctions being produced by rather minor collections of genetic differences, as suggested by the population data analysis discussed above and in Table 2.1. Perhaps a better method to discriminate breed relationships (as compared with species

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relationships) will be provided when we can construct dendrograms based not on breed-based allelic frequency comparisons, but on allelic compositions of individuals within breeds, such as recently presented for cattle by MacHugh et al. (1998). Such studies are more likely to reflect genetic disequilibrium effects that doubtless play a significant role in the comparatively recent formation of breeds.

Addressing questions of introgression between domestic breeds Discussions about horse breed purity have a long-standing history. Indeed, registered horses are often referred to as ‘purebred’, certainly implying a notion of purity, although the genetic meaning of that term is not clear. Parentage verification programmes, designed to address questions concerning firstgeneration pedigree errors, have only been available for about 25 years. Prior to that time, pedigrees were based on sire and dam assignments provided by the owners. Pedigree errors could not have been detected objectively, except when genetic exceptions to coat colour rules occurred (see Chapter 3, this volume) and owners or registries recognized them. As horse owners become knowledgeable about genetics, they are putting pressure on breed registries to address rumours about particular examples in past generations of prescribed introgression from other breeds. Using the well-characterized loci that have been identified to be effective for domestic animal parentage, testing has been used to investigate intra- as well as interbreed similarities and differences, as previously shown in this chapter. Most of the genetic variation within a breed is present in other breeds. Rare variants that are confined to one or a few related breeds are found (e.g. a D system phenogroup in Caspian ponies by Cothran and Long, 1994), but no breed-specific markers (akin to species-specific markers) have been identified. That is to say, no variants have been found that are present only in a single breed and possessed by every horse in that breed. Genetic differences between breeds for the most part are allelic frequency differences, originating from differences in selection schemes, founders or random drift, i.e. differences in breed histories, but not necessarily differences in earliest origins. Certain alternative pedigree proposals involving past generations may be addressed productively using mitochondrial and Y chromosome DNA haplotypes (polymorphisms of linked markers), which would be unaffected by genetic processes of segregation and recombination that confound the tracking of autosomal genes through extended pedigree lines. Mitochondrial haplotypes, strictly maternally inherited in the tail female line of the pedigree, have been used to resolve maternity controversies without access to biological material from the horses in question (Bowling et al., 1998). Assays for Y chromosome haplotypes, inherited strictly in the tail male line of the pedigrees of males, are not yet available for horses, but probably will be developed. Certain pedigree problems in remote generations could thus be addressed by stud book authorities if there were a compelling need to do so. It will be

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important that well-intentioned plans acknowledge the genetic principles that govern the likelihood for the transmission of factors through generations, the lack of biological definition for a breed, the role of crossbreeding in the development of nearly all breeds and the ancient shared origins of domestic horses. All these factors combine to hinder the simple detection of most cases of proscribed crossbreeding in remote generations.

Future Prospects for Domestic Horses All domestic animals in the last 100 years have been subjected to great pressures to evolve to meet the changing needs of a mechanized, commercialized, globalized planet. While horses may still have a working role in today’s society, with the development of combustion engines this role worldwide has become a minor one. Horses are still used for working with beef cattle worldwide, and by nomads and in subsistence agricultural societies, particularly in parts of eastern Europe, Africa, Asia, Central and South America. One of the emerging prominent uses for many breeds of horses is for competitive performance events or as personal sport companions (racing at short distance by trot, pace or gallop; long distance racing; jumping; precision riding; weight pulling; ranch task competition; pleasure riding; pleasure driving, and so forth). Increasingly the horse’s aesthetic value becomes a major aspect of its importance. As with breeds in other domestic species, some horse breeds may be threatened with extinction because they do not appear to meet a current need. Various breed conservation groups have identified threatened breeds and subpopulations and provide active encouragement for breeders to conserve them in order to preserve genetic diversity (see Chapter 14, this volume). The genetic health of breeds and of the species in general appears to be excellent, and the variety of phenotypes and traits available seem sufficient to meet the challenges of the next several centuries.

Acknowledgements Our thanks to C. LaBounty and M. Williams for preparing allelic frequency data and to M.C.T. Penedo for preparing the population data analysis of the ten breeds and Przewalski’s horse. We also thank M. Bowling for editorial assistance with preparation of this chapter.

References Anthony, D., Telegin, D.Y. and Brown, D. (1991) Origin of horseback riding. Scientific American 225, 44–48. Bannikov, A.G. and Flint, V.E. (1989) Order Perissodactyla. In: Sokolov, V.E. (ed.), Life of Animals. Mammals. Vol. 7. Prosveshenie, Moscow, pp. 418–422 (in Russian).

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Belyaev, D.K. (1979) Destabilizing selection as a factor of domestication. Journal of Heredity 70, 301–308. Bixby, D.E., Christman, C.J., Ehrman, C.J. and Sponenberg, D.P. (1994) Taking Stock: the North American Livestock Census. McDonald and Woodward, Blacksburg, Virginia. Bökönyi, S. (1984) Horse. In: Mason, I.L. (ed.), Evolution of Domesticated Animals. Longman, London, pp. 162–173. Bower, B. (1993) Ancient figurine lifts horses’ profile. Science News 143, 22. Bowling, A.T. (1985) The use and efficacy of horse blood typing tests. Equine Veterinary Science 5, 195–199. Bowling, A.T. and Clark, R.S. (1985) Blood group and protein polymorphism gene frequencies for seven breeds of horses in the United States. Animal Blood Groups and Biochemical Genetics 16, 93–108. Bowling, A.T. and Dileanis, S. (1990) Population data and a fourth allele for equine complement component 3 (C3). Animal Genetics 21, 83–86. Bowling, A.T. and Ryder, O.A. (1987) Genetic studies of blood markers in Przewalski’s horses. Journal of Heredity 78, 75–80. Bowling, A.T., Eggleston-Stott, M.L., Byrnes, G., Clark, R.S., Dileanis, S. and Wictum, E. (1997) Validation of microsatellite markers for routine horse parentage testing. Animal Genetics 28, 247–252. Bowling, A.T., DelValle, A. and Bowling, M. (1998) Verification of horse maternal lineage based on mitochondrial DNA sequence. Journal of Animal Breeding and Genetics 115, 351–356. Bradley, R.D. and Wichman, H.A. (1994) Rapidly evolving repetitive DNAs in a conservative genome: a test of factors that affect chromosomal evolution. Chromosome Research 2, 354–360. Brænd, M. (1979) Red cell and serum types of a Przewalskii horse. Animal Blood Groups and Biochemical Genetics 10, 61–62. Breeds of Livestock. Horses. (1996) Oklahoma State University, Stillwater, Oklahoma. http://www.ansi.okstate.edu/breeds/horses/ Breen, M., Downs, P., Irvin, Z. and Bell, K. (1994) Intrageneric amplification of horse microsatellite markers with emphasis on the Przewalski’s horse (E. przewalskii). Animal Genetics 25, 401–405. Budiansky, S. (1997) The Nature of Horses. Their Evolution, Intelligence and Behaviour. Weidenfeld & Nicolson, London. Clutton-Brock, J. (1987) A Natural History of Domesticated Animals. Cambridge University Press, British Museum (Natural History), Stockbridge, Hampshire, UK. Cothran, E.G. and Long, Y.G. (1994) A new phenogroup in the horse D system of red cell alloantigens found in the Caspian pony. Animal Genetics 25, 49–50. Dierenfeld, E.S., Hoppe, P.P., Woodford, M.H., Krilov, N.P., Klimov, V.V. and Yasinetskaya, N.I. (1997) Plasma alpha-tocopherol, beta-carotene, and lipid levels in semi-free-ranging Przewalski horses (Equus przewalskii). Journal of Zoo and Wildlife Medicine 28, 144–147. Dubrovskaya, R.M., Starodumov, I.M. and Bannikova, L.V. (1992) Genetic differentiation between horse breeds for polymorphic blood protein loci. Genetika 28, 152–165 (in Russian). Edwards, E.H. (1980) A Standard Guide to Horse and Pony Breeds. McGraw-Hill, New York. Ellegren, H., Johansson, M., Sandberg, K. and Andersson, L. (1992) Cloning of highly polymorphic microsatellites in the horse. Animal Genetics 23, 133–142.

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A.T. Bowling and A. Ruvinsky Felsenstein J. (1993) PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle. Fincham, D.A., Ellory, J.C. and Young, J.D. (1992) Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski’s horse (Equus przewalskii). Canadian Journal of Physiology and Pharmacology 70, 1117–1127. George, M., Jr and Ryder, O.A. (1986) Mitochondrial DNA evolution in the genus Equus. Molecular Biology and Evolution 3, 535–546. Groves, C.P. (1994) Morphology, habitat and taxonomy. In: Boyd, L. and Houpt, K.A. (eds), Przewalski’s Horse: the History and Biology of an Endangered Species. SUNY Press, Albany, New York, pp. 39–59. Hatami-Monazah, H. and Pandit, R.V. (1979) A cytogenetic study of the Caspian pony. Journal of Reproduction and Fertility 57, 331–333. Haywood, J., with Catchpole, B., Hall, S. and Barratt, E. (1997) The Cassell Atlas of World History. First published by Cassell plc. London. Andromeda Oxford Ltd, Abingdon, UK. Hendricks, B.L. (1995) International Encyclopedia of Horse Breeds. University of Oklahoma Press, Norman. Ishida, N., Oyunsuren, T., Mashima, S., Mukoyama, H. and Saitou, N. (1995) Mitochondrial DNA sequences of various species of the genus Equus with special reference to the phylogenetic relationship between Przewalski’s wild horse and domestic horse. Journal of Molecular Evolution 41, 180–188. Kaminski, M. (1979) The biochemical evolution of the horse. Comparative Biochemistry and Physiology 63B, 175–178. Kus, E. (ed.) (1997) General Studbook of the Przewalski Horse. Zoological Garden Prague, Prague. MacHugh, D.E., Loftus, R.T., Cunningham, P. and Bradley, D.G. (1998) Genetic structure of seven European cattle breeds assessed using 20 microsatellite markers. Animal Genetics 29, 333–340. Marklund, S., Ellegren, H., Eriksson, S., Sandberg, K. and Andersson, L. (1994) Parentage testing and linkage analysis in the horse using a set of highly polymorphic microsatellites. Animal Genetics 25, 19–23. Mason, I.L. (1996) A World Dictionary of Livestock Breeds, Types and Varieties, 4th edn. CAB International, Wallingford, UK. Oakenfull, E.A. and Ryder, O.A. (1998) Mitochondrial control region and 12 SrRNA variation in Przewalski’s horse (Equus przewalskii). Animal Genetics 29, 456–459. Olsen, S.J. (1988) The horse in ancient China and its cultural influence in some other areas. Proceedings of the Academy of Natural Sciences, Philadelphia, 140, 151–189. Ota, T. (1993) DISPAN (Genetic Distance and Phylogenetic Analysis) version 3.5c. Distributed by Institute of Molecular Evolutionary Genetics, Pennsylvania State University, University Park. Patterson, S.D., Bell, K. and Manton, V.J.A. (1990) Equus przewalskii plasma protease inhibitor (Pi) system. Animal Genetics 21, 129–139. Putt, W. and Whitehouse, D.B. (1983) Genetics of four plasma protein loci in Equus przewalskii: new allele at the prealbumin, postalbumin and transferrin loci. Animal Blood Groups and Biochemical Genetics 14, 7–16. Rudenko, S.I. (1970) The Frozen Tombs of Siberia (Thompson, M.W., transl.). J.M. Dent & Sons, London. Ryder, O.A., Trommershausen-Smith, A., Hansen, S.K.,Suzuki, Y., Sparkes, R.S., Clegg, J.B., Oosterhuis, J.E. and Nelson, L.S. (1979) Genetic variation in Przewalski’s

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horse, Equus przewalski, of the Munich line in the United States. In: DeBoer, L.E.M., Bouman, J. and Bouman, I. (eds), Genetics and Hereditary Diseases of the Przewalski’s Horse. Foundation for the Preservation and Protection of the Przewalski Horse, Rotterdam, pp. 41–60. Scott, A.M. (1979) Red-cell groups and serum types in the Przewalski horse (Equus przewalskii). In: DeBoer, L.E.M., Bouman, J. and Bouman, I. (eds), Genetics and Hereditary Diseases of the Przewalski’s Horse. Foundation for the Preservation and Protection of the Przewalski Horse, Rotterdam, pp. 33–40. Tikhonov, V.N., Cothran, E.G. and Knyazev, S.P. (1998) Population genetic parameters of aboriginal Yakut horses as related to the phylogeny of modern breeds of the domestic horse Equus caballus L. Genetika 34, 796–809 (in Russian). The Institute for Ancient Equestrian Studies (1997) Hartwick College, Oneontha, New York. http://www.hartwick.edu/anthropology/iaes.html The International Museum of the Horse (1998) Lexington, Kentucky. http://www.imh. org/imh/imhmain.html Trut, L.N. (1997) D.K. Belyaev’s evolutionary concept – ten years later. Genetika 33, 1060–1068 (in Russian). Trut, L.N. (1999) Early canid domestication: the farm-fox experiment. American Scientist 87, 160–169. Wichman, H.A., Payne, C.T., Ryder, O.A., Hamilton, M.J., Maltbie, M. and Baker, R.J. (1991) Genomic distribution of heterochromatic sequences in equids: implication to rapid chromosomal evolution. Journal of Heredity 82, 526. Wijers, E.R., Zijstra, C. and Lenstra, J.A. (1993) Rapid evolution of horse satellite DNA. Genomics 18, 113–117. Zeuner, F.E. (1963) A History of Domesticated Animals. Hutchinson, London.

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Genetics of Colour Variation A.T. Bowling 3Coat colour A.T.genetics Bowling Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis CA 95616–8744, USA Introduction Basic Colours Grey Chestnut Bay/black (Agouti) Colour Dilution Genes Cream dilution Dun Champagne Silver (dapple) White and White Spotting Genes White Roan Tobiano Overo Leopard (tiger, appaloosa) spotting Conclusions References

53 57 57 58 59 59 59 60 61 62 63 63 64 65 66 67 68 68

Introduction When we compare the individuals of the same variety or sub-variety of our older cultivated plants and animals, one of the first points which strikes us is, that they generally differ more from each other than do the individuals of any one species or variety in a state of nature. (Darwin, 1859, The Origin of Species by Means of Natural Selection or the Preservation of Favoured Races in the Struggle for Life)

Coat colours of domestic horses provide abundant evidence for the aptness of this first sentence of Darwin’s magnum opus. Black, bay, chestnut and grey are the basic colours occurring in nearly all breeds, produced by the interactions of three genes. To account for colour dilution, white and white patterns, at ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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least nine more genes are proposed. No colour in horses appears to be confined to a single breed, suggesting that the mutations producing the colour variants occurred early in the domestication time frame, before the development of modern breeds. Although a genetic definition of some colours has only occurred in the last decade (e.g. champagne and overo, see later), there is no evidence that any colour category has occurred by new mutation within recorded history. Genes controlling horse colours are outlined in Table 3.1. The basic set of horse colours, black, bay, chestnut and grey, can be described by the actions of alleles of the genes Agouti, Extension and Grey (A, E and G). The collection of colours is extended in some breeds with colour dilution genes Cream, Dun, Champagne and Silver (C, D, CH and Z) and with white pattern genes White, Roan, Tobiano, Overo and Leopard Spotting (W, RN, TO, O and LP). Examples of the names (phenotypes) given to the gene combinations (genotypes) are provided in Table 3.2. Among breeds in which nearly all horses can be described by the basic colours alone are Thoroughbred, Lipizzaner and Arabian. Breeds that extend the basic colour set to include colour dilution genes include the Quarter Horse, Morgan and Lusitano. The basic colours, the colour dilutions and white spotting patterns are found among American Saddlebreds, Miniature Horses Tennessee Walking Horses and Paso Finos. Some breed societies (e.g. Paint, Pinto, Palomino and Curly) focus on horses with particular colour, pattern or hair texture traits and may include horses registered with more than one association. Unlike dog and cattle breeds, colour uniformity is seldom a breed trait for horses, the few exceptions being Exmoor (dark bay/brown with mealy muzzle, eye rings, underbelly), Suffolk (chestnut), Haflinger (sorrel), Cleveland Bay (bay) and Friesian (black). The horse has not contributed significantly to the general advancement of the science of genetics. It is a poor candidate to be an experimental organism for genetics due to basic biological characteristics that are limiting to genetic studies, including single births, long gestation period, requirements for substantial physical space and the requirement for handling by experienced personnel. However, horses provided data for genetic analysis significant in the rediscovery of Mendel’s principles. Analysis of coat colour inheritance for grey, black, bay and chestnut Thoroughbred horses recorded in Weatherby’s General Stud Book was presented by Hurst (1906) to the Royal Society to validate the principles of genetics defined by Mendel in 1865. Hurst’s studies were followed by those of prominent early geneticists much better known for their work with species other than horses, such as Sturtevant (1910), Wright (1917), Dobzhansky (1927) and Castle (1948). They confirmed Hurst’s proposals and continued the theme of conducting genetic research on horses using available breeding records, not experimental breeding programmes.

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Coat colour genes and symbols of horses. Locus (symbol)

Alleles (symbol) Description

Any colour, progressively changing to white with age Any colour, not progressively whitening with age Black (eumelanin) pigment in skin and hair Chestnut Extension Black (eumelanin) pigment in skin, red/yellow (E) (phaeomelanin) pigment in hair Black pigment distributed in a points pattern (mane, tail, Bay/black Agouti (A) lower legs) Black pigment uniformly distributed black (a) Red pigment diluted to yellow in heterozygotes; in homoPalomino/ Cream (C) colour dilution zygotes both red and black pigment are diluted to ivory buckskin/ (Ccr) No colour dilution full colour (C) cremello/perlino On body, red pigment diluted to light red and black to Dun Dun (D) colour dilution grey; points not diluted. Colour dilution accompanied by (D) striping pattern on back, upper legs and withers No colour dilution full colour (d) Red pigment diluted to yellow, black pigment to brown or Champagne Champagne colour dilution olive; both with metallic sheen (CH) (CH) full colour (ch) No colour dilution Black pigment diluted to chocolate; minimal effect on red Silver Silver (Z) colour dilution pigment. (Z) No colour dilution full colour (z) Born white (white hair, pink skin, dark eyes). White White (W) white (W) Homozygous lethal Full colour full colour (w) Hair is mixture of white and any other colour. Points Roan Roan (RN) roan (RN) usually dark Full colour not roan (rn) White spotting characterized by large white spots in Tobiano Tobiano tobiano (TO) vertical pattern, (usually) four white legs, white crossing (TO) dorsal line. Homozygotes usually have clusters of small spots in white areas not tobiano (to) No white spotting White spotting characterized by horizontal pattern, Overo Overo (O) overo (O) (usually) dark legs, white not crossing dorsal line. Homozygous lethal (white) No white spotting not overo (o) Leopard spotting Leopard leopard spotting Also known as appaloosa or tiger spotting. Variable pattern of roaning and spotting accompanied by mottled Spotting (LP) skin, eyes showing white sclera. Homozygotes overall (LP) whiter than heterozygotes No white spotting not leopard spotting (lp) Grey

Grey (G)

grey (G) not grey (g) eumelanin (E) phaeomelanin (e) bay (A)

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A.T. Bowling Putting it all together: colour genotypes and phenotypes. Colour genes

Coat colour (phenotype)

White Grey

Black/red

Dilution

Pattern

W

G

E

A

C

CH

D

Z

TO

O

LP

RN

White Grey

Ww ww

~ ~

~ ~

~ ~

~ ~

~ ~

~ ~

~ ~

~ ~

~ ~

~ ~

Bay

ww

~ GG Gg gg

chch

dd

zz

toto

oo

lplp

rnrn

ww

gg

CC

chch

dd

zz

lplp

rnrn

ww

gg

CC

chch

dd

zz

TOto TOTO toto

oo

Bay varnish roan

oo

LPlp

rnrn

Black

ww

gg

AA Aa AA Aa AA Aa aa

CC

Bay tobiano

CC

chch

dd

zz

toto

oo

lplp

rnrn

Champagne (classic) Black roan

ww

gg

aa

CC

zz

toto

oo

lplp

rnrn

gg

aa

CC

CHCH CHch chch

dd

ww

dd

zz

toto

oo

lplp

RNrn

Chestnut Palomino Palomino (champagne) Palomino overo Buckskin tobiano/overo Red few-spot leopard Red dun

ww ww ww

gg gg gg

EE Ee EE Ee EE Ee EE Ee EE Ee EE Ee ee ee ee

~ ~ ~

dd dd dd

zz zz zz

toto toto toto

oo oo oo

lplp lplp lplp

rnrn rnrn rnrn

ww ww

gg gg

zz zz

lplp lplp

rnrn rnrn

CC

chch

dd

zz

toto TOTO TOto toto

Oo Oo

gg

~ AA Aa ~

dd dd

ww

ee EE Ee ee

CC chch CcrC chch CC CHCH CHch CcrC chch CcrC chch

oo

LPLP

rnrn

ww

gg

ee

~

CC

chch

zz

toto

oo

lplp

rnrn

Cremello Cremello Grulla

ww ww ww

gg gg gg

~ ~ aa

~ ~ zz

~ ~ toto

~ ~ oo

~ ~ lplp

~ ~ rnrn

Silver dapple

ww

gg

~ ~ EE Ee EE Ee

DD Dd ~ ~ DD Dd cc

ZZ Zz

toto

oo

lplp

rnrn

aa

CcrCcr chch CcrC CHch CC chch CC

chch

All horses will have a pair of alleles for each of the 12 colour and pattern genes listed here. This chart shows the assignment of genotypes by phenotype observation. For each phenotype with more than one genotype choice and for all boxes with a ‘~’, assignment of genotype requires information from parents or offspring (modified from Bowling, 1996).

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Basic Colours In mammals, melanin is the most important pigment of coat colour. It occurs as pigment granules in melanocytes, which are cells that originate in the early embryo in the neural crest region and migrate ventrally and distally. In adult animals, melanocytes are found in hair follicles, skin, iris and some internal tissues. Coat colour variation is produced by genes that alter the basic pigment type in melanocytes, or the presence, shape, number or arrangement of pigment granules. Melanin occurs in two related forms: eumelanin (black or brown) and phaeomelanin (red or yellow) (Searle, 1968). The biochemistry of pigment production in the horse is homologous to that of other species (Woolf and Swafford, 1988). The candidate genes for controlling the eumelanin/ phaeomelanin switch producing the colours chestnut, bay and black are Extension (E) and Agouti (A). The gene for grey colour, whose action causes progressive destruction of melanin with age, is yet to be identified but conventionally is symbolized as G. The genes controlling the basic colours appear to be unlinked and form an epistatic series from G to E and A.

Grey The inheritance of grey colour as a dominant trait was defined by the early geneticists. A young horse that has the progressive greying allele (G) can be born any colour, depending on the genes present at the other loci controlling coat colour. Soon after birth, a foal with the grey gene will begin to show intermixed white hairs, particularly about the head, that proportionally increase with age over all the body. At maturity, the hair coat will be a clear grey (the horse appears ‘pure’ white with dark skin) or grey with coloured flecks (‘fleabitten’, ‘mosquito-bitten’ or ‘speckled’). If flecking occurs, the small coloured spots will provide clues to the base coat colour that is obscured by the action of the grey allele. Dark pigment remains in the skin and eyes even when the hair colour is completely white. A horse that is not grey is assigned the pair of recessive alleles (gg). The predictable genetic behaviour of the Grey gene has been the basis for breed registrars to flag registration applications for grey foals without a grey parent to review the colours of parents and offspring and to seek information about possible alternative parents. Grey is found in horse breeds throughout the world, from ponies to draught horses. No genetic linkage has been reported between grey and any other trait, and no gene has been defined at the molecular level. Alleles with phenotypically similar effects on colour, three genes of mice warrant study as candidate genes: i.e. the genes for tyrosinase related protein 1 (Tyrp-1) (Brown), mast cell growth factor (Mgf ) (Steel) and Silver (Pmel17). (For information about the mouse or human genes proposed as homologous candidate genes in this chapter, consult the web sites from the Jackson Laboratory (www.informatics.jax.org) or Online Mendelian Inheritance in Man (www3.ncbi.nlm.nih.gov).)

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Chestnut The difference between horses with black hair pigment (blacks, browns and bays; also buckskins, duns and grullas) compared with those with red (chestnuts and sorrels; also palominos and red duns) has long been identified as a dominantly inherited trait. Based on phenotype, trait linkage and a proposed homology with mouse colour, Andersson and Sandberg (1982) suggested Extension as the candidate gene for the horse black/red switch. The dominant allele (E) extends the amount of eumelanin in the coat, accounting for the black-pigmented colours, and the recessive allele (e) diminishes black, accounting for the reds. The genetics of this trait are so robust that many breed registrars routinely flag new registration applications of any ‘not-red’ offspring from matings between two red parents for review of the accuracy of the information supplied. The honour of being the first molecular definition of a horse coat colour belongs to this gene – a mutation in the gene for melanocyte-stimulating hormone receptor (Melanocortin 1 Receptor) (MSHR, MC1R) (Marklund et al., 1996). The protein made by this gene is part of the melanocyte cell membrane and it binds a hormone that stimulates the cell to make eumelanin. Recessive mutants fail to bind the melanocyte-stimulating hormone, so only phaeomelanin, not eumelanin, is made. The difference between the black and red pigment in horses is produced by a single nucleotide change in the DNA sequence of T for C, resulting in an amino acid substitution of phenylalanine for serine in the first transmembrane protein domain. Among horses tested from a variety of domestic horse breeds, only two alternative DNA sequences for MC1R have been identified so far. A proposed allelic variant for dominant black (ED) has not been identified at the molecular level, nor have sequence differences been associated with variation of red hues from very dark to very light. The fox colour (red variant) of Przewalski’s horse has the same base substitution as found in red domestic horses (Bowling, unpublished observation). Chestnut colour occurs in breeds worldwide. Some breeders with specialized colour breeding programmes for black are using a diagnostic test for ‘red factor’ to obtain the genotypic status of young potential breeding stock, without the need for breeding trials. Breeds that embrace black colour as part of their distinctive image may be pressured to require such testing to purge the unwanted ‘red’ alleles from the breed gene pool by proscribing carriers as breeding stock. However, before any such measures are undertaken for the sake of a gene that has no adverse effects on health, the authorities in charge should make certain that they understand the potential of such actions on the breed’s gene pool – particularly if the breed consists of a relatively small number of breeding animals – and should explore less drastic alternatives, such as discouraging the breeding of carriers to each other. Chestnut belongs in the second group of linked genes described for the horse (linkage group II, LG II) (Sandberg and Juneja, 1978; TrommershausenSmith, 1978; Andersson and Sandberg, 1982; Andersson et al., 1983; Sponenberg et al., 1984). This linkage group is an interesting one that includes

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two other coat pattern genes Roan (RN) and Tobiano (TO) as well as genes controlling one mitochondrial and three serum proteins. The chromosomal assignment of LG II is to horse chromosome 3 (ECA3) (Raudsepp et al., 1996; Lear and Bailey, 1997; Godard et al., 1998; Lear et al., 1998).

Bay/black (Agouti) A major feature of black hair in horses is that it may be distributed either uniformly or in a particular ‘points’ pattern (black hair in the mane and tail and on the legs; but reduced or absent on the body). Black hair pattern characteristics in horses are proposed to be due to a gene known in other mammals as Agouti, but homology at the linkage or molecular level has not been established. The gene derives its name from a South American rodent with black-banded hairs. Since Agouti only controls the distribution pattern of eumelanin, its actions are obvious only in the presence of E. The action of the dominant allele (A) in horses is to cause the distribution of hair with eumelanin to be restricted to the points (perhaps reflecting underlying regional temperature differences). The recessive allele (a) does not restrict the distribution of black hair and, when homozygous in the presence of E, produces a uniformly black horse. Alternative alleles of Agouti (A+, At) have been proposed that might be responsible for some of the variation in pigment distribution or colour shades (light or dark bay or brown), but these proposals await confirmation by molecular studies. For most breeds, both bays and blacks are found, although allelic frequencies may vary considerably due either to founder input, selection or both. No gene linkage for Agouti with other traits has been reported, but fluorescence in situ hybridization studies (FISH) have placed the gene for Agouti signalling protein (ASIP) on ECA22 (Godard et al., 1998).

Colour Dilution Genes In association with the genes defining the basic colours, the colour dilution genes substantially augment the extensive array of possible colours for horses. However, compared with the basic colour genes, the colour dilution genes have a more restricted though still extensive breed distribution. The gene homologies for some of the variants currently are not obvious, but with the development of the horse gene map and the opportunities it provides for trait mapping, candidate genes may soon be identified through linkage testing.

Cream dilution Perhaps the most widespread and familiar of the horse colour dilution genes is the one that produces the golden body colour seen on dark-skinned

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palominos and buckskins. Palominos have white (flaxen) manes and tails while buckskins have black manes, tails and legs. Cremellos and perlinos also belong to the palomino/buckskin colour family. Cremellos have pink skin, blue eyes and ivory-coloured hair. Perlinos have the same features except that the mane and tail are slightly darker than the body. Odriozola (1951) proposed that an allele of the Colour (C) gene, which controls tyrosinase action and melanin production in other mammals, is responsible for the golden colour of palominos and buckskins, and the extreme dilution to cremello and perlino. The Ccr (Cream) allele shows incomplete dominance, diluting phaeomelanin (red) to yellow when heterozygous but having little or no effect on eumelanin (black). Heterozygotes for Cream (CCcr) are palomino if the basic colour is red (ee) and buckskin if bay (A–E–). Blacks (aa E–) with a single copy of the dilution gene may show only subtle dilution effects on coat colour (‘smoky’ black) and on eye colour (amber or hazel) (Adalsteinsson, 1974; Sponenberg, 1996). When the dilution allele is homozygous (CcrCcr), both eumelanin and phaeomelanin are diluted to pale ivory, producing the colours known as cremello and perlino. The Cream gene is found in many but not all breeds of horses and ponies. Progress in molecular biology has now identified several colour dilution genes leading to an ‘albino’ phenotype. The Tyrosinase (TYR) and Pink-eye Dilution (PED) genes, in particular, have alleles affecting pigmentation in other mammals (e.g. mice and humans), with graded effects from very slight colour dilution to extreme (albino) that should be considered as candidates for the Cream gene in horses. Dun The dominantly inherited Dun trait dilutes both eumelanin and phaeomelanin. Red body colour is diluted to pale red (claybank or red dun) or yellow red (buckskin dun); black body hair is diluted to mouse-grey (grulla) (Van Vleck and Davitt, 1977). In addition to pigment dilution, the gene produces a coat markings pattern sometimes called ‘primitive’ that includes dark head, dark points, dorsal stripe (list, eel stripe), shoulder stripes and leg bars. The gene symbol D conventionally is assigned to this trait; the alternative allele is assigned the symbol d. The D allelic effects can be confused with those of Ccr, and both breed registries and horse owners may have difficulties making the appropriate colour assignments. Reference to the colours of the parents may help to clarify the appropriate designation for the offspring. Dun affects clumping of pigment granules (Gremmel, 1939), thus providing an optical dilution effect, in contrast to Ccr which probably controls chemical alteration of pigment. D dilutes phaeomelanin pigment on the body to a pinkish-red, yellowish-red or yellow, but does not dilute phaeomelanin to flaxen on the mane and tail, as does Ccr. D dilutes eumelanin body pigment to mouse-grey, while Ccr does not affect it

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conspicuously. A significant characteristic of D is the striping pattern; however, faint striping patterns, which may occur even with non-diluted colours (genetic definition not yet proposed), may on rare occasions confound phenotypic differentiation between Ccr and D. Homozygosity for D does not produce extreme colour dilution nor does compound heterozygosity of Ccr and D. A red horse with both dilution genes may look like a palomino with indistinct dun markings – an analysis of progeny colours may be necessary to confirm that both genes are present. Neither the linkage group nor a candidate gene homology for Dun has been determined. While the current gene symbol suggests a homology with Dilute in mice, the similarity has not been investigated. A prominent breed example with the conspicuous presence of the dun pattern is the Norwegian Fjord. The dun colour is generally found in breeds that also have other dilution genes, so it is important to attempt to distinguish the variants of these genes both alone and in combination.

Champagne Although only recently described in Tennessee Walking Horses (TWHs) (Sponenberg and Bowling, 1996), this coat colour dilution is unlikely to be a new mutation or to be confined to that breed. In addition to colour dilution (eumelanin to olive or chocolate and phaeomelanin to yellow or gold), the action of the gene produces mottled grey skin, a metallic sheen to the hair, and eye colour that is blue at birth but darkens to hazel with age. Depending on the basic colour gene background, the effect of Champagne can be to produce a colour that appears similar to palomino (or buckskin), and the appropriate genotype may fail to be assigned unless the distinctive characteristics of Champagne are recognized. The effect of Champagne with black produces a distinctive olive-hued metallic colour (classic champagne) which is unlikely to have an alternative nomenclature in traditional breed registry colour schemes. The trait is due to the action of a dominant gene for which the gene symbol CH has been proposed (alternative allele ch) (Sponenberg and Bowling, 1996). In combination with Ccr, the genes show additive interaction and the resulting colour appears to be indistinguishable from a ‘cremello’. Cream and Champagne are not allelic, based on the anecdotal evidence that a ‘cremello’ stallion known to be the offspring of palomino and champagne parents sired non-dilute offspring. No linkage information for Champagne is available. The candidate genes for this colour will be similar to those possible for the other dilute colours. Only TWHs and Half-Arabians include the effects of this gene in recorded colour assignments of individual horses, but the gene also occurs in Quarter Horses and Paint horses, and probably in others.

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Silver (dapple) A fourth colour-diluting gene, Silver, presents another confusing story, both because of inaccuracies presented with its early genetic description and because of gene effects that may make genotypic assignment difficult from phenotype alone. The popular name for the colour is based on the first scientific report (Castle and Smith, 1953), but it seems inappropriate to include the term ‘dapple’ as a part of the name of this colour since it is not always an aspect of the phenotype produced. Hence, the colour name at the beginning of this section has the word ‘dapple’ in parentheses. Another confusing point about silver concerns the origin of the colour. Castle and Smith proposed that it originated in the late 1800s among Shetland ponies, an origin that has been widely accepted. However, the occurrence of the colour in Icelandic Horses, likely to share an historical origin with Shetlands, but certainly with no interrelationship in the last several centuries, refutes Castle and Smith’s named founder gene source. The colour also occurs (rarely) in other breeds such as Quarter Horses. The gene effects of Silver are conspicuously seen with aaE– (black) horses in which the coat colour is diluted to a chocolate or black–chocolate, and the mane and tail are diluted to silver grey or flaxen. With AaE– (bay) horses, the gene produces colour dilution such that the horse is usually described as a silver-maned chestnut, i.e. a bay with the Silver gene could appear to be a chestnut (darker points than typically associated with chestnut, but for lack of a better category is often registered as chestnut). An obvious colour name for a bay with the Silver dilution is silver bay, but it is not in common usage. The gene probably has little effect on chestnut (phaeomelanin) coat colour, beyond producing a silver (flaxen) mane and tail. This colour is sometimes called silver sorrel, but it is difficult to distinguish visually from sorrel and, at times, probably because of the flaxen mane and tail, will be called palomino. Castle and Smith (1953) described an interaction of Silver with Grey leading to ‘white-born greys’, i.e. an early greying effect not seen in the interaction of Grey with other colour genes. The silver trait in horses is inherited as a dominant. Currently, Z is often used as the gene symbol for Silver (alternative allele z), but a standard terminology has not been adopted. Silver in other mammals (e.g. cats) describes a different colour dilution effect, so an Si symbol probably is not appropriate. The genetic linkage group of Silver in horses is undetermined and no homologous gene has been identified, but the various previously mentioned colour dilution genes of mice should be considered as well as that for tyrosinase-related protein 2 (Tyrp-2) (Slaty). Silver in horses has a restricted breed distribution compared with the other dilution genes. Its high frequency in breeds that have other dilution genes may lead to difficulties in genotype assignment based on phenotype (Miniature Horses), while in breeds in which Silver occurs at very low frequency, genotype assignment may be incorrect because its presence generally is not recognized as a possible source of phenotypic variation (Quarter Horses).

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White and White Spotting Genes Superimposed on the spectrum of colour variants are patterns of white body spotting. (The common patterns of white markings on the extremities (face and lower legs) that help provide characteristics for individual identification will not be discussed here.) The white pattern can either be in the shape of one big spot (white), a mixture of white and coloured hairs (roan), or discrete white spots of variable size, extent and location (tobiano, overo, leopard spotting). Compared with other coat colour categories, the inheritance of colours in this group may present substantial problems in genetic definition. It is perhaps with this group of colour traits that the failure of the horse to be a reasonable experimental organism for genetic studies is felt most acutely. The recent success (see below) with the molecular definition of an overo mutation and the subsequent understanding of the range of phenotypes it produces may foretell the importance of molecular genetics for sorting out this category of horse colours where breeding data have failed to yield enough information to provide satisfactory genetic hypotheses.

White White is a listed colour in Paints, TWHs and Miniature Horses, but occurs in other breeds as well. A white horse lacks pigment in skin and hair, but the eyes are usually dark brown. White colour in horses is likely to be a genetically heterogeneous category, due either to one or more single genes with major effects or to the additive effects of alleles at two or more white spotting genes. In mouse and pig, white colour is dominantly inherited and the responsible gene is linked to ALB. In mice, W has lethal or deleterious effects in homozygotes, associated with mutations in Kit, a gene encoding the tyrosine kinase transmembrane cellular receptor for the mast/stem cell growth factor. In pigs, white colour does not have deleterious effects in homozygotes and is produced by a duplication of the DNA sequence for KIT (Johansson Moller et al., 1996). The dominant inheritance of white colour in a group of horses was described in a classic colour genetics study (Pulos and Hutt, 1969). These authors provided convincing breeding data that the homozygous white WW genotype was lethal. Taken together, these various points of information lead us to anticipate that some examples of white colour in horses are likely to be genetically homologous to White in mice and pigs. Dark-eyed white horses may occur rarely as offspring of solid (dark) parents, for example in Thoroughbreds, Standardbreds and Arabians. At birth, these horses usually have pigmented hair on and around the ears, in the mane and on the back, but the pigment may disappear with age. White horses from dark parents transmit white as a dominantly inherited trait, although occasionally the ‘white’ offspring are not all-white like the parent, but rather have a substantial proportion of coloured hair patches. No linkage

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analysis has been performed for this trait, and the genetic relationship of these examples to those where white has been transmitted in an unbroken line for generations is not known. The recurrence of white within certain breeds seems significant. It calls to mind maize and mouse models of gene instability in certain genes and genetic backgrounds due to actions of retroposons or gene duplication that can lead to unstable alteration in gene sequence. A satisfactory understanding of this situation in horses probably awaits a molecular-based study. Another genetic scenario for white colour in horses involves the additive interaction of white spotting genes at different loci, especially Tobiano and Overo (Bowling, 1994). Obviously, this type of white could only occur in breeds in which both spotting genes occurred. The genotype of these horses is established most easily from a breeding record since it is not clear that they would necessarily be phenotypically distinguishable from W horses.

Roan The roan colour pattern of horses presents as an admixture of white hairs on any colour background, similar to the phenotype seen in young grey horses, but the coat colour of the roan horse does not whiten progressively with age as does the grey. In its classic expression, the roan horse has a 50% mixture of white and coloured hair on the body, but the head and lower legs are solid colour without mixture. Hair regrowth in areas of skin wounds may not show the white hair mixture, thus accentuating the appearance of scars (and brands) in the roan coat. Roan colour in cattle is associated with heterozygosity for a gene that when homozygous leads to white colour and genital abnormalities in females (white heifer disease). The roan gene of Belgian Blue cattle recently has been mapped and found to be linked to KIT, the same gene responsible for white colour in mice and pigs (Charlier et al., 1996). The roan trait in horses is present in a variety of breeds, but not in all. Roan is inherited as a dominant trait (RN; alternative allele rn), but the basic genetics are still controversial, perhaps because of genetic heterogeneity for this trait. Hintz and Van Vleck (1979) provided stud book data from Belgian horses in the USA suggesting that Roan is a homozygous lethal, although Geurts (1977) had earlier presented evidence supporting the existence of Belgian stallions in Europe homozygous for Roan. Through family studies, Andersson and Sandberg (1982) showed linkage of roan with ALB, GC and E in LG II. Based on phenotypic similarities and comparative mapping data, Marklund et al. (1999) explored the possibility of KIT as the candidate gene for Roan. While the causative mutation was not identified, a DNA sequence polymorphism in KIT was associated with roan colour in 24 of 33 roans in various breeds (Quarter Horse, Belgian, Connemara, New Forest, Shetland, Standardbred, Swedish Halfbred and Welsh), but exceptions were also observed (Belgian, Gotland, Shetland and Welsh). This study provided molecular evidence supporting KIT as a candidate gene for Roan and for genetic

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heterogeneity, but no definitive evidence to address the conflicting evidence concerning lethality of homozygotes. Neither TBs nor Arabians have the Roan gene discussed above, although roan is recognized as a colour category by registration authorities for both of these breeds. In TBs, the phenotype called roan is ascribed to a chestnut horse turning to grey (the bay or brown horse turning grey is the phenotype labelled grey). Roan in Arabians is probably associated with a currently undefined white markings pattern, perhaps an entity called sabino. Unlike with the traditional Roan gene, in Arabians a roan offspring can be produced by non-roan parents. Again, this colour category provides examples for which molecular definition may be needed to understand complex situations that cannot be sorted out easily by phenotypic observations.

Tobiano Tobiano is a dominantly inherited pattern of white spotting on any colour background found in certain breeds worldwide. The allelic symbol is TO (alternative allele is to). The pink-skinned pattern is present at birth, and is characterized by four white legs, white areas crossing the spine between the withers and croup, with minimal extent of white facial markings. White areas on the body generally are large, but are highly variable in extent, and only minimally symmetrical, probably due to actions of genes at other loci. The gene has been identified as belonging to LG II (Trommershausen-Smith, 1978), allowing a tentative proposal of candidate genes to be from the receptor tyrosine gene family, especially KIT (piebaldism). Notice that Tobiano is the third colour in this discussion to be linked to and possibly representing alleles of KIT (the others being White and Roan), perhaps not surprising considering the importance of this gene or gene region to colour genetics in other mammals including humans, mice, pigs and cattle. The tobiano trait has two unusual genetic features: (i) the occurrence of ‘secondary spotting’ in the white areas of most homozygotes, and (ii) phase conservation of the tobiano trait with its linked genes. The secondary spotting feature of homozygotes is called by breeders ‘ink spots’ or ‘paw prints’. Characteristically, Tobiano homozygotes have clusters of small spots of colour within the areas of white on the body. While minimal expression of this feature overlaps with small spots occasionally found in the white areas of heterozygotes, a dramatic association is seen between prolific spotting and homozygosity for the tobiano trait. The second unusual genetic feature of Tobiano is the linkage phase association of Tobiano with certain alleles of its linked genes and failure to detect evidence of recombination. From non-tobiano family studies, recombination between ALB and GC is estimated to be about 1% (Sandberg and Juneja, 1978). In Paint horses, nearly all tobianos have TO in linkage phase with a certain marker phase (haplotype) of the linked serum protein genes (ALB-B with GC-S), polymorphisms which are commonly found in non-tobiano horses

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as well (Bowling, 1987). The three other possible haplotypes account in aggregate only for about 2% of the Tobiano-bearing chromosomes (Bowling, unpublished data). The same predominant phase association of TO with ALB-B and GC-S apparently is also true for other breeds. This effect is clearly an example of linkage disequilibrium related to founder effect, but there is no documented or even substantial anecdotal evidence relating all tobiano horses. The genetic mechanism that is preventing recombination and tending to preserve a single marker phase association for Tobiano is yet to be defined. Although the molecular definition of Tobiano is not yet available, this dramatic example of phase association allows the application of marker-assisted selection to help breeders identify homozygotes (Bowling, 1987; Duffield and Goldie, 1998). However, marker analysis depends on the presence of heterozygous marker genotypes in the heterozygous tobiano parents. About 86% of matings between obligate heterozygous Paint horses are informative for zygosity analysis using ALB and GC markers (Bowling, unpublished observations). Progeny testing using a test cross (breeding to a non-tobiano mate) is an alternative option for zygosity diagnosis in situations where the linked markers are uninformative. Progeny testing is a reasonable proposition for stallions, but is not feasible for mares.

Overo Overo is a term applied to variable white spotting that is neither tobiano nor leopard spotting. Horses to which this colour term is applied may be recognized at birth as having moderate to extensive white markings, particularly on the face, and asymmetrical white patches on the sides of the neck and barrel. Also, this colour category may include horses with high white leg markings and extensive ventral white, either as a distinctive class or combined with characteristics more fitting the first description. Overo is likely to be a genetically heterogeneous colour category and, not surprisingly, the genetics literature about it provides inconsistent conclusions. Initially, Overo was said to be a recessive trait, due to the occurrence of ‘cropout’ foals in Quarter Horse matings that were valued as foundation stock for the Paint horse breed. The inheritance of overo colour under the recessive trait definition must have been confusing for breeders since it was clear that many overo Paint stallions sired foals as though heterozygous for a dominant gene (Bowling, 1994), and lethal white foals (presumptive homozygotes) were associated with breedings between overos (Smith, 1977; Vonderfecht et al., 1983; McCabe et al., 1990). The pathology of the lethal white foals included lack of intestinal ganglia along with the lack of pigment, similar to pathological findings in piebald lethal mice and in people with Hirschsprung disease. Molecular level analysis has defined the gene responsible for at least one of the overo patterns, providing a solid foundation of information for overo breeding programmes. Heterozygosity for a 2 bp substitution in the endothelin receptor B gene (EDNRB) is found in parents of lethal white foals,

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while the foals are homozygous for the mutation (Metallinos et al., 1998; Santschi et al., 1998; Yang et al., 1998). The dinucleotide mutation is TC to AG, which results in a substitution of lysine for isoleucine at amino acid 118 in the first transmembrane domain of the EDNRB protein. Among the non-overo Quarter Horse parents of cropout overos, in each case studied one parent was heterozygous for the Overo mutation, despite having markings that would not qualify it for registration as an overo (Metallinos et al., 1998). Thus, molecular definition has clarified the genetics of at least one category of overo. Breeders now have the option of using results of a diagnostic test to help breed horses with the overo pattern, while avoiding the risk of producing lethal white foals. The EDNRB mutant gene also occurs in Miniature Horses and Thoroughbreds. Not all colours called overo are defined by the mutation detected in EDNRB. This is to be expected since heterogeneity was already phenotypically obvious and breeding data supported the occurrence of more than one gene (Bowling, 1994). Additional candidate genes to be considered can be chosen from white spotting mutants in humans such as endothelin 3 (EDN3), RET proto-oncogene (RET) and PAX3 (Waardenburg syndrome).

Leopard (tiger, appaloosa) spotting This complex of spotting and diffuse roaning patterns of variable extent is known variously as leopard, tiger or appaloosa spotting. The pattern is dispersed relatively symmetrically over the hips, down and forward. Mottled skin, white sclera and striped hooves are also characteristic. The white pattern of an individual horse may increase in extent to about age 5, but at least some pattern manifestations are present at birth, such as mottled skin. The patterns can occur with any basic coat colour and with the other major spotting genes (Tobiano and Overo). The basic colour may be slightly diluted or darkened along with the spotting effects. Often the spots have a different texture from the surrounding coat, especially noticeable in winter coat. The origin of this pattern pre-dates written history, but is spread worldwide in pony, draught and light horse breeds such as Appaloosa, Miniature Horse, Pony of the Americas, Mongolian pony, Knabstrup and Noriker. Probably a single major gene acting as an incomplete dominant is responsible for all the patterns, with pattern diversity attributed to modifying genes (Sponenberg, 1982; Sponenberg et al., 1990). Homozygotes for the spotting gene usually have a greater extent of white than heterozygotes, and are popularly called few-spot leopards. The gene symbol LP has been proposed (alternative allele lp). Definition of gene linkage within a family or breed could help clarify whether a single gene produces the variety of patterns seen, but no linkage studies have been reported. A possible candidate gene to be considered for this spotting pattern is that for microphthalmia-associated transcription factor (MITF) (Vitiligo).

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Conclusions The early reports of the basic inheritance of coat colour variants of horses, although lacking in definition of colour dilution and white pattern genes, have proven to be substantially correct. The recent focus of horse coat colour research efforts includes the definition of horse colours in the context of mammalian gene homologies. Application of molecular techniques that identify the DNA sequence of genes is providing information that clearly ties the colour genes of horses with those of humans, mice and other mammals. The rapidly developing human–horse comparative chromosomal map (Raudsepp et al., 1996; and see Chapter 9) and the availability of chromosomeassociated gene markers for linkage mapping of traits (Shiue et al., 1999; and see Chapter 10) will allow the more efficient identification of homologous candidate genes. When the DNA sequence differences between alleles are known, diagnostic tests can be developed to define a horse’s colour genotype should an owner be interested in knowing the zygosity status of breeding animals or should a breed registrar need to have an objective colour definition. Horse coat colour genetics is likely to be entering a new era of rapid progress.

References Adalsteinsson, S. (1974) Inheritance of the palomino color in Icelandic Horses. Journal of Heredity 65, 15–20. Andersson, L. and Sandberg, K. (1982) A linkage group composed of three coat color genes and three serum protein loci in horses. Journal of Heredity 73, 91–94. Andersson, L., Sandberg, K., Adalsteinsson, S. and Gunnarsson, E. (1983) Linkage of the equine serum esterase (Es) and mitochondrial glutamate oxaloacetate transaminase (GOTm) loci. Journal of Heredity 74, 361–364. Bowling, A.T. (1987) Equine linkage group II: phase conservation of To with Al B and GC S. Journal of Heredity 78, 248–250. Bowling, A.T. (1994) Dominant inheritance of overo spotting in Paint horses. Journal of Heredity 85, 222–224. Bowling, A.T. (1996) Horse Genetics. CAB International, Wallingford, UK. Castle, W.E. (1948) The ABC of color inheritance in horses. Genetics 33, 22–35. Castle, W.E. and Smith, F.H. (1953) Silver dapple, a unique color variety among Shetland ponies. Journal of Heredity 44, 139–145. Charlier, C., Denys, B., Belanche, J.I., Coppieters, W., Grobet, L., Mni, M., Womack, J., Hanset, R. and Georges, M. (1996) Microsatellite mapping of the bovine roan locus: a major determinant of White Heifer disease. Mammalian Genome 7, 138–142. Dobzhanzky, T.K. (1927) K voprosi o nasledovanii mastei u Kirghizskoi loshadi (Russian). Bulletin of the Bureau of Genetics 5, 79–108. Duffield, D.A. and Goldie, P.L. (1998) Tobiano spotting pattern in horses: linkage of To with AlA and linkage disequilibrium. Journal of Heredity 89, 104–106. Geurts, R. (1977) Hair Colour in the Horse. J.A. Allen, London.

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Godard, S., Schibler, L., Oustry, A., Cribiu, E.P. and Guérin, G. (1998) Construction of a horse BAC library and cytogenetical assignment of 20 type I and type II markers. Mammalian Genome 9, 633–637. Gremmel, F. (1939) Coat color in horses. Journal of Heredity 30, 437–445. Hintz, H.F. and Van Vleck, L.D. (1979) Lethal dominant roan in horses. Journal of Heredity 70, 145–146. Hurst, C.C. (1906) On the inheritance of coat colour in horses. Proceedings of the Royal Society, Series B 77, 388–394. Johansson Moller, M., Chaudhary, R., Hellmen, E., Hoyheim, B., Chowdhary B. and Andersson, L. (1996) Pigs with dominant white coat color phenotype carry a duplication of the KIT gene encoding the mast/stem cell growth factor receptor. Mammalian Genome 7, 822–830. Lear, T.L. and Bailey, E. (1997) Localization of the U2 linkage group of horses to ECA 3 using chromosome painting. Journal of Heredity 88, 162–164. Lear, T.L., Coogle, L.D. and Bailey, E. (1998) Assignment of the horse mitochondrial glutamate oxaloacetate transaminase 2 (GOT2) and v-kit Hardy–Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) to horse chromosome 3 by in situ hybridization. Cytogenetics and Cell Genetics 82, 112–113. Marklund, L., Johanssonn Moller, M., Sandberg, K. and Andersson, L. (1996) A missense mutation in the gene for melanocyte-stimulating hormone receptor (MC1R) is associated with the chestnut coat color in horses. Mammalian Genome 7, 895–899. Marklund, S., Moller, M., Sandberg, K. and Andersson, L. (1999) Close association between sequence polymorphism in the KIT gene and the roan coat color in horses. Mammalian Genome 10, 283–288. McCabe, L., Griffin, L.D., Kinzer, A., Chandler, M., Beckwith, J.B. and McCabe, R.B. (1990) Overo lethal white foal syndrome: equine model of aganglionic megacolon (Hirschsprung disease). American Journal of Medical Genetics 36, 336–340. Metallinos, D.L., Bowling, A.T. and Rine, J. (1998) A missense mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome: an equine version of Hirschsprung disease. Mammalian Genome 9, 426–431. Odriozola, M. (1951) A los Colores del Caballo. Publicaciones del Sindicato Nacional de Ganaderia, Madrid. Pulos, W.L. and Hutt, F.B. (1969) Lethal dominant white in horses. Journal of Heredity 60, 59–63. Raudsepp, T., Frönicke, L., Scherthan, H., Gustavsson, I. and Chowdhary, B.P. (1996) Zoo-FISH delineates conserved chromosomal segments in horse and man. Chromosome Research 4, 1–8. Sandberg, K. and Juneja, R.K. (1978) Close linkage between the albumin and Gc loci in the horse. Animal Blood Groups and Biochemical Genetics 9, 169–173. Santschi, E.M., Purdy, A.K., Valberg, S.J., Vrotsos, P.D., Kaese, H. and Mickelson, J.R. (1998) Endothelin receptor B polymorphism associated with lethal white foal syndrome in horses. Mammalian Genome 9, 306–309. Searle, A.G. (1968) Comparative Genetics of Coat Colour in Mammals. Logos Press, London. Shiue, Y.-L, Bickel, L.A., Caetano, A.R., Millon, L.V, Clark, R., Eggleston, M.L., Michelmore, R., Bailey, E., Guerin, G., Godard, S., Mickelson, J.R., Valberg, S.J., Murray, J.D. and Bowling, A.T. (1999) A synteny map of the horse genome comprised of 240 microsatellite and RAPD markers. Animal Genetics 30, 1–9. Smith, A.T. (1977) Lethal white foals in matings of overo spotted horses. Theriogenology 8, 303–312.

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A.T. Bowling Sponenberg, D.P. (1982) The inheritance of leopard spotting in the Noriker horse. Journal of Heredity 73, 357–359. Sponenberg, D.P. (1996) Equine Coat Color Genetics. Iowa State University Press, Ames. Sponenberg, D.P. and Bowling, A.T. (1996) Champagne, a dominant colour dilution of horses. Genetics, Selection, and Evolution 28, 457–462. Sponenberg, D.P., Harper, H.T. and Harper, A.L. (1984) Direct evidence for linkage of roan and extension loci in Belgian horses. Journal of Heredity 75, 413–414. Sponenberg, D.P., Carr, G., Simak, E. and Schwink, K. (1990) The inheritance of the Leopard complex of spotting patterns in horses. Journal of Heredity 81, 323–331. Sturtevant, A.H. (1910) On the inheritance of colour in the American harness horse. Biological Bulletin 19, 204–216. Trommershausen-Smith, A. (1978) Linkage of tobiano coat spotting and albumin markers in a pony family. Journal of Heredity 69, 214–216. Van Vleck, L.D. and Davitt, M. (1977) Confirmation of a gene for dominant dilution of horse colors. Journal of Heredity 68, 280–282. Vonderfecht, S.L., Bowling, A.T. and Cohen, M. (1983) Congenital intestinal aganglionosis in white foals. Veterinary Pathology 20, 65–70. Woolf, C.M. and Swafford, J.R. (1988) Evidence for eumelanin and phaeomelanin producing genotypes in the Arabian horse. Journal of Heredity 79, 100–106. Wright, S. (1917) Color inheritance in mammals. VII: the horse. Journal of Heredity 8, 561–564. Yang, G.C., Croaker, D., Zhang, A.L., Manglick, P., Cartmill, T. and Cass, D. (1998) A dinucleotide mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome (LWFS); a horse variant of Hirschsprung disease (HSCR). Human Molecular Genetics 7, 1047–1052.

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Genetics of Morphological Traits and Inherited Disorders F.W. Nicholas 4Morphological F.W. Nicholas Traits and Inherited Disorders Department of Animal Science, University of Sydney, NSW 2006, Australia Introduction The Range of Possibilities Previous Reviews Current Sources of Information An Overview Conclusion Acknowledgements References and Reviews

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Introduction Less is known about the inheritance of morphological traits and inherited disorders in horses than in the other major domesticated animal species, primarily because of the relatively high cost and long time involved in investigating inheritance patterns in horses. However, some important discoveries have been made, most notably at the molecular level in recent years. The main aim of this chapter is to present a summary of equine morphological traits and inherited disorders for which there is substantial evidence of single-gene inheritance. A brief mention is also made of other traits for which there is strong evidence of familial occurrence, but for which single-locus inheritance has not been established. Further discussion on inherited disorders associated with coat colour is presented in Chapter 3, and additional detail on other inherited disorders can be found in Chapter 8. With the molecular revolution now in full swing, and, in particular, with the development of gene markers covering all regions of all bovine chromosomes (see Chapter 10), knowledge of the inheritance of morphological traits and disorders in horses will increase rapidly in the decades ahead. As described below, regularly updated information on the subject matter of this chapter is available on the Internet. By this means, it is possible for readers ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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throughout the world to obtain the latest information on any single-locus trait or inherited disorder in horses.

The Range of Possibilities The spectrum of morphological traits and inherited disorders ranges from those that are definitely due to the action of just one gene, to those that are due to the combined action of many genes and many non-genetic (environmental) factors. In between these two extremes are many traits and disorders which appear to run in families, but for which there is insufficient information to enable a conclusion to be drawn about whether one or more genes are involved. Unfortunately, the literature abounds with examples of traits and disorders that have been claimed to be due to just one gene, despite the data being so sparse that such a claim cannot be justified. Similar problems exist with claims of inheritance being recessive or dominant; in most cases, there is insufficient information to justify the claims that have been made. In the fullness of time, of course, additional data might support the initial claims. However, we must be careful not to jump the gun. This scarcity of reliable data on the inheritance of traits and disorders poses a challenge to those who are asked to compile lists of such traits – as required for this chapter. No two reviewers will interpret the evidence in exactly the same way, and we must expect that lists of single-locus traits and disorders compiled by different authors will differ at the margins. As more data become available, these differences will be resolved.

Previous Reviews Several comprehensive reviews of inherited traits and disorders in horses have been published over the years. The first major summary was by Wriedt (1926). Since then, there have been many surveys published – these are marked with an asterisk in the list of references. It should be noted that some of these reviews are concerned with congenital traits and disorders, i.e. traits and disorders that are present at birth. Not all such traits and disorders are inherited.

Current Sources of Information While a list of reviews is useful, it is even more useful to have a single catalogue of morphological traits and inherited disorders that is updated regularly, and which is made available both electronically (on the Internet) and in hardcover (book) form on a regular basis. Human geneticists have long had access to such a resource – McKusick’s Mendelian Inheritance in Man (MIM). Now in its 12th edition as a book (McKusick, 1998), and accessible on the Internet as Online Mendelian Inheritance in Man (OMIM) at

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http://www3.ncbi.nlm.nih.gov:80/omim/, this catalogue contains a wealth of information on thousands of morphological traits and inherited disorders in humans. In 1978, the present author commenced compiling a catalogue of inherited traits and disorders in a wide range of animal species. Being modelled on, and complementary to, McKusick’s catalogue, this catalogue is called Mendelian Inheritance in Animals (MIA). It is accessible on the Internet as Online Mendelian Inheritance in Animals (OMIA) at http://www.angis.su.oz.au/ Databases/BIRX/omia/ in the same format as OMIM, and at http://ars-genome. cornell.edu/aboutomia.html in a different format. OMIA includes entries for all inherited disorders in horses, together with other traits in horses for which single-locus inheritance has been claimed, however dubiously. Each entry consists of a list of references arranged chronologically, so as to present a convenient history of knowledge about each disorder or trait. For some entries, there is additional information on inheritance or molecular genetics. If the disorder or trait has a human homologue, the relevant MIM numbers are included, providing a direct hyperlink to the relevant entry in McKusick’s online catalogue OMIM. OMIA is updated regularly.

An Overview Table 4.1 provides a list of single-locus morphological traits and inherited disorders that have been reported in horses – a total of 17 – together with the earliest report plus the most recent reference, extracted from MIA. The entries have been divided into two categories: those that have been characterized at the molecular level, and the rest. Obviously, each of these lists will expand as new discoveries are made. A complete set of up-to-date references for all entries for horses, including the many traits and disorders for which information is still too incomplete to signify single-locus inheritance, can be obtained by searching for horse in OMIA on the Internet. Table 4.2 presents a list of disorders that are familial, but for which there is insufficient evidence at present to justify their inclusion in Table 4.1. It is readily acknowledged that the information in OMIA is incomplete, and that it included errors of omission and commission. However, one of the advantages of having this type of information stored in a database is that errors can be rectified easily as soon as they are spotted. The author would therefore be very grateful to any readers who identify errors in the information supplied in this chapter.

Conclusion The list of inherited morphological traits and disorders presented in this chapter provides an indication of the range of such traits and disorders that (Cont’d on p. 80)

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Megacolon

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Also known as overo lethal white foal syndrome (LWFS) and overo spotting. In this disorder, the large Trommershausen-Smith (1977); Metallinos et al. (1998) intestine (caecum, colon and sometimes the rectum) undergoes a large dilation and fills with faecal mass. Associated with incomplete migration of nerve cells to the intestine during embryonic development, resulting in lack of peristalsis and hence a build-up of faecal material. Also associated with incomplete migration of melanoblasts: as with white spotting, the overo coat colour pattern is characterized by pigment spreading down both sides from the dorsal midline, giving way to lack of pigment (i.e. white) primarily on the ventral surfaces. Unfortunately, homozygosity for the overo allele results in white or nearly white foals which die within a few days of birth: the so-called lethal white foal syndrome (LWFS). The cause of death is intestinal obstruction resulting from a lack of nerve cells in the distal portion of the large intestine (aganglionic megacolon), which is thought to be due to a fault in the proliferation and/or migration of nerve stem cells from the neural crest of the developing embryo. Noting that mutations in the genes for endothelin 3 (EDN3) or its receptor (EDNRB) are responsible for similar disorders in humans and rodents, Santschi et al. (1998), from the University of Minnesota, sequenced cDNA from 22 affected foals, their parents and some solid-colour normal controls, all registered with the American Paint Horse Association. There was no variation in the EDN3 gene, but a dinucleotide substitution (TC→AG) at nucleotides 353–354 of the gene EDNRB, resulting in an Ile118Lys amino acid substitution, segregates perfectly with LWFS, thereby providing a molecular explanation for the disorder in American Paint horses. In quick succession, two other research groups reported similar findings: Metallinos et al. (1998), from the University of California, found the same mutation in other US horses; and Yang et al. (1998) found the same mutation causing the same disorder in Australian horses. Interestingly, both American studies found that some non-overo horses are carriers, suggesting that the mutation has incomplete penetrance. The overo coat colour is dominant to normal solid colour, but LWFS is recessive (i.e. the only horses to show this disorder are homozygotes for the overo allele). (See Chapter 3.)

Summary

A list of single-locus morphological traits and inherited disorders in horses (from Online Mendelian Inheritance in Animals).

Molecular defect known

Table 4.1.

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Cox (1985); Reynolds et al. Also known as hyperkalaemic periodic paralysis (HYPP) adynamia episodica hereditaria, Gamstorp disease and Impressive syndrome (after the sire from whom all recorded cases are direct descendants). (1998) First described only in the mid-1980s (Cox, 1985; Steiss and Naylor, 1986), this disorder provides an excellent example of how knowledge of comparative genetics can be put to good use in elucidating the molecular basis of disorders. In this particular case, the cause of the problem is a missense mutation in the gene encoding the α-chain of the adult skeletal muscle sodium channel, resulting in increased sodium permeability across the skeletal muscle cell membrane. Characterized by muscle fasciculation and spasm, weakness and recumbency. McGuire and Poppie (1973); Also known as agammaglobulinaemia, Swiss or alymphocytotic type; combined immunodeficiency Severe combined Bernoco and Bailey (1998) immunodeficiency disease; CID; and SCID. After many years of frustration for many researchers, the molecular basis of disease, autosomal SCID in horses was discovered by Shin et al. (1997), who used the candidate gene approach (based on work with SCID mice). They showed that SCID in horses is due to a frameshift mutation in the gene for DNA-dependent protein kinase catalytic subunit (DNA-PK), resulting in the lack of full-length kinase, and absence of kinase activity. Bailey et al. (1997) showed that the gene for DNA-PK is part of Kentucky synteny group 3. By FISH analysis, they also showed that this group physically maps to horse chromosome Eca9p12. Kent et al. (1986); Pailhoux et al. Sex reversal: Also known as gonadal hypoplasia; gonadal dysgenesis, XY female type; and Swyer syndrome. This (1995) XY female disorder is characterized by apparently normal female secondary sexual characteristics, but an XY karyotype in every cell. Pailhoux et al. (1995) showed that in the only case of this syndrome investigated at the molecular level in horses to date, the DNA-binding portion of the X-linked SRY gene was absent. Information gleaned from other species suggests that the SRY peptide is the initial signal causing the undifferentiated gonads in the developing embryo to go down the male path. In the absence of this trigger, the embryonic gonads develop down the female path, giving rise to female secondary sexual characteristics.

Periodic paralysis II

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Other single-locus traits and disorders Eriksson (1955) Aniridia with Absence of iris, plus presence of cataract, giving rise to a form of blindness reported in a Swedish cataract population of Belgian draught horses by Eriksson (1955), who provided convincing segregation data supporting autosomal dominant inheritance. Epitheliogenesis Congenital absence of the skin in certain parts of the body. Also known as aplasia cutis. Butz and Butz and Meyer (1957); Crowell imperfecta Meyer (1957) provided convincing evidence of autosomal recessive inheritance. et al. (1976) Exostosis, A disease in which benign tumours occur in many different bones, sometimes being present at birth. Morgan et al. (1962); Leone et al. multiple Gardner et al. (1975) presented pedigree data strongly suggesting autosomal dominant inheritance. (1987) Stockham et al. (1994) Stockham et al. (1994) provided convincing biochemical, pathological and clinical evidence of the Glucose-6deficiency of this enzyme in an American Saddlebred colt. Since this disorder is inherited as an X-linked phosphate recessive trait in other mammals, the application of Ohno’s law justifies the conclusion that this disorder dehydrogenase is X-linked in horses. deficiency Archer (1961); Littlewood et al. Haemophilia A Also known as classic haemophilia and HEMA. Characterized by impaired coagulability of the blood, (1991) with a consequential strong tendency to bleed, due to a deficiency of the clotting factor VIII. Since the gene for this factor is located on the X chromosome, the disorder is expected to be X-linked. As with haemophilia B, this expectation has been confirmed in all species of mammal in which the disorder has been reported. In humans, there are hundreds of different mutations that give rise to haemophilia A. In domesticated animals, the disorder has been reported in a large range of species, but in no case has the molecular basis yet been determined. Deprez et al. (1995) Hyperbilirubinaemia Also known as Gilbert’s syndrome. Bilirubin is an orange bile pigment produced by the breakdown of I haem and reduction of biliverdinin. An excess of bilirubin in the blood, resulting from a defect in uptake of organic anions by the liver, is called hyperbilirubinaemia I or Gilbert’s syndrome. Another anion unable to be taken up by affected livers is phylloerythrin, a metabolite of chlorophyll. The resultant high blood levels of phylloerythrin lead to photosensitive dermatitis.

Summary

Continued.

Molecular defect known

Table 4.1.

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Rhabdomyolysis

Lethal dominant white Myoclonus

Lethal dominant roan

Roan coat colour occurs at a low frequency (generally < 5%) in many horse breeds. Hintz and van Vleck (1979) provided convincing evidence that roan coat colour in Belgian horses is due to heterozygosity for an autosomal gene that is lethal when homozygous. Building on the results of recent work showing that dominant white coat colour in pigs is due to a mutation at the KIT gene (a proto-oncogene), Marklund et al. (1999) reported very strong evidence that the KIT gene in horses is the gene for roan coat colour: a linkage study revealed zero recombination between Roan and KIT, and a 79 bp insertion between exons 1 and 2 of the KIT gene, creating a frameshift mutation, was associated with the roan phenotype. However, definitive evidence of a causative mutation is still lacking. (See Chapter 3.) There are several different causes of white coat colour in horses (see Chapter 3). Pulos and Hutt (1969) provided convincing evidence that homozygous for dominant white results in embryonic lethality. Also known as myoclonus epilepsy of Unverricht and Lundborg, Baltic myoclonus epilepsy and progressive myoclonus epilepsy. This disorder is characterized by myoclonic jerks of the skeletal musculature, occurring spontaneously or in response to certain stimuli, due to the lack of inhibitory glycine receptors. Although definitive segregation data are yet to be published for horses, the similarity of the syndrome to inherited myoclonus in other mammals is sufficient to justify a conclusion of single-locus inheritance. Disintegration of striated muscle fibres, with excretion of myoglobin in the urine. The disorder is definitely familial, and there have been inclusive suggestions of autosomal recessive inheritance (Valberg et al., 1996). A recent analysis by MacLeay et al. (1999) supported autosomal dominant inheritance, with variable expressivity.

Valberg et al. (1993); MacLeay et al. (1999)

Castle (1954); Pulos and Hutt (1969) Gundlach et al. (1993)

Hintz and Van Vleck (1979); Marklund et al. (1999)

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von Willebrand disease

Ulnar and tibial malformation

Testicular feminization

Earliest reference; latest reference (if different from earliest reference)

Kieffer et al. (1976); Crabbe et al. Also known as androgen insensitivity syndrome, androgen receptor deficiency; dihydrotesterone (1992) receptor deficiency; and DHTR deficiency. This is an abnormality of sexual development in which affected individuals have an XY chromosomal constitution, undescended testes and female secondary sexual characteristics (including female external genitalia). Also, instead of normally developed Mullerian duct derivatives (Fallopian tubes, uterus, cervix and upper portion of the vagina), they have under-developed Wolffian duct derivatives (epididymis, vas deferens and seminal vesicle). In all species so far investigated, the inheritance is X-linked recessive. In humans and mice, this disorder is known to be due to a deficiency of an androgen receptor encoded by a gene on the X chromosome. The presence of a Y chromosome induces the undifferentiated embryonic gonads to develop as testes, but, in the absence of androgen receptor, the androgens produced by the testes cannot exert any effect. The result is that the embryo follows the default path of development, which is female. Speed (1958); Martens (1995) A disorder characterized by congenital malformation of ulnar and tibia, resulting in splayed legs and severe locomotion problems. Hermans (1970) provided evidence for autosomal recessive inheritance in Shetland ponies. Brooks et al. (1991); Smith et al. Also known as pseudohaemophilia and vascular haemophilia. von Willebrand factor (vWF) is a (1997) multimeric form of a plasma protein encoded by an autosomal gene (not yet mapped in pigs). vWF plays a vital role in platelet adhesion and clot formation. It also combines with factor VIIIC (the product of the X-linked haemophilia A locus), forming factor VIII. vWF accounts for 99% of the mass of factor VIII, its role is to protect factor VIIIC from degradation. von Willebrand disease (also called pseudohaemophilia or vascular haemophilia) is an autosomal bleeding disorder resulting from deficient or defective vWF.

Summary

Continued.

Molecular defect known

Table 4.1.

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Narcolepsy Osteochondrosis Patellar dislocation Stringhalt

Hemeralopia (night blindness) Laryngeal paralysis (roaring)

Degenerative myeloencephalopathy Epidermolysis bullosa, junctionalis

Degeneration of the laryngeal nerves (either unilateral or bilateral) leads to paralysis of the larynx, resulting in ‘snoring’ during exercise. Affected horses are unable to exercise to their full capacity Abrupt loss of voluntary muscular function Abnormal differentiation of growth cartilage Luxation (dislocation) of the patella, leading to lameness Involuntary repetitive exaggerated flexion of a hock

Blisters in and around the mouth and on the limbs, leading in the latter case to separation of the hoof from the corium (dermis), resulting in inability to stand Also known as red foot disease Impaired vision at night

Uncoordinated muscle movement resulting in unsteady and/or irregular gait. Many studies have shown familial occurrence Also known as occipital-atlanto-axial malformation (OAAM) A range of disorders of the cerebellum, including cerebellar abiotrophy, cerebellar degeneration and cerebellar hypoplasia Also known as collagen defect Both dominant and recessive inheritance have been reported, but the evidence is inconclusive Degenerative disease of the brain and spinal cord

Ataxia (wobbler disease, wobbles) Atlanto-occipital fusion Cerebellar disorders

Connective tissue disease Curly coat

Summary

Disorder

Lunn et al. (1993), Mignot and Dement (1993) Hultgren et al. (1988), Henson et al. (1997) Fischer and Helbig (1951), Engelbert et al. (1993) Hitenkov (1941)

Schaper (1939), Christley et al. (1997)

Witzel et al. (1977a), Witzel et al. (1977b)

Frame et al. (1988)

Hultgren et al. (1988), Miller and Collatos (1997)

Hardy et al. (1988) Blakeslee et al. (1943), Sponenberg (1990)

Dimock and Errington (1939), Tomizawa et al. (1994) Leipold et al. (1974), Mayhew et al. (1978) Palmer et al. (1973), Gerber et al. (1995)

Earliest reference; latest reference (if different from earliest reference)

Table 4.2. Disorders that show familial occurrence in horses, but for which there is insufficient evidence to justify a claim of single-locus inheritance (from Online Mendelian Inheritance in Animals, where further details can be found).

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have been observed and studied in horses. The molecular and gene mapping revolutions now underway will lead to an explosion of knowledge in this area in the years ahead. To exploit fully the genetic variation that does occur, breeders and researchers need continually to be on the look-out for unusual animals, saving them where possible. If DNA can be sampled from several generations of a family in which a particular morphological trait or disorder occurs, and if careful records on the occurrence of the trait or disorder in that family have been kept, it will be an increasingly straightforward matter to identify the gene responsible.

Acknowledgements Some of the information on hyperbilirubinaemia I was compiled by Philippa Beard. Some of the clinical descriptions were drawn from Blood and Studdert (1988).

References and Reviews (marked with an asterisk) *Agar, N.S. and Board, P.G. (eds) (1983) Red Blood Cells of Domestic Mammals, 1st edn. Elsevier, Amsterdam. Archer, R.K. (1961) True haemophilia (haemophilia A) in a thoroughbred foal. Veterinary Record 73, 338–340. Bailey, E., Reid, R.C., Skow, L.C., Mathiason, K., Lear, T.L. and McGuire, T.C. (1997) Linkage of the gene for equine combined immunodeficiency disease to microsatellite markers HTG8 and HTG4 – synteny and FISH mapping to ECA9. Animal Genetics 28, 268–273. *Ballarini, G. (1977) Hereditary diseases in veterinary practice. Rivista di Zootecnia e Veterinaria 2, 177–186. Bernoco, D. and Bailey, E. (1998) Frequency of the SCID gene among Arabian horses in the USA. Animal Genetics 29, 41–42. Blakeslee, L.H., Hudson, R.S. and Hunt, H.R. (1943) Curly coat in horses. Journal of Heredity 34, 115–118. Blood, D.C. and Studdert, V.P. (1988) Baillière’s Comprehensive Veterinary Dictionary. Baillière Tindall, London. *Bowling, A.T. (1996) Horse Genetics, 1st edn. CAB International, Wallingford, UK. Brooks, M., Leith, G.S., Allen, A.K., Woods, P.R., Benson, R.E. and Dodds, W.J. (1991) Bleeding disorder (von Willebrand disease) in a quarter horse. Journal of the American Veterinary Medical Association 198, 114–116. Butz, H. and Meyer, H. (1957) Epitheliogenesis imperfecta in foals. Deutsche Tierarztliche Wochenschrift 64, 555–559. Castle, W.E. (1954) Coat colour inheritance in horses and other mammals. Genetics 39, 35–44. Christley, R.M., Hodgson, D.R., Evans, D.L. and Rose, R.J. (1997) Cardiorespiratory responses to exercise in horses with different grades of idiopathic laryngeal hemiplegia. Equine Veterinary Journal 29, 6–10. *Cook, W.R. and Kirk, N.W. (1991) Hereditary diseases of the horse and their prevention. Irish Veterinary Journal 44, 59–66.

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Cox, J.H. (1985) An episodic weakness in four horses associated with intermittent serum hyperkalemia and the similarity of the disease to hyperkalemic periodic paralysis in man. Proceedings of the American Association of Equine Practitioners 21, 383–391. Crabbe, B.G., Freeman, D.A., Grant, B.D., Kennedy, P., Whitlatch, L. and Macrae, K. (1992) Testicular feminization syndrome in a mare. Journal of the American Veterinary Medical Association 200, 1689–1691. *Crew, F.A.E. and Buchanan-Smith, A.D. (1930) The genetics of the horse. Bibliographia Genetica 6, 123–170. Crowell, W.A., Stephenson, C. and Gosser, H.S. (1976) Epitheliogenesis imperfecta in a foal. Journal of the American Veterinary Medical Association 168, 56–58. Deprez, P., Sustronck, B., Vanloon, G. and Muylle, E. (1995) Persistent hyperbilirubinemia in a horse – a case report (Dutch). Vlaams Diergeneeskundig Tijdschrift 64, 212–215. Dimock, W.W. and Errington, B.J. (1939) Incoordination of equidae: wobblers. Journal of the American Veterinary Medical Association 95, 261–267. *Eaton, O.N. (1955) A summary of lethal characters in animals and man. Journal of Heredity 28, 320–326. Engelbert, T.A., Tate, L.P., Richardson, D.C., Honore, E.K. and Little, E.D.E. (1993) Lateral patellar luxation in miniature horses. Veterinary Surgery 22, 293–297. Eriksson, K. (1955) Hereditary aniridia with secondary cataract in horses. Nordisk Veterinaermedicin 7, 773–793. *Fischer, H. (1954) Lethal factors in horse and pigs. Tierarztliche Umschau 9, 50–51. Fischer, H. and Helbig, K. (1951) A contribution to the question of the inheritance of patella dislocation in the horse. Tierzucht 5, 105. Frame, S.R., Harrington, D.D., Fessler, J. and Frame, P.F. (1988) Hereditary junctional mechanobullous disease in a foal. Journal of the American Veterinary Medical Association 193, 1420–1424. Gardner, E.J., Shupe, J.L., Leone, N.C. and Olson, A.E. (1975) Hereditary multiple exostosis: a comparative genetic evaluation in man and horses. Journal of Heredity 66, 318–322. Gerber, H., Gaillard, C., Fatzer, R., Marti, E., Pfistner, B., Sustronck, B., Ueltschi, G., Meier, H.P., Herholz, C., Straub, R., Geissbuhler, U. and Gerber, V. (1995) Cerebellar abiotrophy in pure-bred Arabians. Pferdeheilkunde 11, 423–431. *Granier, P. (1955) Hereditary of Disease in Horses. Thesis, University, Paris (Alfort). *Grootenhuis, G. (1956) Horsebreeding and heredity: abortion, stillbirth and disease in foals. Proceedings of the Third International Congress of Animal Reproduction, Cambridge, Section II, 88–90. Gundlach, A.L., Kortz, G., Burazin, T.C.D., Madigan, J. and Higgins, R.J. (1993) Deficit of inhibitory glycine receptors in spinal cord from Peruvian Pasos – evidence for an equine form of inherited myoclonus. Brain Research 628, 263–270. Hardy, M.H., Fisher, K.R.S., Vrablic, O.E., Yager, J.A., Nimmo-Wilkie, J.S., Parker, W. and Keeley, F.W. (1988) An inherited connective tissue disease in the horse. Laboratory Investigation 59, 253–262. Henson, F.M.D., Davies, M.E. and Jeffcott, L.B. (1997) Equine dyschondroplasia (osteochondrosis) – histological findings and type VI collagen localization. Veterinary Journal 154, 53–62. Hermans, W.A. (1970) A hereditary anomaly in Shetland ponies. Netherlands Journal of Veterinary Science 3, 55–63.

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F.W. Nicholas Hintz, H.F. and Van Vleck, L.D. (1979) Lethal dominant roan in horses. Journal of Heredity 70, 145–146. Hitenkov, G.G. (1941) Stringhalt in horses and its inheritance. Vestnik Sel’skohozjaistvennoi Nauki, Zivotnovodstvo 2, 64. *Hollander, M. (1959) [Examples of the inheritance of defects in the horse]. Przeglad Hodowlany 27, 68–70. Hultgren, B.D., Appell, L.H., Wagner, P.C., Blythe, L.L., Watrous, B.J., Slizeski, M.L., Duffield, D.A., Goldie, P., Clarkson, D. and Shell, D. (1988) Current research topics in equine genetics, part 2. Equine Practice 10, 19–22. *Huston, R., Saperstein, G. and Leipold, H.W. (1977) Congenital defects in foals. Journal of Equine Medicine and Surgery 1, 146–162. *Jones, W.E. (1994) Genetics and disease in the horse. Journal of Equine Veterinary Science 14, 479–482. *Jones, W.E. and Bogart, R. (1971) Genetics of the Horse. Callabus Publishing, East Lansing, Michigan. Kent, M.G., Shoffner, R.N., Bouen, L. and Weber, A.F. (1986) XY sex-reversal syndrome in the domestic horse. Cytogenetics and Cell Genetics 42, 8–18. Kieffer, N.M., Burns, S.J. and Judge, N.C. (1976) Male pseudohermaphroditism of the testicular feminizing type in a horse. Equine Veterinary Journal 8, 38–41. *Koch, W. (1936) Some hereditary diseases in the horse and their practical significance. Munchener Tierarztliche Wochenschrift 87, 181. Leipold, H.W., Brandt, G.W., Guffy, M. and Blauch, B. (1974) Congenital atlanto occipital fusion in a foal. Veterinary Medicine and Small Animal Clinician 69, 1312–1316. Leone, N.C., Shupe, J.L., Gardner, E.J., Millar, E.A., Olson, A.E. and Phillips, E.C. (1987) Hereditary multiple exostosis. A comparative human–equine epidemiological study. Journal of Heredity 78, 171–177. *Lerner, I.M. (1944) Lethal and sublethal characters in farm animals. Journal of Heredity 35, 219. Littlewood, J.D., Bevan, S.A. and Corke, M.J. (1991) Haemophilia-A (classic haemophilia, factor-VIII deficiency) in a thoroughbred colt foal. Equine Veterinary Journal 23, 70–72. Lunn, D.P., Cuddon, P.A., Shaftoe, S. and Archer, R.M. (1993) Familial occurrence of narcolepsy in miniature horses. Equine Veterinary Journal 25, 483–487. MacLeay, J.M., Valberg, S.J., Sorum, S.A., Sorum, M.D., Kassube, T., Santshi, E.M., Mickelson, J.R. and Geyer, C.J. (1999) Heritability of recurrent exertional rhabdolyolysis in Thoroughbred racehorses. American Journal of Veterinary Research 60, 250–256. Marklund, S., Moller, M., Sandberg, K. and Andersson L. (1999) Close association between sequence polymorphism in the KIT gene and the roan coat colour in horses. Mammalian Genome 10, 283–288. Martens, P. (1995) Limb deviation in a Shetland pony foal. Equine Practice 17, 24–26. *Mauderer, H. (1938) Hereditary defects in the horse. Deutsche Tierarztliche Wochenschrift 46, 469. Mayhew, I.G., Watson, A.G. and Heissan, J.A. (1978) Congenital occipitoatlantoaxial malformations in the horse. Equine Veterinary Journal 10, 103–113. McGuire, T.C. and Poppie, M.J. (1973) Hypogammaglobulinemia and thymic hypoplasia in horses: a primary combined immunodeficiency disorder. Infection and Immunity 8, 272–277.

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McKusick, V.A. (1998) Mendelian Inheritance in Man. Catalogs of Human Genes and Genetic Disorders, 12th edn. Johns Hopkins University Press, Baltimore, Maryland. Metallinos, D.L., Bowling, A.T. and Rine, J. (1998) A missense mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome – an equine version of Hirschsprung-disease. Mammalian Genome 9, 426–431. Mignot, E.J.M. and Dement, W.C. (1993) Narcolepsy in animals and man. Equine Veterinary Journal 25, 476–477. Miller, M.M. and Collatos, C. (1997) Equine degenerative myeloencephalopathy. Veterinary Clinics of North America – Equine Practice 13, 43. Morgan, J.P., Carlson, W.D. and Adams, O.R. (1962) Hereditary multiple exostosis in the horse. Journal of the American Veterinary Medical Association 140, 1320–1322. Pailhoux, E., Cribiu, E.P., Parma, P. and Cotinot, C. (1995) Molecular analysis of an XY mare with gonadal dysgenesis. Hereditas 122, 109–112. Palmer, A.C., Blakemore, W.F., Cook, W.R., Platt, H. and Whitwell, K.E. (1973) Cerebellar hypoplasia and degeneration in the young Arab horse: clinical and neuropathological features. Veterinary Record 93, 62–66. *Priester, W.A. (1972) Congenital ocular defects in cattle, horses, cats and dogs. Journal of the American Veterinary Medical Association 160, 1504–1511. Pulos, W.L. and Hutt, F.B. (1969) Lethal dominant white in horses. Journal of Heredity 60, 59–63. *Rasbech, N.O. (1964) Fertility and reproductive disorders of various species of farm livestock in Denmark. British Veterinary Journal 120, 415–430. Reynolds, J.A., Potter, G.D., Greene, L.W., Wu, G., Carter, G.K., Martin, M.T., Peterson, T.V., Murraygerzik, M., Moss, G. and Erkert, R.S. (1998) Genetic–diet interactions in the hyperkalemic periodic paralysis syndrome in quarter horses fed varying amounts of potassium. I. Potassium and sodium balance, packed cell volume and plasma potassium and sodium concentrations. Journal of Equine Veterinary Science 18, 591–600. *Robinson, R. (1989) Genetic defects in the horse. Zeitschrift für Tierzuchtung und Zuchtungsbiologie 106, 475–478. Santschi, E.M., Purdy, A.K., Valberg, S.J., Vrotsos, P.D., Kaese, H. and Mickelson, J.R. (1998) Endothelin receptor B polymorphism associated with lethal white foal syndrome in horses. Mammalian Genome 9, 306–309. *Saunders, L.Z. (1952) A check list of hereditary and familial disease of the central nervous system in domestic animals. Cornell Veterinarian 42, 592–600. Schaper, U. (1939) On the inheritance of roaring in the horse. Deutsche Tierarztliche Wochenschrift 47, 385. Shin, E.K., Perryman, L.E. and Meek, K. (1997) A kinase-negative mutation of DNAPKCS in equine SCID results in defective coding and signal joint formation. Journal of Immunology 158, 3565–3569. Smith, J.M., Meyers, K.M., Barbee, D.D., Schott, H. and Bayly, W.M. (1997) Plasma von Willebrand factor in thoroughbreds in response to high-intensity treadmill exercise. American Journal of Veterinary Research 58, 71–76. Speed, J.G. (1958) A cause of malformation of the limbs of Shetland ponies with a note on its phylogenic significance. British Veterinary Journal 114, 18–22. Sponenberg, D.P. (1990) Dominant curly coat in horses. Genetics Selection Evolution 22, 257–260. Steiss, J.E. and Naylor, J.M. (1986) Episodic muscle tremors in a quarter horse: resemblance to hyperkalemic periodic paralysis. Canadian Veterinary Journal 27, 332–335.

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F.W. Nicholas Stockham, S.L., Harvey, J.W. and Kinden, D.A. (1994) Equine glucose-6-phosphate dehydrogenase deficiency. Veterinary Pathology 31, 518–527. *Stormont, C. (1958) Genetics and disease. Advances in Veterinary Science 4, 137–162. Tomizawa, N., Nishimura, R., Sasaki, N., Nakayama, H., Kadosawa, T., Senba, H. and Takeuchi, A. (1994) Relationships between radiography of cervical vertebrae and histopathology of the cervical cord in 19 wobbling foals. Journal of Veterinary Medical Science 56, 227–233. *Trommershausen Bowling, A. (1980) Counselling for genetic diseases of horses. Veterinary Clinics of North America – Large Animal Practice 2, 377–389. Trommershausen-Smith, A. (1977) Lethal white foals in matings of overo spotted horses. Theriogenology 8, 303–311. *Trommershausen-Smith, A. (1980) Aspects of genetics and disease in the horse. Journal of Animal Science 51, 1087–1095. *Tuff, P. (1948) The inheritance of a number of defects in the joints, bones and ligaments of the foot of the horse. Norsk Veterinaertidsskrift 60, 385. Valberg, S., Jonsson, L., Lindholm, A. and Holmgren, N. (1993) Muscle histopathology and plasma aspartate aminotransferase, creatine kinase and myoglobin changes with exercise in horses with recurrent exertional rhabdomyolysis. Equine Veterinary Journal 25, 255. Valberg, S.J., Geyer, C., Sorum, S.A. and Cardinet, G.H. (1996) Familial basis of exertional rhabdomyolysis in quarter horse-related breeds. American Journal of Veterinary Research 57, 286–290. Witzel, D.A., Joyce, J.R. and Smith, E.L. (1977a) Electroretinography of congenital night blindness in a filly. Journal of Equine Medicine and Surgery 1, 226. Witzel, D.A., Riis, R.C., Rebhun, W.C. and Hillman, R.B. (1977b) Night blindness in the Appaloosa: sibling occurrence. Journal of Equine Medicine and Surgery 1, 383–386. *Wriedt, C. (1926) The genetics of the horse. Bibliographia Genetica 6, 123–170. Yang, G.C., Croaker, D., Zhang, A.L., Manglick, P., Cartmill, T. and Cass, D. (1998) A dinucleotide mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome (LWFS) – a horse variant of Hirschsprung-disease (HSCR). Human Molecular Genetics 7, 1047–1052.

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Blood Groups and Biochemical Polymorphisms 1 and E.G. K. Sandberg 5Blood Groups K. Sandberg and andE.G. Biochemical Cothran Polymorphisms Cothran2 1Department of Animal Breeding and Genetics, Swedish University for Agricultural Sciences, Box 7023, S-750 07 Uppsala, Sweden; 2Department of Veterinary Science, University of Kentucky, Lexington, KY 40546–0099, USA

Introduction Blood Groups Early blood group studies Blood group systems (erythrocyte antigens) Blood cell chimaerism Neonatal isoerythrolysis Blood transfusion Biochemical Polymorphism Plasma and red cell proteins Milk proteins Utilization of the Blood Markers Parentage and identity testing Population studies References

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Introduction The finding that haemolytic disease of the newborn foal (neonatal isoerythrolysis, NI) is caused by transplacental immunization of the mare was a significant impetus for the growing interest in horse blood groups in the middle of this century (Caroli and Bessis, 1947). Also the introduction of immune sera as the main source of antibodies for blood group reagents at this time (Ferguson, 1941) gave support to studies of blood groups in horses and in other domestic animals. The advent of starch gel electrophoresis (Smithies, 1955) in combination with histochemical stains for proteins and enzymes opened up a new field of research in genetics. The technique made it possible to separate protein molecules differing in one or a few amino acids. The differences in amino acid ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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composition between protein molecules are the consequence of substitutions of nucleotides in the DNA molecule. Hence they are under strict genetic control. Studies on blood groups and protein polymorphisms soon revealed an unimagined amount of genetic variation between individuals in horses and in other animal species. Blood groups and protein polymorphisms found important applications in the horse breeding industry for parentage testing, for solving questions of alternative paternity and for the identification of individual horses. Blood typing gradually has become a routine procedure in horse breeding in several countries. The blood markers have also been utilized for the construction of genetic maps (Chapter 10), and for studies of breed relationships, origin of breeds and breed structure in horses. This chapter will give an overview of blood groups and protein polymorphisms in horses and of the various applications of these genetic markers.

Blood Groups Early blood group studies Since the classical discovery of the ABO blood group system of man (Landsteiner, 1901), numerous red cell antigens have been identified both in man and in animals. During the first half of this century, most studies on animal blood groups seemed to be based on the incorrect hypothesis that they had to fit into systems built up like the human ABO system. Therefore, the studies usually were confined to the use of naturally occurring antibodies. Furthermore, there was no comparison of antisera produced by different workers and no unified nomenclature was established. In horses, normally occurring isoantibodies that react with red cell antigens are quite rare. When present in a serum, they usually have a low titre and most often are of the specificities anti-Aa or anti-Ca (Stormont et al., 1964). Reagents prepared from normal sera give weak reactions, sometimes difficult to interpret. Early equine blood group studies have been reviewed by Podliachouk (1957), Adams (1958) and Bell (1983). The real impetus to animal blood group research came in the 1940s when pioneering work on cattle blood groups was carried out at the University of Wisconsin, Madison, USA (Ferguson, 1941; Ferguson et al., 1942; Stormont et al., 1951). Contemporary studies on equine blood groups began with the work of Podliachouk (1957). Using mainly naturally occurring antibodies, she was able to identify ten blood group factors in horses, designated A, C, D, E, F, G, H, I, J and K. She also initiated comparison tests in which the same panel of red cells was tested against reagents produced in different laboratories (Podliachouk et al., 1960; Podliachouk and Hesselholt, 1962). Franks (1959, 1962) produced 11 reagents using antisera from transplacentally immunized mares as his main source of antibodies. Based on their vast experience from studies on cattle blood groups, Stormont et al. (1964) and Stormont and Suzuki (1964) carried out fundamental

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work on the serology and genetics of blood groups in horses. They produced 16 reagents using horses and rabbits for immunizations. Six of these reagents were identical to reagents previously produced by other workers, while ten reagents did not have any known duplicates in other laboratories. By comprehensive family studies, they were able to assign the 16 blood group factors to eight blood group systems designated A, C, D, K, P, Q, T and U. The systems C, K, T and U were simple, one-factor, two-allele systems, while the systems A, D, P and Q involved two or more factors and proved to be controlled by multiple alleles. Today, all these systems, except the T system, are officially recognized. Since there are no known duplicates produced in any other laboratory of the reagents in the T system, it is not considered officially recognized (see below). Blood group studies in horses including the production of reagents have been published by several other workers, for example Hesselholt (1966), Schmid (1965), Sandberg (1970, 1979a, b), Watanabe and Noda (1970), Scott (1973), Suzuki (1978) and Meriaux and Podliachouk (1981). Every 2 years since the early 1970s, an international equine blood typing comparison test has been carried out under the auspices of the ISAG (International Society for Animal Genetics). The results of the test are discussed at the subsequent conference of the ISAG. All established laboratories in the world providing an equine blood typing service are members of ISAG and participate regularly in the comparison test. The function of the test is to compare and standardize results and nomenclature and to give international recognition to blood group factors. A blood group factor is considered officially recognized when at least two different laboratories, independently, have produced the antibody by which the factor is identified and identical test results are reported by the laboratories for at least 40 red blood cells (RBCs). Originally the horse blood group factors were designated by upper case letters with or without subscripts (e.g. A1) or superscripts (e.g. E ′) like the nomenclature still in use in bovine blood groups. In 1974 (Anonymous, 1974), a nomenclature based on the one used for porcine blood groups was adopted for horses. Each factor is designated by an upper case letter indicating the blood group system to which it belongs followed by a lower case letter indicating the individual factor (e.g. Aa) or by a dash (e.g. A–) when no factor is detected in the system.

Blood group systems (erythrocyte antigens) EAA system When first described, the A system comprised the factors A1, A′ and H (Aa, Ab and Ac, respectively, in the present nomenclature) controlled by five alleles Aa, Ab, Ac, Abc and A− (Stormont and Suzuki, 1964). Subsequently, four additional factors (Ad, Ae, Af and Ag) were identified (Suzuki, 1978; Meriaux and Podliachouk, 1981). For a long time, the factors Aa and Ab were considered to be controlled by allelic genes as are the factors Af and Ag (i.e. they were not

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thought to be transmitted together from parent to offspring). However, Bowling and Ewalt-Evans (1988) observed the allele Aabdg in the Arab and the Icelandic horse breeds and Bowling and Nickel (1992) found the allele Aabdf in the Paso Fino breed. Currently, seven factors altogether appear to define 12 alleles in the EAA (erythrocyte antigen A) system (Table 5.1). Anomalous transmission of the Ae factor was observed by Bowling and Ewalt-Evans (1988). The Aa factor is the most potent antigen among the equine blood group factors and the factor involved in a majority of NI cases in horses (Stormont, 1975). EAC, EAK and EAU systems Each one of these systems is a one-factor, two-alleles, two-phenotypes system comprising the alleles C a and C −, Ka and K− and U a and U −, respectively. Recently antisera were produced which define three subtypes of the Ua factor (Nogaj et al., 1997). EAD system The D system was described initially as a two-factor (D and J corresponding to Da and De, respectively, in the present nomenclature), three-allelic system (Stormont and Suzuki, 1964). Subsequently, no less than 15 additional factors have been detected which has expanded the EAD system to the most complex system in the horse (Sandberg, 1973b; Podliachouk and Meriaux, 1977, 1979; Bouquet et al., 1981; Scott, 1985; Bowling, 1987; Bowling and Williams, 1991) Table 5.1.

Blood group systems in horses.

Systems

Factors

Recognized alleles

EAA

a, b, c, d, e, f, g

EAC EAD

a a, b, c, d, e, f, g, h, i, k, l, m, n, o, p q, r

EAK EAP

a a, b, c, d

EAQ

a, b, c

EAU

a

Aa Aabdg Ac Ca Dadl Dbcmq Dcfgkm Dcgmp Dcgmr Ddelq Ddghmq Ddlnq Dq Ka Pa Pb Qa Qbc Ua

Aadf Ab Ace C−

Aadg Abc Ae Dadlnr Dcefgmq Dcfmqr Dcgmq Ddeklqr Ddfklr Ddghmqr Ddlnqr (D−)

K− Pac Pbd Qabc Qc U−

Aabdf Abce A− Dadlr Dcegimnq Dcgm Dcgmqr Ddeloq Ddghmp Ddkl Ddlqr

Pacd Pd Qac Q−

Pad P− Qb

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controlled by at least 25 alleles (Table 5.1). As new populations of horses are studied, new alleles (phenogroups) are found (e.g. Cothran and Long, 1994). After the detection of the Dq factor (Bowling and Williams, 1991), it is unsettled whether there is a null allele (D−) or not in the EAD system, but certainly the EAD system behaves as a closed system through the factors Dc and Dd, with only rare exceptions. Hence it is the most efficient blood group system for parentage control and for solving problems of disputed paternity in the horse (Bowling and Clark, 1985). EAP system The two factors P1 and P′ (Pa and Pb, respectively, in the present nomenclature) originally constituted the P system (Stormont and Suzuki, 1964). The four phenotypes Pa, Pb, PaPb and P– (no Pa or Pb) were shown to be controlled by the three alleles Pa, Pb and P−. A third factor X (Pd in the present nomenclature) was described by Suzuki (1978). Bowling and Williams (1985) reported family data and allele frequency data from seven horse breeds involving the recognized factors Pa, Pb, Pc (NF29) and Pd (X) and an additional factor (NF13) which has not yet had an official designation. The four factors Pa–Pd seemed to be controlled by eight alleles (Table 5.1), while two more alleles could be identified when NF13 was considered. EAQ system The horse blood group Q system originally comprised the three factors Q (Qa in the present nomenclature), R and S (Stormont and Suzuki, 1964). R and S turned out to be difficult to work with as the antibodies defining them were incomplete antibodies giving subthreshold reactions. Therefore, the anti-R and anti-S reagents have not been used extensively in studies of horse blood groups and the two factors have not been included in the official nomenclature. Two additional factors designated Qb and Qc were described by Sandberg (1979a). Currently the EAQ system seems to be controlled by seven alleles (Table 5.1). The Qa factor is a potent antigen and it is the second most common factor involved in NI in horses (Stormont, 1975). Blood cell chimaerism In about 40% of twin pregnancies in horses, vascular anastomosis (i.e. connections between blood vessels) between the two fetuses develop (Vandeplassche et al., 1970). A reciprocal exchange of primordial blood-forming cells takes place between the two fetuses, with these cells becoming established in the tissue of the co-twin and persisting into adult life. Such twins are called chimaeras and are known to occur also in cattle, sheep and other species. A blood cell chimaera has an admixture of two populations of blood cells; one that corresponds to its own genotype and one that corresponds to the genotype of its co-twin. For obvious reasons, this complicates the interpretation of the blood typing test in chimaeras.

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When both populations of cells are present in detectable quantities, a chimaera is revealed by the red cell test as incomplete reactions or in the electrophoretic test of red cell proteins as a mixture of patterns. However, in some cases, one of the red cell types is so greatly displaced that it escapes detection by the blood typing test. This may cause mistakes in the interpretation of the test. There are, however, methods which can be used to detect whether an individual is a chimaera or not. When the twins are of different sex, karyotyping may provide useful information. Both female (XX) and male (XY) sex chromosome sets may be found in a chimaera (Sandberg, 1980). Analyses of DNA from both blood and hair bulbs can also reveal a suspected chimaera (Bowling et al., 1993a). Solid tissue, such as hair bulbs, does not seem to be affected by the vascular anastomosis in utero. In cattle, a heifer born twin to a bull is sterile (freemartin) when there have been connections between the circulatory systems. A female foal is not affected in this way under the same circumstances (Bouters and Vandeplassche, 1972).

Neonatal isoerythrolysis (NI) NI was first described in the 1940s (Caroli and Bessis, 1947; Bruner et al., 1948; Coombs et al., 1948). The disease in the horse is comparable with the transplacental (Rh) immunization in humans. The sire transmits to the offspring red cell antigens (blood group factors) not present in the dam. At the end of the pregnancy, some red cells of the fetus enter the circulation of the dam. The dam’s immune system reacts by forming antibodies against the antigens on the red cells of the fetus. The most potent immunogens are the Aa and Qa factors (Stormont, 1975). A great majority of cases of NI are caused by these two factors, but other factors may also be involved (Scott and Jeffcott, 1978). Usually the antibodies do not reach a concentration that is harmful to the foal in the first 1–3 pregnancies. It is not until the mare has been exposed repeatedly to the same antigen, foreign to herself, that the antibodies reach a titre likely to lead to severe anaemia in the newborn foal. Far from all Aanegative mares develop anti-Aa antibodies even when they have given birth to Aa-positive foals. The reason for this is not known. One theory is that mares which are prone to develop retroplacental haemorrhages are more likely to get foals with NI. In some cases where mares are negative for both factors Aa and Ca, they may be protected by naturally produced anti-Ca antibodies that destroy the fetal red cells before they sensitize the mare (Bailey et al.,1988). There are marked differences between man and the horse in the way in which passive immunity is transferred from mother to offspring. In humans, antibodies enter the blood circulation of the fetus in utero and lead to red cell destruction, causing anaemia in the fetus if no preventive treatment is undertaken. In horses, the antibodies are concentrated in the mare’s colostrum and reach the foal’s blood circulation only after the foal has ingested colostrum. Hence the foal is born quite healthy but develops clinical signs of anaemia,

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including jaundice, lethargy and elevated pulse and respiration rates, within 2–5 days of birth (Scott and Jeffcott, 1978). The foal may recover spontaneously or after blood transfusion, but it may also die from severe anaemia. NI is diagnosed reliably by screening the serum of the mare for antibodies against horse RBCs. This is done by testing the serum against a panel of red cells representing as many blood group factors as possible including Aa and Qa. If possible, red cells from the sire of the foal should also be included in the panel. Such a test can be carried out by a laboratory providing a horse blood typing service. Once a mare has had a foal with NI, there is a considerable risk that subsequent foals will develop the disease. From mares at risk, a serum sample should be taken about a week before the expected parturition and tested against a red cell panel. If antibodies of a measurable titre are found in the serum, the foal must not suck the mare during the first 36 h after birth. Colostrum from another mare then has to be fed to the foal as a source of passive immunity (Scott and Jeffcott, 1978).

Blood transfusion In humans, antibodies regularly occur in blood serum as part of the ABO blood group system. Therefore, blood typing is of vital importance to make sure that donor and recipient have compatible ABO blood types. As already stated, naturally occurring isoantibodies are rarely found in horses. When they occur, they usually have a low titre. Hence blood transfusion between horses initially may be carried out disregarding their blood types. If, however, blood typing facilities are at hand, it is desirable that recipient and available donors are tested and that the recipient’s serum is analysed for isoantibodies. If antibodies against red cells are found in the recipient, a donor whose red cells do not react with these antibodies is selected. Also an Aa-negative recipient should not be given blood from an Aa-positive donor as this may stimulate production of anti-Aa antibodies in high titre even at a first-time transfusion and thus shorten the life span of the donor erythrocytes. In general, transfusion reactions are less likely to occur if the donor and the recipient are closely related. Blood transfusion between horses of different breeds should be avoided unless blood tests are carried out. Horses of different breeds are more likely to diverge in their make-up of red cell antigens, with an increasing risk of transfusion reactions.

Biochemical Polymorphism Plasma and red cell proteins The loci examined by biochemical methods in the horse are primarily those of blood plasma or serum and red or white blood cells. A wide variety of

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electrophoretic techniques has been used to examine variation at these loci and, for some loci, refinement of techniques has revealed greater variation, i.e. for the protease inhibitor and transferrin. Some loci have been examined in only a small number of breeds and individuals, while others, especially those routinely used in parentage testing (A1B, ALB, ES, GC, GPI, HBA, PGM, 6-PGD, PI and TF), have been studied in a wide variety of breeds (Bowling and Clark, 1985; Cothran and Kovac, 1997; Cothran et al., 1993; Cothran and van Dyk, 1998). The numbers of alleles so far discovered at some biochemical loci are shown in Table 5.2. A1B-Glycoprotein (A1B) A1B was initially reported as an unknown serum protein polymorphism called XK, detectable on acid starch gels (pH 4.8) used for electrophoretic detection Table 5.2.

The number of alleles at biochemical loci in horses. Number of alleles

Locus name A1B-Glycoprotein Aspartate aminotransferase Albumin Acid phosphatase Complement component 3 Carbonic anhydrase Catalase Ceruloplasmin NADH diaphorase Serum carboxylesterase Fucosidase alpha Vitamin D-binding protein Glucosephosphate isomerase Haptoglobin Haemoglobin alpha Malic enzyme 1 Mannosephosphate isomerase Peptidase A Plasminogen Phosphoglucomutase 6-Phosphogluconate dehydrogenase Protease inhibitor Red cell protein Serum protein 3 Transferrin Lactoglobulin beta II

Locus symbol

Common

Rarea

A1B AAT ALB AP C3 CA CAT CP DIA ES FUCA GC GPI HP HBA ME1 MPI PEPA PLG PGM 6-PGD PI RCP SP3 TF BLG-II

3 2 3 2 4 6 2 2 2 10 3 2 4 2 4 2 3 2 2 3 3 25 2 5 15 5

(4)

(+) (+) (2) (4)

(2) (+) (+)

aRare,

observed at very low frequency in one or only a few breeds; +, several examples have been observed but not reported.

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of pre-albumins (protease inhibitor; Braend, 1967b, 1970). The XK system was shown to be the same as the post-albumin system, detected on pH 8.1 starch gels, by Trommershausen-Smith and Suzuki (1978). This protein was identified as A1B by immunoblotting with antiserum to human A1BG (Juneja et al., 1987) and N-terminal amino acid sequence analysis of horse and pig A1B confirmed this assignment (Van de Weghe et al., 1988). A1B functions as a metalloproteinase inhibitor (Catanese and Kress, 1992). In horses, there are three alleles (F, K and S) found in a wide variety of horse breeds. Additionally, up to four other rare alleles have been described (Patterson et al., 1991; Nickel et al., 1992; Cristofalo et al., 1992). Aspartate aminotransferase-2 (mitochondrial) (AAT-2) AAT-2 (previously known as glutamate oxaloacetic transaminase or GOT-2) was shown to be polymorphic in an electrophoretic analysis of 79 horses of three breeds by Putt and Fisher (1979). Two alleles were seen in two of the three breeds, with the more anodal of the cathodally migrating protein (in starch gel at pH 7.4) being relatively rare (P = 0.09) in Mongolian ponies while the reverse was true (P = 0.88) in Icelandic ponies (Putt and Fisher, 1979). Albumin (ALB) The serum albumin locus was one of the first polymorphisms described in the horse (Stormont and Suzuki, 1963). Initially, two codominant alleles, ALB-A and ALB-B, were observed. A third allele, very rare in the few breeds where it has been found, was described by Sandberg (1972). Two separate studies (Suzuki and Stormont, 1974; Bowling and Clark, 1988) showed statistically significant segregation distortion at the ALB locus. Acid phosphatase (AP) Polymorphism at the horse AP locus was first described by Podliachouk et al. (1972). These results were later confirmed when Bengtsson and Sandberg (1973) reported on a starch gel electrophoresis procedure to detect four red cell enzymes (including AP) simultaneously. Variation in AP was based upon two co-dominant alleles. Complement component 3 (C3) Kay et al. (1986) reported polymorphism of horse plasma C3 using agarose immunofixation electrophoresis. Initially, three electrophoretically detected alleles were observed. Further study of C3 variation in 25 breeds of horses (Bowling and Dileanis, 1990) showed widespread polymorphism of this locus and also revealed a fourth allele that was very rare in the horse breeds but occurred at a frequency of 0.43 in Przewalski’s horse. Carbonic anhydrase (CA) Polymorphism of horse CA was first described by Sandberg (1968) using starch gel (pH 8.5) electrophoresis. Five codominant alleles were confirmed in an examination of about 600 Swedish horses. Further work by Deutsch et al.

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(1972) on Japanese farm horses, Deutsch and Bray (1975) on American ponies, and Jabusch and Deutsch (1984) were consistent with the initial study but provided additional information on enzymatic characteristics of the equine form of the red cell enzyme. Bowling et al. (1990) reported a sixth allele of this locus and provided populational data on CA variation from 27 domestic horse breeds and seven feral horse populations. Although six alleles exist in horses, the CA-I allele was most common in all populations and the level of overall polymorphism was not especially high. Catalase (CAT) Initial description of variation at the horse red cell catalase locus was given by Kelly et al. (1971). Using starch gel (pH 8.0) electrophoresis, two codominant alleles were found in Quarter Horses, Thoroughbreds and Shetlands. Schleger et al. (1972) also found two alleles in Austrian horses. Bowling et al. (1990) examined CAT variation in 27 domestic horse breeds and seven feral horse populations. Again, two alleles were observed, with the CAT-S allele the most common in all populations. Ceruloplasmin (CP) There has been little work on CP in horses. Schleger et al. (1972) reported the presence of two CP phenotypes in Austrian horses presumed to be controlled by two alleles, CP-A and CP-a, with CP-A dominant to CP-a. Juneja et al. (1984) examined CP variation in horses using two-dimensional electrophoresis. A low level of polymorphism, based upon two codominant, autosomal alleles, was observed in the Icelandic horse breed only. NADH diaphorase (DIA) Sandberg (1974a) reported on variation at the DIA locus of Swedish horses using starch gel electrophoresis. Two alleles were observed; however, it also was found that fresh RBCs should be examined. Serum carboxylesterase (ES) Polymorphism at the serum esterase locus of the horse was first recognized by Kaminski and Gajos (1964) and Gahne (1966), who found three codominant alleles plus a suggestion of a silent or null allele. Extensive work on horses and other equids by Kaminski and coworkers (e.g. Kaminski, 1970, 1978; Kaminski and Podliachouk, 1970) further refined the esterase system of equids, confirmed the existence of the null allele and showed the complete lack of a product from this locus in some equid species. Further work and different methodologies (such as isoelectric focusing) revealed additional variation (see Fisher and Scott, 1978). There are now at least ten recognized alleles and there have been several reports of observed instances of mutation (Sandberg, 1976; Sandberg et al., 1987; Bell et al., 1995). The horse ES system is difficult to type in that the ES-F and ES-G alleles are electrophoretically undistinguished at basic pH, while the ES-I and ES-S alleles have the same electrophoretic migration at acidic pH.

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Fucosidase alpha (FUCA) Variation at the horse FUCA locus from white cells was reported by Putt and Fisher (1979). Using flat bed isoelectric focusing (IEF), these authors observed three alleles in Mongolian and Icelandic ponies. No family studies were conducted in these breeds. Vitamin D-binding protein (GC) Vitamin D-binding protein, also known as group-specific component, was first recognized specifically and variation described by three separate reports in 1978 (Gahne and Juneja, 1978; Juneja et al., 1978; Weitkamp, 1978). Initially, two codominant alleles were recognized either by polyacrylamide (Gahne et al., 1977) or agarose immunofixation electrophoresis (Weitkamp, 1978). Two additional rare alleles were later reported (Cleve and Schmid, 1991; Ouragh et al., 1992; Ouragh and Juneja, 1994). Glucosephosphate isomerase (GPI) Sandberg (1973a) and Bengtsson and Sandberg (1973) first reported variation of GPI (also known as phosphohexose isomerase (PHI) or phosphoglucose isomerase (PGI)) from horse red cells using starch gel electrophoresis. Initially, three codominant alleles were observed. A fourth allele was reported by Bowling and Wictum (1988). Haptoglobin (HP) Weitkamp et al. (1985) described variation at the equine haptoglobin locus using thin layer agarose IEF (pH 4–6.5). Two alleles were observed. Haemoglobin alpha (HBA) Studies of haemoglobin in horses has a long history. Variation was first reported by Cabannes and Serain (1955) who observed two forms by use of paper electrophoresis. Braend (1967a) first reviewed the research on equine haemoglobin, and later Braend and Johansen (1983) reviewed it again. Most of the work detailing the molecular structure of the variation of horse haemoglobin was done by Clegg and coworkers (i.e. Kilmartin and Clegg, 1967; Clegg et al., 1984; Clegg, 1987). Basically, the variation in horse haemoglobin, as revealed by IEF (Ryder et al., 1979), is due to duplicated α-globin loci, which results in as many as four different protein bands by IEF. As reviewed by Bowling et al. (1988), normal horses have an α-1-globin with a lysine (Lys) in position 60 and the α-2 has a glutamine (Gln) in position 60. The two major alleles (or haplotypes) differ in the amino acids at position 24 of each of the linked genes, tyrosine (Tyr) for the BI allele and phenylalanine (Phe) for the BII allele. The BI/BII haplotype then has four protein bands resulting from the four different α-chains; α24 Tyr60Gln, α24 Tyr60Lys (BI) and α24 Phe60Gln, α24 Phe60Lys (BII). A third allele or haplotype (A) has only the α24 Tyr60Gln, while the AII variant has only the α24 Phe60Gln. There are four other variants that have been recognized (Bowling et al., 1988).

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Lactate dehydrogenase (LDH) Rauch (1968) reported a mutant form of the B locus controlling synthesis of LDH in the horse; however, this does not appear to be a real polymorphism in the horse. Malic enzyme 1 (ME1) Polymorphism at the soluble erythrocytic ME was reported by Guttormsen and Weitkamp (1980, 1981) using starch gel electrophoresis. Two codominant alleles were observed. Mannosephosphate isomerase (MPI) Putt and Fisher (1979) described variability of the horse MPI locus from leucocytes separated by starch gel electrophoresis. Two codominant alleles were found. Hall et al. (1991) reported levels of variation at MPI from 14 horse breeds and the presence of a third allele. Peptidase A (PEPA) Yut and Weitkamp (1979) examined horse peptidases and reported polymorphism for peptidase A from leucocytes. Two codominant alleles were found using starch gel electrophoresis (pH 7.2). Plasminogen (PLG) Horse plasminogen variation was first described by Weitkamp et al. (1983) by use of agarose IEF and immunofixation with a goat anti-human plasminogen antibody. Two alleles were found in American Standardbred horses. Bowling et al. (1993b) examined PLG variation in 23 breeds of domestic horse and Equus przewalskii and found limited variation in most breeds. Phosphoglucomutase (PGM) Bengtsson and Sandberg (1972) described variation at the PGM locus in Swedish horses based upon starch gel electrophoresis. Two codominant alleles were reported. Scott (1974) observed a third allele at this locus. 6-Phosphogluconate dehydrogenase (6-PGD) Genetic variation at the 6-PGD locus of the horse was reported by Sandberg and Bengtsson (1972) and Op’t Hof and Osterhoff (1973). Three codominant alleles were reported based upon starch gel electrophoresis. Janzen and Cothran (1991) reported a fourth allele in feral horses and Farndale et al. (1992) described a fifth variant at this locus from Thoroughbreds. Protease inhibitor (PI) The protease inhibitor system is one of the most variable genetic systems of the horse and has been the subject of a great deal of study. Gahne (1966) first demonstrated that the most anodal series of pre-albumins in acid starch gel represented a single genetic system. Four codominant alleles were recognized at that time. Braend (1970) refined the typing of this system and showed

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additional alleles. Ek (1979) was the first to use two-dimensional electrophoresis to examine variation in the PI system, while Matthews (1979) first reported IEF of PI. Ek (1977) showed that these pre-albumin bands corresponded to human PI on the basis of antitrypsin activity and molecular weight. Juneja et al. (1979), using two-dimensional electrophoresis, first showed evidence that more than one locus was involved in the PI phenotypes observed. Pollitt and Bell (1980) first described a polyacrylamide gradient gel system that revolutionized PI typing, and later refined this method (Pollitt and Bell, 1983a,b). By 1984, 18 alleles were recognized and formally described (Bell et al., 1984) and evidence of recombination within the PI system was reported by Patterson and Bell (1986). Bell and Patterson (1987) provided evidence that the PI system was controlled by at least two tightly linked loci. Further research showed that the PI system actually was a multigene family of four loci (Patterson et al. 1989). At this time, well over 25 alleles (haplotypes) have been reported for this system, and many others have been observed in single individuals. Red cell protein (RCP) Weitkamp (1987) reported polymorphism for a minor red cell protein of unknown function and identity demonstrated by agarose IEF (pH 4–6.5). Two alleles were observed. No further work on this system has been reported. Serum proteins (SP1, SP2 and SP3) Juneja et al. (1984, 1987) reported on variation of unidentified protein polymorphisms from horse serum using non-denaturing two-dimensional electrophoresis. SP2 was identified to be the same as human apolipoprotein A-IV (Juneja et al., 1989). SP3 appears to be controlled by five alleles (Juneja et al., 1989) Transferrin (TF) Serum transferrin is among the most variable genetic systems of the horse, and a large number of studies of TF have been reported (see Bell et al., 1988). Early work was reported by Braend and Stormont (1964), Gahne (1966) and Scott (1970). Later work focused on using thin-layer polyacrylamide gel electrophoresis (pH 7.9) (Bell et al., 1988). At least 15 variants at the equine TF locus have been reported. This does not include reported silent variants (Schmid et al., 1990) and other atypical variants (Stratil and Glasnak, 1981; Bowling, 1991). Recent mutations also have been reported (Farndale et al., 1991; Bell et al., 1995; Niini et al., 1997).

Milk proteins Lactoglobulin beta II (BLG-II) Bell et al. (1981) reported variation at the BLG-II locus. Five alleles were observed. Little other work on milk proteins of horses has been conducted.

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Utilization of the Blood Markers Parentage and identity testing Blood group factors and blood protein variants which are used for parentage testing, for identification and for solving problems of questionable paternity have the following characteristics in common: (i) They have a simple and direct inheritance which implies that they are transmitted from one generation to the next as dominant or codominant characters. (ii) They are fully developed at birth or shortly thereafter and remain unchanged throughout life. (iii) They are controlled only by heredity and are not influenced by environment. In the hands of persons who are fully qualified through education and experience the test methods by which the blood markers are determined are safe and reliable. The efficacy of a genetic system in solving parentage problems depends on the number of alleles in the system, their frequencies and whether the genotypes can be inferred directly from the phenotypes (Stormont and Suzuki, 1965). The efficiency of a single blood marker system as well as of the combination of several systems in a blood typing test usually is expressed as a percentage. This percentage indicates the probability of detecting incorrect parentage by the individual system or by all systems combined in a test, respectively. In most laboratories providing a horse blood typing service, the conventional test is composed of about 15 systems of blood markers (i.e. blood group systems EAA, EAC, EAD, EAK, EAP, EAQ and EAU and protein polymorphic systems ALB, A1B, ES, GC, HBA, PGD, PI and TF). The efficiency of the test in revealing an erroneously assigned stallion (or mare) is in the range 90–97% (Sandberg, 1974b; Bowling and Clark, 1985) depending on the breed and the composition of the test. In several laboratories, the conventional test is being replaced gradually by DNA analysis (microsatellites; see Chapter 6). The blood type is also an efficient means of identifying an individual horse. The number of possible blood types when all systems in the blood typing test and all phenotypes within each system are taken into account is extremely large. In a study of two Swedish horse breeds, Sandberg (1974b) estimated the probability that two unrelated horses within either breed had identical blood types at about 1/100,000. Hence, in breeds where parentage control of foals is compulsory before registration, the identity of an individual horse can be checked by repeated blood typing, whenever there is a need for it.

Population studies In addition to parentage analysis, blood group and biochemical polymorphisms can be used to estimate genetic variability within populations and relationship among populations. Genetic variation can be measured as

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heterozygosity (H) or some measure of populational diversity such as effective number of alleles (Ae). In almost all cases, studies of genetic variation of horses have only utilized loci known to be polymorphic in horses rather than a survey of a number of loci of which some will be variable and some not (for a review of genetic variation in natural populations, see Nevo, 1978). Thus, most estimates of genetic variability in domestic horses are not comparable with those for natural populations of other mammalian species. However, they do provide estimates of comparative levels of variability among horse populations or breeds. Early studies examined only a few loci (Bengtsson et al., 1968; Schleger and Mayrhofer, 1973; Podliachouk et al., 1975; Kaminski, 1984) and/or a small number of breeds (Kaminski and Urbanska-Nicolas, 1979; Romagnoli et al., 1984). Also, early genetic studies of horses frequently concentrated on a single region or county (Nozawa et al., 1976; Blokhuis and Buis, 1979; Lubas et al., 1984). These studies tended to focus upon variation at particular loci rather than comparative variability when the focus was on genetic variation at all. More often, the primary goal of these studies was to examine genetic relationships among a limited number of breeds within a given geographic region. It seems that the most comprehensive study of genetic variation within horse breeds was that of Bowling and Clark (1985). They examined 20 loci (seven blood group and 13 biochemical) in seven North American horse breeds. Variability was measured as Hardy–Weinberg heterozygosity, number of alleles per locus and efficacy for parentage testing. Heterozygosity ranged from 0.378 (± 0.069) for the Thoroughbred to 0.481 (± 0.066) for the Peruvian Paso breed. The emphasis of the variability analysis of the 20 loci was their usefulness in parentage testing. Genetic relationship also was examined. Other recent studies of blood group and biochemical genetic variation of horses have looked at genetic variation and reproductive performance in American Standardbred horses (Weitkamp et al., 1982; Cothran et al., 1986), genetic differentiation associated with gait within American Standardbreds (Cothran et al., 1987) and research on the genetic make-up of wild (feral) horse populations from the western USA (Bowling, 1994). In this later study, the feral populations were found to have heterozygosities, based upon 19 loci, within the range of that of 16 domestic horse breeds (mean H of 0.402 ± 0.009 for the feral herds compared with a mean H of 0.402 ± 0.045 for the domestic breeds). Examination of the genetic relationships of the feral herds to domestic breeds suggested origins from Iberian type horses and from American saddle and draught horse breeds (Bowling, 1994). Moureaux et al. (1996) examined genetic variability based upon both genealogical data from pedigrees and 16 blood marker polymorphisms in five French populations of horse breeds. They found that the relationship of genealogical estimates of inbreeding to genetic variation based upon blood marker data was complex and not always as predicted from theoretical expectations. Analysis of genetic variation can also provide data with potential significance to the preservation of rare breeds. Due to small population size, the loss of variation is a major concern for rare breeds. Also, it is important to know if a

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rare breed represents a unique genetic population before major conservation efforts are undertaken. Comparative genetic analyses can provide input into both these issues. Few recent studies of rare horse breeds have been reported to date. Cothran et al. (1993) found levels of variation near the average for the Chilote Horse of Chile. This rare breed from the island of Chiloe was clearly related to Iberian breeds. Similarly, the Posavina heavy horse breed from Croatia had heterozygosity that was relatively high (Cothran and Kovac, 1997). The Posavina showed the closest relationship with other heavy horse breeds. Another rare breed from South America, the Pantaneiro Horse of Brazil, also showed no evidence of a loss of genetic variation, with heterozygosity levels near the average for domestic breeds (Cothran et al., 1998). This breed also showed clear evidence of Iberian ancestry. The reasons why these rare breeds did not show reduced variation were not clear. Possibly, the reductions in population size were too recent to have resulted in reduced variation.

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K. Sandberg and E.G. Cothran polymorphism of ceruloplasmin and two other serum proteins. Animal Blood Groups and Biochemical Genetics 15, 237–250. Juneja, R.K., Gahne, B. and Stratil, A. (1987) Polymorphic plasma postalbumins of some domestic animals (pig PO2, horse Xk and dog Pa proteins) identified as homologous to human plasma a1B-glycoprotein. Animal Genetics 18, 119–124. Juneja, R.K., Sandberg, K., Kuryl, J. and Gahne, B. (1989) Genetic polymorphism of horse serum protein 3 (SP3). Animal Genetics 20, 43–49. Kaminski, M. (1970) Common and species-specific serum esterases of Equidae – II. Horse, donkey, zebra and their hybrids. Comparative Biochemistry and Physiology 35, 631–638. Kaminski, M. (1978) The null allele in the horse esterase (Es) system detected by enzyme assay and rocket immunoelectrophoresis in heterozygous animals. Animal Blood Groups and Biochemical Genetics 9, 18–19. Kaminski, M. (1984) Genetic diversity in horses inferred from distribution of hemotypes – II. Genetic structure of mixed breed populations. Comparative Biochemistry and Physiology 79B, 61–66. Kaminski, M. and Gajos, E. (1964) Comparative examination of carboxylic esterases in sera of horse, donkey and their hybrids. Nature 201, 716–718. Kaminski, M. and Podliachouk, L. (1970) Serum esterases of Equids: truly or apparently negative phenotypes. Comparative Biochemistry and Physiology 36, 207–209. Kaminski, M. and Urbanska-Nicolas, H. (1979) Electrophoretic polymorphism of proteins in the blood of horses: studies of eleven pony breeds or populations. Biochemical Systematics and Ecology 7, 229–237. Kay, P.H., Dawkins, R.L., Bowling, A.T. and Bernoco, D. (1986) Electrophoretic polymorphism and molecular structure of equine C3. Animal Genetics 17, 209–215. Kelly, E.P., Stormont, C. and Suzuki, Y. (1971) Catalase polymorphism in the red cells of horses. Animal Blood Groups and Biochemical Genetics 2, 135–143. Kilmartin, J.V. and Clegg, J.B. (1967) Amino acid replacements in horse hemoglobin. Nature 213, 269–271. Landsteiner, K. (1901) Über Agglutinations-erscheinungen normalen menschlichen Blutes. Wiener Klinische Wochenschrift 14, 1132–1134. Lubas, G., Gugliucci, B., Mengozzi, G. and De Berardinis, T. (1984) Genetic markers in the blood of four Italian horse breeds. Animal Blood Groups and Biochemical Genetics 15, 133–135. Matthews, A.G. (1979) Isoelectric focusing of horse acidic prealbumins of thin-layer polyacrylamide gels. Animal Blood Groups and Biochemical Genetics 10, 219–226. Meriaux, J.C. and Podliachouk, L. (1981) Additional factors Af and Ag in the A blood group system of the horse. Animal Blood Groups and Biochemical Genetics 12, 75–77. Moureaux, S., Verrier, E., Richard, A. and Meriaux, J.C. (1996) Genetic variability within French race and riding horse breeds from genealogical data and blood marker polymorphism. Genetics, Selection, Evolution 28, 83–102. Nevo, E. (1978) Genetic variation in natural populations: patterns and theory. Theoretical Population Biology 13, 121–177. Nickel, L., Bowling, A. and Wictum, E. (1992) Rare variant alleles of A1B in Shires and American Quarter horses. Animal Genetics 23 (Suppl. 1), 23. Niini, T., Stratil, A., Cizova-Schröffelova, D. and Sandberg, K. (1997) A partially deficient and atypical equine transferrin variant, TF N. Animal Genetics 28, 233–234. Nogaj, A., Duniec, M.J., Slota, E. and Duniec, M. (1997) Three newly detected alloantigens in the U blood group system of horses. Animal Genetics 28, 313–314.

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Nozawa, K., Shotake, T. and Ohkura, Y. (1976) Blood protein variations within and between the east Asian and European horse populations. Zeitschrift für Tierzuchtung und Zuchtungsbiologie 93, 60–74. Op’t Hof, J. and Osterhoff, D.R. (1973) Isoenzyme polymorphism of 6-phosphogluconate dehydrogenase (EC 1.1.1.44) in the family Equide. Animal Blood Groups and Biochemical Genetics 4, 111–113. Ouragh, L. and Juneja, R.K. (1994) A new allele in the horse Gc system. Animal Genetics 25 (Suppl. 2), 15. Ouragh, L., Braun, J.P. and Meriaux, J.C. (1992) A new phenotype in the horse Gc system. Animal Genetics 23 (Suppl. 1), 21. Patterson, S.D. and Bell, K. (1986) The equine protease inhibitory system (Pi): Abnormal expression of PiF, PiL, and PiS1. Biochemical Genetics 24, 529–543 Patterson, S., Bell, K. and Shaw, D. (1989) Equine Pi system: a multigene family of four loci. Animal Genetics 20 (Suppl. 1), 99–100. Patterson, S.D., Bell, K. and Shaw, D.C. (1991) Donkey and horse a1B-glycoprotein: partial characterization and new alleles. Comparative Biochemistry and Physiology 98B, 523–528. Podliachouk, L. (1957) Les Antigènes de Groupes Sanguins des Équidés et Leur Transmission Héréditaire. Thesis, Université Paris. Podliachouk, L. and Hesselholt, M. (1962) Les groupes sanguins des équidés. Les sérums de réferénce. Immunogenetics Letter, 2, 6971. Podliachouk, L. and Meriaux, J.C. (1977) A new factor (Dg) of the D blood group system of the horse. Animal Blood Groups and Biochemical Genetics 8, 179–181. Podliachouk, L. and Meriaux, J.C. (1979) Two new factors of the D blood group system of the horse. In: Proceedings of the 16th International Conference on Animal Blood Groups and Biochemical Genetics. Leningrad, 1978, IV, pp. 117–123. Podliachouk, L., Sirbu, Z., Kownacki, M. and Szeniawska, D. (1960) Les groupes sanguins des chevaux. Etude comparative des sérums de référence. Annales de l’Institut Pasteur 98, 861–867. Podliachouk, L., Balbierz, H., Kaminski, M., Nikolajczuk, M. and Strzelecka, A. (1972) Immunogenetic study of the Mur-Insulan horses. In: Proceedings of the 12th European Conference on Animal Blood Groups and Biochemical Polymorphism. Budapest, 1970, pp. 533–536. Podliachouk, L., Kaminski, M., Van de Weghe, A., Bouquet, Y., Zwolinski, J. and Siudzinski, S. (1975) Marquers genetiques sanguins chez les chevaux de course. Annales de Genetique et de Selection Animale 7, 339–355. Pollitt, C.C. and Bell, K. (1980) Protease inhibitor system in horses: classification and detection of a new allele. Animal Blood Groups and Biochemical Genetics 11, 235–244. Pollitt, C.C. and Bell, K. (1983a) Characterization of the α1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 1. Protein staining. Animal Blood Groups and Biochemical Genetics 14, 83–105. Pollitt, C.C. and Bell, K. (1983b) Characterization of the α1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 2. Protease inhibition. Animal Blood Groups and Biochemical Genetics 14, 107–118. Putt, W. and Fisher, R.A. (1979) An investigation of seven enzymes as possible genetic markers in horse leukocytes. Animal Blood Groups and Biochemical Genetics 10, 191–197.

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K. Sandberg and E.G. Cothran Rauch, N. (1968) A mutant form of lactate dehydrogenase in the horse. Annals of the New York Academy of Science 151, 672–677. Romagnoli, A., Lubas, G., Mengozzi, G. and Guidi, G. (1984) Genetic markers in the blood of the Italian Standardbred Trotter horse. Animal Blood Groups and Biochemical Genetics 15, 137–146. Ryder, O.A., Sparkes, R.S., Sparkes, M.C. and Clegg, J.B. (1979) Hemoglobin polymorphism in Equus przewalski and E. caballlus analyzed by isoelectric focusing. Comparative Biochemistry and Physiology 62B, 305–308. Sandberg, K. (1968) Genetic polymorphism in carbonic anhydrase from horse erythrocytes. Hereditas 60, 411–412. Sandberg, K. (1970) Blood group factors and erythrocyte protein polymorphism in Swedish horses. In: Proceedings of the 11th European Conference on Animal Blood Groups and Biochemical Polymorphism. Warsaw, 1968, pp. 447–452. Sandberg, K. (1972) A third allele in the horse albumin system. Animal Blood Groups and Biochemical Genetics 3, 207–210. Sandberg, K. (1973a) Phosphohexose isomerase polymorphism in horse erythrocytes. Animal Blood Groups and Biochemical Genetics 4, 79–82. Sandberg, K. (1973b) The D blood group system of the horse. Animal Blood Groups and Biochemical Genetics 4, 193–205. Sandberg, K. (1974a) Genetically controlled variants of NADH diaphorase from horse red cells. Animal Blood Groups and Biochemical Genetics 5 (Suppl. 1), 23–24. Sandberg, K. (1974b) Blood typing of horses: current status and application to identification problems. In: Proceedings of the 1st Congress on Genetics Applied to Livestock Production. Madrid, 1974, pp. 253–265. Sandberg, K. (1976) An apparent case of mutation in the horse esterase (Es) system. Animal Blood Groups and Biochemical Genetics 8 (Suppl. 1), 18–19. Sandberg, K. (1979a) On the Q blood group system of the horse. In: Proceedings of the 16th International Conference on Animal Blood Groups and Biochemical Genetics, Leningrad, 1978, IV, pp. 141–146. Sandberg, K. (1979b) Studies on Blood Groups and Genetic Protein Polymorphisms of the Horse. Thesis, Swedish University of Agricultural Sciences. Sandberg, K. (1980) Horse chimaera detected by investigation of progeny. Animal Blood Groups and Biochemical Genetics 11 (Suppl. 1), 20. Sandberg, K. and Bengtsson, S. (1972) Polymorphism of hemoglobin and 6-phosphogluconate-dehydrogenase in horse erythrocytes. In: Proceedings of the 12th European Conference on Animal Blood Groups and Biochemical Polymorphism. Budapest, 1970, pp. 527–531. Sandberg, K., Andersson, L. and Bengtsson, S. (1987) Another case of mutation in the equine serum esterase (Es) system. Animal Genetics 18 (Suppl. 1), 66–67. Schleger, W. and Mayrhofer, G. (1973) Genetic relationships between Lipizzan horses, Haflingers, Noriker and Austrian Trotters. Animal Blood Groups and Biochemical Genetics 4, 3–10. Schleger, W., Kramser, P. and Dworak, E. (1972) Catalase and ceruloplasmin polymorphism in three Austrian horse breeds. Animal Blood Groups and Biochemical Genetics 3 (Suppl. 1), 48. Schmid, D.O. (1965) Blood group studies in horses. In: Proceedings of the 9th European Conference on Animal Blood Groups. Prague, 1964, pp. 237–243. Schmid, D.O., Ek, N. and Braend, M. (1990) Further evidence for a silent allele in the transferrin locus of the horse. Animal Genetics 21, 423–426.

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Scott, A.M. (1970) A single gel for the separation of albumins and transferrins in horses. Animal Blood Groups and Biochemical Genetics 1, 253–254. Scott, A. M. (1973) Red cell groups of horses. In: Proceedings of the 3rd International Conference on Equine Infectious Diseases. Paris, 1972, pp. 384–393. Scott, A.M. (1974) Evidence for a third allele in the phosphoglucomutase (PGM) system in horses. Animal Blood Groups and Biochemical Genetics 5 (Suppl. 1), 24–25. Scott, A.M. (1985) The D red-cell system in horses: the factor Dl. Animal Blood Groups and Biochemical Genetics 16 (supplement 1), 23. Scott, A.M. and Jeffcott, L.B. (1978) Hemolytic disease of the newborn foal. Veterinary Record 103, 71–74. Smithies, O. (1955) Zone electrophoresis in starch gels; group variations in serum proteins of normal human adults. Biochemical Journal 61, 629–641. Stormont, C. (1975) Neonatal isoerythrolysis in domestic animals: a comparative review. Advances in Veterinary Science 19, 23–46. Stormont, C. and Suzuki, Y. (1963) Genetic control of albumin phenotypes in horses. In: Proceedings of the Society for Experimental Biology and Medicine 114, 673–675. Stormont, C. and Suzuki, Y. (1964) Genetic systems of blood groups in horses. Genetics 50, 915–929. Stormont, C. and Suzuki, Y. (1965) Paternity tests in horses. Cornell Veterinary 55, 365–377. Stormont, C., Owen, R.D. and Irwin, M.R. (1951) The B and C systems of bovine blood groups. Genetics 36, 134–161. Stormont, C., Suzuki, Y. and Rhode, E.A. (1964) Serology of horse blood groups. Cornell Veterinary 54, 439–452. Stratil, A. and Glasnak, V. (1981) Partial characterization of horse transferrin heterogeneity with respect to the atypical type, TF C. Animal Blood Groups and Biochemical Genetics 12, 113–122. Suzuki, Y. (1978) Studies on blood groups of horses. Thesis. Memoirs of the Tokyo University of Agriculture 20, 1–50. Suzuki, Y. and Stormont, C. (1974) Deviation of observed from expected albumin types in the offspring of heterozygous matings in horses. Animal Blood Groups and Biochemical Genetics 5 (Suppl. 1), 31. Trommershausen-Smith, A. and Suzuki, Y. (1978) Identity of Xk and Pa systems in equine serum. Animal Blood Groups and Biochemical Genetics 9, 127–128 Vandeplassche, M., Podliachouk, L. and Beaud, R. (1970) Some aspects of twingestation in the mare. Canadian Journal of Comparative Medicine 34, 218–226. Van de Weghe, A., Coppieters, W., Bauw, G., Vandekerckhove, J. and Bouquet, Y. (1988) The homology between the serum proteins Po2 in pig, Xk in horse and a1B-glycoprotein in humans. Comparative Biochemistry and Physiology 90B, 751–756. Watanabe, Y. and Noda, H. (1970) Preparation of equine blood typing reagents. Japanese Journal of Zootechnological Science 41, 649–654. Weitkamp, L.R. (1978) Equine marker genes. Polymorphism for group-specific component (Gc). Animal Blood Groups and Biochemical Genetics 9, 123–126. Weitkamp, L.R. (1987) Equine marker genes: polymorphism for a minor red cell protein, RCP. Animal Genetics 18 (Suppl. 1), 73–74. Weitkamp, L.R., MacCluer, J.W., Guttormsen, S., McKnight, J., Wert, N., Witmer, J., Boyce, P. and Egloff, J. (1982) Genetics of Standardbred stallion reproductive performance. Journal of Reproduction and Fertility 32 (Suppl.), 135–142.

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K. Sandberg and E.G. Cothran Weitkamp, L.R., Costello-Leary, P. and Guttormsen, S.A. (1983) Equine marker genes: polymorphism for plasminogen. Animal Blood Groups and Biochemical Genetics 14, 219–223. Weitkamp, L.R., Costello-Leary, P. and Guttormsen, S.A. (1985) Equine marker genes: polymorphism for haptoglobin and assignment of the locus for haptoglobin to equine linkage group II. Animal Blood Groups and Biochemical Genetics 16, (Suppl. 1), 78. Yut, J. and Weitkamp, L.R. (1979) Equine peptidases: correspondence with human peptidases and polymorphism for erythrocyte peptidase A. Biochemical Genetics 17, 987–994.

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Molecular Genetics of the Horse M. BinnsMatthew 6Molecular etGenetics al.

Binns, June E. Swinburne and Matthew Breen

Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, UK Introduction Minisatellites, Microsatellites and Single Nucleotide Polymorphisms (SNPs) Minisatellites and DNA fingerprinting Microsatellites Other satellite sequences SNPs and other polymorphisms DNA-based Parentage Analysis Mitochondrial DNA Genes and Expressed Sequence Tags (ESTs) Resources for Molecular Genetics in the Horse References

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Introduction The equine genome, like those of other mammals, is estimated to contain approximately 3000 megabases (Mb) of DNA. This DNA is distributed on 31 pairs of autosomes and the X and Y sex chromosomes. In common with other mammals, the horse is estimated to have between 50,000 and 70,000 genes, with the majority of the DNA being made up of repeated sequences, which whilst not encoding proteins may have a structural role. The repeated sequences comprise many different types that have been subclassified into minisatellite, microsatellite, α-satellite, SINE, LINE and telomeric amongst others. These repeat sequences have proven invaluable for genetic studies of individual identification, parentage analysis, genetic mapping and the study of the evolution of horse breeds. This chapter will review molecular genetics in the horse including the use of DNA-based parentage analysis. While the focus is on the horse, Equus caballus (L.), it should be noted that the different Equidae have very different chromosome numbers but do share a very high ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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level of DNA identity. This means that molecular reagents developed for use in the horse usually can be used in other members of the Equidae, thereby greatly facilitating conservation studies.

Minisatellites, Microsatellites and Single Nucleotide Polymorphisms (SNPs) DNA fingerprinting was the first sensitive DNA-based method for identifying individuals and for studying genetic relationships (Jeffreys et al., 1985a, b). Human and equine minisatellites were therefore used in early fingerprinting studies to assess relationships within and between members of different horse breeds. This technique, which requires comparatively large amounts of DNA for Southern blotting, has largely been replaced by polymerase chain reaction (PCR)-based methods using microsatellites, which require smaller amounts of DNA. Methods utilizing SNPs for human genetic studies are now becoming common following the development of assay systems based on ‘chips’ that enable large numbers of such polymorphisms to be tested simultaneously. An enzyme-linked immunosorbent assay (ELISA)-based system using SNPs has been described in the horse (Nikiforov et al., 1994), and it is likely that SNPs will play an increasing role in future equine genetic studies.

Minisatellites and DNA fingerprinting Minisatellites are tandemly repeated sequences that generally are not transcribed, and are present on all chromosomes. The individual repeated minisatellite unit varies in length (commonly 9–24 bp), and tandem arrays of minisatellite sequences can be up to 20 kb long. The significance of minisatellites is not clear although they have been reported to be ‘hotspots’ for homologous recombination. Georges et al. (1988) first examined the use of minisatellites in the horse (and a range of other domestic animals) using four different minisatellite probes on a family of Belgian half-bred horses. The probes were found to be highly effective in distinguishing individuals, with a combined probability that two individuals would share the same fingerprint being estimated as 3.2 ×10−12. Troyer et al. (1989) used a human variable number tandem repeat (VNTR) probe to reveal a highly polymorphic pattern of restriction fragments on Southern blots of Quarter Horse DNAs digested with HinfI, and suggested that such a system would be useful for individual identification and pedigree analysis. The widely used human minisatellite probes 33.15 and 33.6 were used by Hopkins et al. (1991) to solve a paternity case in closely related Exmoor ponies. Guerin et al. (1993) also used the 33.6 minisatellite in a variety of breeds and concluded that the loci identified by the minisatellites were polymorphic and comparatively stable, as no mutations were seen in the three half-sibling families examined. DNA fingerprinting has also been carried out

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using a synthetic (TG)n polynucleotide to compare populations of Swedish trotters, North Swedish trotters, Thoroughbreds and Arabians (Ellegren et al., 1992a) and Japanese native horses (Yamashita et al., 1994). Whilst the studies described above used non-equine minisatellite or synthetic polynucleotide probes, minisatellites have been characterized in the horse. Tandem copies of a 14-bp sequence related to a family of human minisatellite sequences was identified in the first introns of both the zeta and psi-zeta (pseudogene) globin genes when the horse zeta-globin locus was sequenced (Flint et al., 1988). Horse minisatellites have also been cloned by Anglana et al. (1996a) who showed that the clones containing the minisatellites gave highly polymorphic fingerprints using Lipizzaner DNAs. The sequences of the minisatellite-containing clones were not determined although the probe used to isolate them, STR16C4, contains tandem repeats of TTTGAGGTGGATGGAC.

Microsatellites Microsatellites are short arrays of simple sequence repeats, typically 2–4 bp, which are interspersed throughout the genome. This class of repeats is common in mammalian genomes and it has been estimated that approximately 100,000 copies of the CA/GT microsatellite repeat (the commonest sequence found) is present in the typical mammalian genome. Microsatellites are often highly polymorphic and easily assayed using PCR-based methods, and this has led to them being widely adopted as the markers of choice for genetic studies. Microsatellites are usually specific to species within the same genus so it has not been feasible to use microsatellites generated from human, mouse or cattle projects for horse studies. Horse microsatellites were first characterized by Ellegren et al. (1992b) and Marklund et al. (1994) who isolated sets of CA/GT microsatellites and demonstrated that they were highly polymorphic in horses, such that they could be used for parentage analysis and for genetic linkage studies. A combined exclusion probability for a parentage test based on eight of these microsatellites was reported to be 0.96–0.99, which demonstrated the power of microsatellites for this work. Subsequently, many reports have described the isolation of equine microsatellites and their use in parentage studies, in the generation of a genetic linkage map of the horse and in their linkage to inherited diseases. A comprehensive list of these publications is beyond the scope of this chapter but can be compiled easily by searching the common databases. At a recent meeting in January 1999, it was estimated that approximately 500 horse microsatellites have been sequenced, and this number continues to grow rapidly as molecular genetic studies in the horse expand. The vast majority of the microsatellites isolated have been CA/GT dinucleotide repeats from small insert libraries. A subset of microsatellites has been isolated from cosmid, phage or bacterial artificial chromosome (BAC) clones containing larger inserts (Sakagami et al., 1995; Tozaki et al., 1995; Breen et al., 1997; Godard et al., 1997, 1998; Hirota

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et al., 1997; Marti et al., 1998), and these have been particularly useful in allowing the integration of genetic and physical maps of the equine genome. At present, microsatellite markers have been physically assigned to the majority of equine chromosomes, with the ‘missing’ chromosomes being mainly the smaller acrocentric ones. By typing 182 microsatellites on a somatic cell hybrid panel, Shiue et al. (1999) were able to assign microsatellites into 32 syntenic groups, of which 21 could be assigned to known chromosomes. It appears that the non-assigned groups do represent separate chromosomes such that microsatellites have been isolated from every equine chromosome except the Y chromosome. Breen et al. (1994b) demonstrated that microsatellites isolated from the horse frequently amplified polymorphic sequences in the other species of the genus, suggesting that the markers developed for horse studies could be used for conservation genetics in these species. Several of these species have a narrow genetic base, and the ability to maximize outbreeding in captive breeding programmes through the use of genetic monitoring should improve these programmes. In other species, such as humans, tetranucleotide repeats have been used extensively in mapping (Murray et al., 1994) and forensic studies (Gill et al., 1995) as they have advantages in the automated scoring of alleles, due to the increased size differences between alleles and the reduced ‘stutter’ bands that are seen with these markers. In dogs, at least one class of tetranucleotide repeats, (GAAA)n, has been found to possess very high levels of polymorphism which on average exceed those of dinucleotide repeats (Francisco et al., 1996). Whilst tetranucleotide repeats have been described in the horse (e.g. Breen et al., 1994a), there is a general perception that the tetranucleotide microsatellites so far examined have not been as informative as most dinucleotide repeats.

Other satellite sequences Other classes of satellite repeats, which correspond to human α-satellite sequences, have been characterized from the horse genome. Wijers et al. (1993) estimate that approximately 1 million copies of a 221-bp repeat sequence are present in horse DNA, which represents about 5–10% of the total equine genome. This repeat comprises the major horse satellite repeat, and is localized to the centromeres of 29 of the 33 horse chromosomes. The chromosome pairs to which the repeats failed to hybridize were metacentric or submetacentric, and were thought to include chromosomes 7 and 10, which have been reported not to contain heterochromatin (Ryder et al., 1978). The repeats are 90–100% identical to each other and are not related to any other sequences in the database. Interestingly, this repeat sequence was found to hybridize to donkey DNA but not to DNA from Grevy’s zebra. Given that the divergence of the present-day Equidae is thought to have occurred 3–5 million years ago, the failure to hybridize to Grevy’s zebra DNA indicates that an

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extremely rapid evolution of this major satellite sequence has taken place. A second 221-bp satellite sequence which did not share sequence similarity with the repeat described above was cloned by Sakagami et al. (1994a). This sequence localized to the centromeres of all equine chromosomes except chromosomes 2, 9 and 11, and was estimated to represent 3.7–11% of the horse genome. Broad et al. (1995b), subsequently cloned two repeats of about 80 bp, one of which showed high levels of identity with the repeat sequenced by Wijers et al. (1993) and the other of which showed high levels of identity to that characterized by Sakagami et al. (1994a). Another repeat sequence of 21–22 bp was identified by Broad et al. (1995a), who demonstrated by in situ hybridization that this element was localized in the centromeres of acrocentric but not metacentric horse chromosomes. This repeat, which shows no sequence identity with those described above, hybridized to horse, donkey and zebra DNA, but not to DNA from cattle, sheep, goats or pigs, suggesting that it is specific to the Equidae. Other repetitive DNA components have been called dispersed elements as they are not restricted to the centromeres or telomeres of chromosomes. Two major classes of dispersed repeats exist, long interspersed elements (LINEs) and short interspersed elements (SINEs). Both LINEs and SINEs are retroposons that have reintegrated into the genome via cDNA intermediates by reverse transcription. SINEs have been classified into two groups depending on their putative origins from either 7SL RNA or tRNAs. Most mammalian SINEs are thought to have derived from tRNALys, whilst the equine SINE element appears to derive from tRNASer (Sakagami et al., 1994b). Equine SINE elements recently have been shown to form two families, ERE-1 and ERE-2, which whilst sharing some sequence similarity have a different subunit structure. Common subunits, defined as either tRNASer-derived, I (60 bp), II (70 bp) and III (50 bp), were identified. ERE-1 elements have a structure based on tRNASer-I–II, whilst ERE-2 has a subunit structure based on I–III–III (Gallagher et al., 1999). In many mammals, SINE and LINE elements frequently are associated with microsatellites and this has been observed for equine microsatellites (Breen et al., 1997; Hiromura et al., 1997; Gallagher et al., 1999). In a recent survey, 3.4% of 293 microsatellites examined had an associated ERE-1 element, whilst 6.5% has an associated ERE-2 element (Gallagher et al., 1999). Some of the ERE-1 and ERE-2 microsatellite markers amplified sequences from other equids, as well as from rhinoceros, whilst no amplification was seen from human or cattle DNA. This suggests that these sequences are perissodactyl specific. The ERE-2 element was also found to be associated with functional genes, being present in an intron of the DNA-PK gene, within the promoter region of the Pghs gene and within the coding region of the Pam gene (Gallagher et al., 1999).

SNPs and other polymorphisms With the development of improved detection systems for assaying SNPs, based on microarrays/chip technology, the use of SNPs in genetic studies in humans

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and other species has mushroomed in the past 2 years. Whilst the development of the microarray/chip technology is recent, and has yet to prove itself fully in practical situations, it does have real potential to revolutionize the amount of genetic data that can be accessed easily for large numbers of individuals. Relatively few reports describe SNPs in horses, and no large-scale efforts to discover equine SNPs have been undertaken. Nikiforov et al. (1994) describe a collection of SNPs and a method of assaying them called genetic bit analysis (GBA) based on an ELISA format. They propose the use of the GBA system for large-scale parentage verification. However, due to the reduced information content of each SNP compared with highly polymorphic microsatellites, 25–50 diallelic polymorphisms must be assayed to achieve levels of exclusion probabilities comparable with those seen with microsatellites. The mutations underlying several phenotypes in the horse have been identified and these are described in more detail in Chapters 3 and 8. These polymorphisms include those responsible for hyperkalaemic periodic paralysis (Rudolph et al., 1992), severe combined immunodeficiency (Shin et al. 1997), lethal white foal syndrome (Santschi et al., 1998; Metallinos et al., 1998; Yang et al., 1998) and chestnut coat colour (Marklund et al., 1996). Polymorphisms have been identified in the transferrin gene, which encodes one of the proteins routinely used as part of the parentage testing system using protein polymorphisms (Bailey et al., 1991; Carpenter and Broad, 1994). Restriction fragment length polymorphisms (RFLPs) have also been described in the acetylcholine receptor and complement C4 genes (Kay et al., 1987a, b) and in the non-transcribed spacer region of horse rDNA (Anglana et al., 1996b). Random amplified polymorphic DNA (RAPD) markers have been mapped on to a somatic cell hybrid panel (Shiue et al., 1999), but RAPD markers have not found widespread acceptance due to reservations about the repeatability of PCRs involving these markers.

DNA-based Parentage Analysis Parentage verification validates the horse pedigrees that make up the stud books and as such is an important function of the breed registries. Until recently, testing was based routinely on a set of 15 blood group and protein markers that could detect an incorrectly presented sire or dam with an exclusion probability of between 97 and 99%. Due to increased international movement of horses, it is important that laboratories in different countries use the same set of markers for verification so that data from different countries can be used. Whilst the blood typing system has worked well, it is limited to blood samples, requires sets of specialized reagents and is relatively time consuming, in that several of the typing systems have to be analysed separately. DNA-based methods offer several potential advantages over conventional parentage assays in terms of their accuracy and specificity, the ability to use a range of easily obtainable sample tissues including hair, and in the ease

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of laboratory analysis with commonly available molecular biological reagents. Parentage studies based on the use of minisatellite ‘fingerprinting’ are mentioned above, although this technique is unlikely to be adopted for routine analysis in horses. The technique is labour intensive and requires large amounts of DNA for Southern blotting, compared with the small amounts of DNA that can be used in PCR-based systems. Microsatellite markers have been chosen as the markers of choice, due to the high levels of polymorphism they exhibit and the ease with which they can be scored. Other groups are developing parentage analysis methods based on SNPs which, although they are less informative and thus require more markers to be analysed, do have potential advantages in the automation of the techniques used and in the scoring of results. Ideally, for parentage verification, sets of microsatellite markers that are unlinked, are highly polymorphic, show low levels of mutation, are easily scoreable and can be multiplexed into a single PCR reaction would be selected from the available microsatellites to generate an optimum panel of markers. The microsatellite panel for parentage verification in the horse was assembled at an early stage of the horse genome mapping project when relatively few microsatellites were available and their map locations were largely unknown. The current commercially available Stockmarks for Horses kit (PE Applied Biosystems) comprises a panel of 12 microsatellite markers which can be amplified in two multiplex PCR reactions, comprising one eight-plex reaction and one four-plex reaction. One of the primers for each of the markers is labelled with one of three different fluorophores (FAM, JOE or TAMRA) to facilitate the multiplex analysis of the 12 markers in a single lane. Products from the two reactions can be pooled together with molecular size standards labelled with a fourth fluorophore (ROX) which provides an internal lane size standard to increase greatly the accuracy of allele size calling. The markers selected, VHL20, HTG4, HTG6, HTG7, HTG10, AHT4, AHT5, HMS2, HMS3, HMS6, HMS7 and ASB2 have a combined exclusion probability of approximately 99% in a variety of horse breeds including Thoroughbreds, Bavarian Warmbloods, Tennessee Walking Horses, Friesians, Fjord horses and Standardbreds. Recent mapping data (Shiue et al., 1999) have indicated that the 12 markers used in the panel are not all on different chromosomes, with HMS6 and HTG7 mapping to chromosome 4, HMS3 and HTG4 mapping to chromosome 9, and ASB2 and HTG6 mapping to chromosome 15. There is thus a slight danger that these markers might not segregate totally independently, which could have an effect on the real exclusion probabilities obtained. In practice, the panel has performed effectively, and DNA-based testing has already been adopted by several breed registries (e.g. the Quarter Horse Association). It is highly likely that DNA-based typing will replace blood group and protein marker typing in the near future, although the adoption of a new system will require breeding animals that previously had been blood typed to be re-tested using DNA markers. Interestingly, the molecular bases for some of the different alleles of transferrin, which is one of the most polymorphic

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markers used in blood typing, have been characterized recently (Carpenter and Broad, 1994). The identification of the mutations underlying the variation in the other markers used for blood typing could permit the transition from blood typing to an SNP-based system that assayed these mutations to take place without extensive re-testing.

Mitochondrial DNA The mitochondrial genome has been used extensively in studies of molecular evolution in many species as it has an approximately fivefold higher rate of nucleotide substitution than nuclear DNA. It also has the unique property of being inherited only through the maternal line. Mitochondrial DNA (mtDNA) is therefore useful for studying the evolution of closely related species, and many studies have focused on the mitochondrial D-loop which has a higher substitution rate than the rest of the mtDNA. The horse mtDNA sequence was determined by Xu and Arnason (1994) who demonstrated that it comprised approximately 16,660 bp, but that this figure varied due to the presence of variable numbers of GTGCACCT motifs in the control region. The number of repeats was found to vary between 2 and 29 copies, although the majority were in the 22–27 range. Several other groups (Marklund et al., 1995; Ishida et al., 1995, 1996) have identified other variations in mtDNA sequences that have been used to study the evolutionary relationships between breeds of horses and for tracing maternal lineages. These polymorphisms have been examined using a variety of techniques. Fifteen distinct variants were found in an approximately 440-bp fragment from the hypervariable D-loop region when 78 maternally unrelated horses of five breeds were examined by singlestrand conformation polymorphism (SSCP) analysis (Marklund et al., 1995). PCR-based restriction fragment length polymorphism (PCR-RFLP) of the mitochondrial cytochrome b gene enabled four mitochondrial types to be identified amongst 140 horses of six breeds (Ishida et al., 1996). In contrast, direct sequencing of a 270-bp region of the D-loop was used to investigate phylogenetic relationships between various E. caballus (L.) breeds and other equid species (Ishida et al., 1995). Recently, variation in the mitochondrial control region and 12S rRNA was investigated in all surviving mitochondrial lineages of the Przewalski’s horse (E. ferus przewalskii) and found to be very low, despite individuals apparently originating from three distinct geographical regions (Oakenfull and Ryder, 1998).

Genes and Expressed Sequence Tags (ESTs) Comparatively few genes have been sequenced in the horse, compared with the wealth of data which exist for most domesticated species. A recent search of the EMBL Database revealed approximately 700 horse entries. Many of these entries, however, are duplicate sequences for a variety of major

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histocompatibility complex (MHC) genes (e.g. class II DQBeta) and many entries refer to microsatellite sequences, the majority of which are present in non-coding regions of the genome. The horse globin genes have been well characterized and the structure of the different loci compared with those in man and other mammals (Clegg, 1987; Flint et al., 1988). The alpha 1, alpha 2 and theta-globin genes recently have been sequenced from six equid species to investigate relationships within the genus Equus (Oakenfull and Clegg, 1998), thus providing a useful addition to the mitochondrial studies described above. The globin studies suggest that horses diverged from the zebra/ass ancestor about 2.4 million years ago (mya) and that the zebra and ass species arose in a rapid radiation about 0.9 mya. Mitochondrial studies suggest a more gradual set of speciation events, and more data obviously are required to resolve the discrepancies. A comparison of the percentage identity of the equine Scya5 gene (McManus, 1997) with that of some other mammals is presented in Table 6.1. The results are typical of comparisons of equine genes with other species in that the highest homology tends to be seen with human and bovine genes, whilst the homology to rodent genes tends to be lower. One study has reported the results of a pilot EST sequencing project in which clones from an equine peripheral blood lymphocyte cDNA library (Tavernor et al., 1993) were sequenced and compared with the DNA and protein databases (Binns et al., 1994). From approximately 100 random clones sequenced, the authors were able to identify homologues to at least 19 previously characterized genes, including several which reflected the immunological origin of the cells used to construct the cDNA library. These included genes for MHC class I, class II and beta 2-microglobulin. This work subsequently was extended (McManus,1997) and from 182 clones, 89% were found to share significant homology to database entries. The increased number of matches observed in the latter study probably reflects the explosion of EST sequences present in the database at the latter date. The construction of further cDNA libraries from alternative tissues followed by extensive EST sequencing is likely to be a valuable approach to increase gene information in the horse. In turn, it would provide valuable markers for radiation hybrid mapping and allow useful comparative studies to be performed. Table 6.1.

Horse Human Cow Guinea-pig Mouse Rat

Homology (%) of the horse Scya5 coding sequence with other species. Horse

Human

Cow

Guinea-pig

Mouse

Rat

100 87 86 86 83 82

84 100 86 88 85 86

82 80 100 84 82 81

85 87 81 100 85 84

77 79 75 83 100 95

77 79 75 81 96 100

DNA comparisons are on the left of the table, below the diagonal, whilst amino acid comparisons are on the right of the table, above the diagonal.

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Resources for Molecular Genetics in the Horse Many of the molecular resources for carrying out genetic studies in the horse are now available. These include both genomic (Godard et al., 1998; Marti et al., 1998) and cDNA libraries (Tavernor et al., 1993), somatic cell (Shiue et al., 1999) and radiation hybrid (E.A. Oakenfull and R. Chowdhary, personal communications) panels, genetic markers and maps (Lindgren et al., 1998; Guerin et al., 1999) and a rapidly growing number of sequenced genes. There is also a good framework understanding of the syntenic relationship between the genes present on individual horse chromosomes and those on the human genome. This knowledge, which is described in detail in Chapters 9 and 10, enables reasonable predictions to be made concerning gene organization in the horse. It is thus becoming increasingly feasible to carry out molecular studies on the many interesting and unique traits which the horse possesses.

References Anglana, M., Vigoni, M.T. and Giulotto, E. (1996a) Four horse genomic fragments containing minisatellites detect highly polymorphic DNA fingerprints. Animal Genetics 27, 286. Anglana, M., Bertoni, L. and Giulotto, E. (1996b) Cloning of a polymorphic sequence from the non-transcribed spacer of horse rDNA. Mammalian Genome 7, 539–541. Bailey, E., Lear, T.L. and Cothran, E.G. (1991) Association of MspI restriction length polymorphism with transferrin in horses. Animal Genetics 22, 436. Binns, M.M., Smith, K., McManus, A., Tavernor, A., Butcher, G. and Mumford, J.A. (1994) Identification of equine genes by characterisation of clones from an equine peripheral blood lymphocyte cDNA library. In: Nakajima, H. and Plowright, W. (eds), Equine Infectious Diseases VII: Proceedings of the Seventh International Conference. R&W Publications, Newmarket, UK, pp. 153–156. Breen, M., Downs, P., Irvin, Z. and Bell, K. (1994a) An equine tetranucleotide repeat: microsatellite MPZ001. Animal Genetics 25, 123. Breen, M., Downs, P., Irvin, Z. and Bell, K. (1994b) Intrageneric amplification of horse microsatellite markers with emphasis on the Przewalski’s horse (E. przewalskii). Animal Genetics 25, 401–405. Breen, M., Lindgren, G., Binns, M.M., Norman, J., Irvin, Z., Bell, K., Sandberg, K. and Ellegren, H. (1997) Genetical and physical assignments of equine microsatellites – first integration of anchored markers in horse genome mapping. Mammalian Genome 8, 267–273. Broad, T.E., Forrest, J.W., Lewis, P.E., Pearce, P.D., Phua, S.H., Pugh, P.A. and StewartScott, I.A. (1995a) Cloning of a DNA repeat element from horse: DNA sequence and chromosomal localization. Genome 38, 1132–1138. Broad, T.E., Ede, A.J., Forrest, J.W., Lewis, P.E., Phua, S.H. and Pugh, P.A. (1995b) Families of tandemly repeated DNA elements from horse: cloning, nucleotide sequence and organization. Genome 38, 1285–1289. Carpenter, M.A. and Broad, T.E. (1994) Polymorphism in the coding sequence of the horse transferrin gene. Genome 37, 157–165. Clegg, J.B. (1987) Gene conversions in the horse α-globin gene complex. Molecular Biology Evolution 4, 492–503.

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Ellegren, H., Andersson, L., Johansson, M. and Sandberg, K. (1992a) DNA fingerprinting in horses using simple (TG)n probe and its application to population comparisons. Animal Genetics 23, 1–9. Ellegren, H.,Johansson, M., Sandberg, K. and Andersson, L. (1992b) Cloning of highly polymorphic microsatellites in the horse. Animal Genetics 23, 133–142. Flint, J., Taylor, A.M. and Clegg, J.B. (1988) Structure and evolution of the horse zeta globin locus. Journal of Molecular Biology 199, 427–437. Francisco, L.V., Langston, A.A., Mellersh, C.S., Neal, C.L. and Ostrander, E.A. (1996) A class of highly polymorphic tetranucleotide repeats for canine genetic mapping. Mammalian Genome 7, 359–362. Gallagher, P.C., Lear, T.L., Coogle, L.D. and Bailey, E. (1999) Two SINE families associated with equine microsatellite loci. Mammalian Genome 10, 140–144. Georges, M., Lequarre, A.S., Castelli, M., Hanset, R. and Vassart, G. (1988) DNA fingerprinting in domestic animals using four different minisatellite probes. Cytogenetics and Cell Genetics 47, 127–131. Gill, P., Kimpton, C.P., Urquhart, A., Oldroyd, N., Millican, E.S., Watson, S.K. and Downes, T.J. (1995) Automated short tandem repeat (STR) analysis in forensic casework – a strategy for the future. Electrophoresis 16, 1543–1552. Godard, S., Vaiman, D., Oustry, A., Nocart, M., Bertaud, M., Guzylack, S., Meriaux, J.C., Cribiu, E.P. and Guerin, G. (1997) Characterization, genetic and physical mapping analysis of 36 horse plasmid and cosmid-derived microsatellites. Animal Genetics 8, 745–750. Godard, S., Schibler, L., Oustry, A., Cribiu, E.P. and Guerin, G. (1998) Construction of a horse BAC library and cytogenetical assignment of 20 type I and type II markers. Animal Genetics 9, 633–637. Guerin, G., Bertand, M., Billoud, B. and Meriaux, J.C. (1993) A genetic analysis of variable number tandem repeat (VNTR) polymorphism in the horse. Genetics Selection and Evolution 25, 435–445. Guerin, G., Bailey, E., Bernoco, D., Anderson, I., Antczak, D.F., Bell, K., Binns, M.M., Bowling, A.T., Brandon, R., Cholewinski, G., Cothran, E.G., Ellegren, H., Forster, M., Godard, S., Horin, P., Ketchum, M., Lindgren, G., McPartlan, H., Meriaux, J.-C., Mickelson, J.R., Millon, L.V., Murray, J., Neau, A., Roed, K., Sandberg, K., Shiue, Y.-L, Skow, L.C., Stott, M., Swinburne, J., Valberg, S.J., Van Haeringen, W.A. and Ziegle, J. (1999) Report of the International Equine Gene Mapping Workshop: Male Linkage Map. Animal Genetics 30, 341–354. Hiromura, K. Sakagami, M., Mashima, S. and Mukoyama, H. (1997) Mononucleotide repeat polymorphisms within the poly-A region of equine SINEs. Animal Genetics 28, 238–246. Hirota, K., Mashima, S., Tozaki, T., Sakagami, M., Mukoyama, H. and Miura, N. (1997) Sequence tagged sites on horse chromosomes. Archivos de Zootecnia 46, 3–7. Hopkins, B., O’Connell, F.M. and Hopkins, J. (1991) Use of DNA fingerprinting in paternity analysis of closely related Exmoor ponies. Equine Veterinary Journal 23, 277–279. Ishida, N., Oyunsuren, T., Mashima, S., Mukoyama, H. and Saitou, N. (1995) Mitochondrial DNA sequences of various species of the genus Equus with special reference to the phylogenic relationship between Przewalski’s wild horse and domestic horse. Journal of Molecular Evolution 41, 180–188. Ishida, N., Hasegawa, T., Oyunsuren, T. and Mukoyama, H. (1996) PCR-RFLP analysis of the cytochrome b gene in horse mitochondrial DNA. Animal Genetics 27, 359–363.

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M. Binns et al. Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985a) Hypervariable ‘minisatellite’ regions in human DNA. Nature 314, 67–73. Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985b) Individual-specific ‘fingerprints’ of human DNA. Nature 316, 76–79. Kay, P.H., Dawkins, R.L., Bowling, A.T. and Bernoco, D. (1987a) Heterogeneity and linkage of equine C4 and steroid 21-hydroxylase genes. Journal of Immunogenetics 14, 247–253. Kay, P.H., Dawkins, R.L., Bowling, A.T. and Bernoco, D. (1987b) Polymorphism of the acetylcholine receptor in the horse. Veterinary Record 120, 363–365. Lindgren, G., Sandberg, K., Persson, H., Marklund, S., Breen, M., Sandgren, B., Carlsten, J. and Ellegren, H. (1998) A primary male autosomal linkage map of the horse genome. Genome Research 8, 951–966. McManus, A. (1997) The identification and characterization of equine genes. PhD thesis, University of Cambridge, UK. Marklund, L., Johansson-Moller, M., Sandberg, K. and Andersson, L. (1996) A missense mutation in the gene for melanocyte-stimulating hormone receptor (MC1R) is associated with the chestnut coat colour in horses. Mammalian Genome 7, 895–899. Marklund, S., Ellegren, H., Eriksson, S., Sandberg, K. and Andersson, L. (1994) Parentage testing and linkage analysis in the horse using a set of highly polymorphic horse microsatellites. Animal Genetics 25, 19–23. Marklund, S., Chaudhary, R, Marklund, L., Sandberg, K. and Andersson, L. (1995) Extensive mtDNA diversity in horses revealed by PCR-SSCP analysis. Animal Genetics 26, 193–196. Marti, E., Breen, M., Fischer, P. and Binns, M.M. (1998) Isolation, characterisation and physical mapping of cosmid derived microsatellites from the horse. Animal Genetics 29, 236–238. Metallinos, D.L., Bowling, A.T. and Rine, J. (1998) A missense mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome: an equine version of Hirschsprung disease. Mammalian Genome 9, 426–431. Murray, J.C., Buetow, K.H., Weber, J.L., Ludwigsen, S., Scherpbier-Heddema, T., Manion, F., Quillen, J., Sheffield, V.C., Sunden, S., Duyk, G.M. et al. (1994) A comprehensive human linkage map with centimorgan density. Cooperative Human Linkage Centre (CHLC). Science 265, 2049–2054. Nikiforov, T.T., Rendle, R.B., Goelet, P., Rogers, Y.-H., Kotewicz, M.L., Anderson, S., Trainor, G.L. and Knapp, M.R. (1994) Genetic bit analysis: a solid phase method for typing single nucleotide polymorphisms. Nucleic Acids Research 22, 4167–4175. Oakenfull, E.A. and Clegg, J.B. (1998) Phylogenetic relationships within the genus Equus and the evolution of α and θ globin genes. Journal of Molecular Evolution 47, 772–783. Oakenfull, E.A. and Ryder, O.A. (1998) Mitochondrial control region and 12S rRNA variation in Przewalski’s horse (Equus przewalskii). Animal Genetics 29, 456–459. Rudolph, J.A., Spier, S.J., Byrns, G., Rojass, C.V., Bernoco, D. and Hoffman, E.P. (1992) Periodic paralysis in Quarter Horses: a sodium channel mutation disseminated by selective breeding. Nature Genetics 2, 144–147. Ryder, O.A., Epel, N.C. and Benirschke, K. (1978) Chromosome banding studies of the Equidae. Cytogenetics and Cell Genetics 20, 323–350. Sakagami, M., Hirota, K., Awata, T. and Yasue, H. (1994a) Molecular cloning of an equine satellite-type DNA sequence and its chromosomal location. Cytogenetics and Cell Genetics 66, 27–30.

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Sakagami, M., Ohshima, K., Mukoyama, H., Yasue, H. and Okada, N. (1994b) A novel tRNA species as an origin of short interspersed repetitive elements (SINEs): equine SINEs may have originated from tRNASer. Journal of Molecular Biology 239, 731–735. Sakagami, M., Tozaki, T., Mashima, S., Hirota, K. and Mukoyama, H. (1995) Equine parentage testing by microsatellite at chromosome 1q2.1. Animal Genetics 26, 123–124. Santschi, E.M., Purdy, A.K., Valberg, S.J., Vrotsos, P.D., Kaese, H. and Mickelson, J.R. (1998) Endothelin receptor B polymorphism associated with lethal white foal syndrome in horses. Mammalian Genome 9, 306–309. Shiue, Y.-L., Bickel, L.A., Caetano, A.R., Millon, L.V., Clark, R.S., Eggleston, M.L., Michelmore, R., Bailey, E., Guerin, G., Godard, S., Mickelson, J.R., Valberg, S.J., Murray, J.D. and Bowling, A.T. (1999) A synteny map of the horse genome comprised of 240 microsatellite and RAPD markers. Animal Genetics 30, 1–9. Shin, E.K., Perryman, L.E. and Meek, K. (1997) A kinase-negative mutation of DNA-PK(CS) in equine SCID results in defective coding and signal joint formation. Journal of Immunology 158, 3565–3569. Tavernor, A.S., Deverson, E.V., Coadwell, W.J., Lunn, D.P., Zhang, C., Davis, W. and Butcher, G.W. (1993) Molecular cloning of equine CD44 cDNA by a COS cell expression system. Immunogenetics 37, 474–477. Tozaki, T., Sakagammi, M., Mashima, S., Hirota, K. and Mukoyama, H. (1995) ECA3: Equine (CA) repeat polymorphism at chromosome 2p1.3–4. Animal Genetics 26, 283. Troyer, D., Leipold, H.W., Howard, D. and Smith, J.E. (1989) A human minisatellite sequence reveals DNA polymorphism in the equine species. Journal of Veterinary Medicine A36, 81–83. Wijers, E.R., Zijlstra, C. and Lenstra, J.A. (1993) Rapid evolution of horse satellite DNA. Genomics 18, 113–117. Xu, X. and Arnason, U. (1994) The complete mitochondrial DNA sequence of the horse Equus caballus: extensive heteroplasmy of the control region. Gene 148, 357–362. Yamashita, H., Murata, S., Komura, K., Okamoto, S., Maeda, Y. and Hashiguchi, T. (1994) Population differentiation of Japanese native horses by DNA fingerprinting. Journal of Equine Science 5, 115–120. Yang, G.C., Croaker, D., Zhang, A.L., Manglick, P., Cartmill, T. and Cass, D. (1998) A dinucleotide mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome (LWFS): a horse variant of Hirschsprung disease. Human Molecular Genetics 7, 1047–1052.

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Immunogenetics of the Horse E. BaileyE. 7Immunogenetics et Bailey al. of 1the , E.Horse Marti2,

and S.

Lazary2

D.G. Fraser3, D.F. Antczak4

1Department

of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA; 2Institute of Animal Breeding, University of Berne, Bremgartenstrasse 109a, Berne 3012, Switzerland; 3Department of Veterinary Microbiology and Pathology, Bustad 402, Washington State University, Pullman, WA 99164–7040, USA; 4James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Introduction Major Histocompatibility Genetics MHC Gene Map and Comparison with Other Species Discovery of the MHC in Horses and the International Workshop Characterization of MHC Class I Genes Serological studies Molecular studies: Southern blotting and RFLPs Equine soluble class I substance (ESCI) Molecular studies: DNA sequencing Characterization of MHC Class II Genes Structure of class II genes Mixed lymphocyte culture and serological tests Molecular studies: Southern blotting and RFLPs Molecular studies: DNA sequencing Characterization of MHC Class III Genes Characterization of Non-MHC Lymphocyte Alloantigens MHC and Bone Marrow Transplantation MHC and Reproduction Association of MHC with Antibody Production MHC and Disease Associations Equine sarcoid tumours Insect bite dermal hypersensitivity ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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Immunoglobulin Genetics Organization of immunoglobulin heavy chain loci Immunoglobulin light chains T-cell Receptor Genetics Severe Combined Immunodeficiency Disease Conclusions Acknowledgements References

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Introduction The word ‘immunogenetics’ was coined to describe detection of blood group antigens by immunoglobulins (Irwin and Cole, 1936). Subsequently, one of the blood groups, specifically the H-2 blood group system of mice, was found to have a profound impact on the immune response and to be part of a major gene family that included histocompatibility genes, immunoglobulins and T-cell receptors (Snell et al., 1976). During the 1960s and 1970s, the field of immunogenetics evolved to focus on this particular gene family. This gene family initially was considered remarkable because of its genetic diversity within each species. Later, the truly remarkable aspect of this gene family was discovered to be its capacity specifically to bind a wide array of molecules in connection with the immune response. The function, DNA sequences and resulting protein structures of the genes in this family show many similarities. Figures 7.1 and 7.2 show schematic representations of these molecules. Each protein is composed of a series of domains formed by disulphide bonds cross-linking a group of 220 amino acids. This common tertiary structure and amino acid sequence homology suggest that these molecules are related to a common but distant evolutionary precursor. Each of these molecules is found as a glycoprotein on the cell surface, although some may be secreted into the plasma, specifically immunoglobulins and sometimes major histocompatibility complex (MHC) class I molecules. The immune function of this gene family appears to be recognition of foreign substances and elicitation of an immune response. The cytoplasmic, N-terminal domains are products of unusual somatic recombination and other mutational events that generate a wide diversity of sequences within binding domains. As a consequence, these molecules have the capacity to bind to a wide array of molecules, including those produced by viruses, bacteria and parasites. Usually the immune response provides protection to infectious or invasive diseases, although this response can also cause allergies and autoimmune diseases. The immune system of horses recently was reviewed elsewhere (Lunn et al., 1998). The genes discussed in this chapter are related to a single genetic precursor dating to the dawn of vertebrate evolution. The evolution of this molecule resulted in a system that enabled a complex life form to recognize self and

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Fig. 7.1. Schematic representations of the prototypical structures of the MHC class I, MHC class II and T-cell receptor molecules are shown. Each of the circular domains represents approximately 200–220 amino acids and ‘–S S–’ represents the placement of disulphide bonds. The most cytoplasmic domains exhibit the greatest genetic variability. The MHC class I gene encodes a single molecule composed of three domains (1, 2 and 3). It is functionally complete in association with the β2-microglobulin molecule (2), a protein unrelated to this gene family. The MHC class II molecule results by association of two MHC gene products, the α-chain and the β-chain, each exhibiting two domains. The T-cell receptor has a similar structure with an α- and β-chain, as shown here, or a γ- and δ-chain. The dotted lines for the T-cell receptor indicate the ‘V’ region that has variation as the result of somatic recombination or other mutational events.

non-self and to evolve a system of specific immune responses. Here we review the work conducted to characterize these genes, their genomic organization in the horse and especially their diversity among horses.

Major Histocompatibility Genetics The genes of the MHC are among the most polymorphic found in any species and they have been subject to more investigation than any other set of genes. Their study has been important to understanding the biology of evolution, immunology, transplantation biology and disease associations, as well as in forensic applications including parentage testing. Therefore, the major focus of MHC research in the horse has been: (i) identification of homology between the horse MHC and the MHC of other species; (ii) characterization of horse MHC genes; and (iii) investigation of associations between some equine diseases and MHC genes.

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Fig. 7.2. Schematic representation of the structure of an immunoglobulin molecule is shown. It also is composed of a series of 220 amino acid domains. The heavy chain determines function of the molecule. The variable region, denoted by dotted lines, determines the antigenic specificity. The variable region is the product of somatic recombination and other mutational events. The heavy chain constant domains are denoted CH1, CH2 and CH3 while the variable domain is identified as VH. Likewise, the constant region of the light chain is denoted as CL and the variable region as VL.

A common set of genes defines the MHC. However, no common mechanism has yet explained the wide variety of health problems that appear to have a hereditary component linked to the MHC. For the horse, MHC genes were implicated in such health problems as the occurrence of sarcoid tumours and ‘sweet itch’ or insect bite dermal hypersensitivity, and they also appear to have an impact on response to immunization with commercial vaccines. These diseases clearly have an immunological component, as do many other MHCassociated diseases.

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Clearly the MHC is important in connection with health and welfare of horses. The equine MHC system was reviewed previously in a volume dedicated to infectious diseases of the horse (Antczak, 1991) and more recently in a volume reviewing the MHC of domestic animal species (Marti et al., 1996). Here we update those reviews and describe the essential aspects of this important system for the horse in the context of genetics. This review will describe the approaches used for characterization of the MHC genes, beginning with the initial serological studies and including the more recently developed molecular tests.

MHC Gene Map and Comparison with Other Species The MHC for most species is quite similar in organization. Figure 7.1 shows a schematic representation of the MHCs found in humans, mice, cattle and horses. The DNA sequences are completely known for the human and mouse MHCs while for other species even the order of genes has not been established definitively. The MHC in all species is characterized as possessing three classes of genes (I, II and III) and, in most species, is located within a chromosome region of about 4 centiMorgans (cM) or about 4 ×106 bp. Class I genes encode proteins which are responsible for directing immune responses to pathogens which invade the cell, for example viruses, and are also primarily responsible for organ and tissue transplant rejection. Class I molecules bind intracellular peptides and present them at the cell surface to cytotoxic T lymphocytes. Class II genes encode two types of proteins, an α molecule and a β molecule, which form a dimer molecule that binds soluble antigen, internalized by the antigen-presenting cell, and presents these peptides to helper T lymphocytes to elicit immune responses. Class III genes include a diverse array of genes that are not obviously related to class I or class II genes in sequence or structure, yet they occur within the MHC region of all species. The class III genes often play a role in some aspect of the immune response and include genes for complement, tumour necrosis factor and other genes involved in antigen processing. The horse MHC, or equine lymphocyte antigen (ELA) system, was localized to horse chromosome 20q14–q22 on the basis of in situ hybridization using porcine and human class I MHC DNA probes (Ansari et al., 1988; Mäkinen et al., 1989). In family studies, the ELA system was shown to be approximately 1.4 cM from the A blood group locus (Bailey et al., 1979) with the F13A locus (coagulation factor XIII, A1 polypeptide locus) another 10–12 cM further along the chromosome (Weitkamp et al., 1989). F13A is not considered part of the MHC. However, this linkage relationship appears to be conserved in many species, as indicated in Fig. 7.3. F13A was localized to chromosome region 20q13 (Godard et al., 1998). This suggests that the gene order along chromosome 20 should be: centromere–F13A–A blood group system–ELA–telomere. This order, with F13A centromeric to the MHC, is different from that seen in other species and reflects obvious chromosomal

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Fig. 7.3. Structure and linear organization of the MHC region for several species. The organization of MHC class I, II and III loci is illustrated for humans, mice, cattle and horses. The thick horizontal lines represent the chromosome and the large attached circles represent the centromeres. The locations of loci are denoted by vertical lines intersecting the chromosome line. The class I, II and III loci are identified below the chromosomes. The diagram is not to scale, since the MHC regions are approximately 4 cM long and the chromosomes are several hundred cM in length. The interruption (//) of the cattle chromosome denotes a possible distance of 17 cM between the DYA locus and the rest of the cattle MHC.

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rearrangement. Although multiple class I and class II loci have been identified within the ELA system, the order is unknown and probably cannot be determined until the entire region is sequenced. Horse class III genes were also mapped to the ELA region. Kay et al. (1987) mapped the complement gene, C4, and steroid 21-hydroxylase (CYP21) to the MHC of the horse. Additionally, studies with somatic cell hybrid panels indicated that the gene for tumour necrosis factor-α (TNFA) is syntenic with the MHC (Bailey et al., 1995).

Discovery of the MHC in Horses and the International Workshop Even before the importance of the MHC was understood, scientists investigated the genetic variation of antigens on lymphocytes. Extensive variation was already known for red blood cell antigens (see Chapter 5) and it was natural to pursue investigations of genetic variation detectable on lymphocytes. Lymphocytes are produced in the bone marrow and belong to the same cell lineage as red blood cells. Indeed, they are sometimes referred to as white blood cells because isolated cells appear white in colour. Lymphocytes are primarily responsible for the immune response. Lymphocyte typing was used to investigate genetic variation among horses in several laboratories using a wide array of techniques. Every laboratory used a different nomenclature to describe their results (Lazary et al., 1975, 1980a; Schmid et al., 1975; Bright et al., 1978; deWeck et al., 1978; Bailey et al., 1979; Bailey, 1980; Mottironi et al., 1981; Antczak et al., 1982). On the basis of family studies demonstrating linkage of a system of lymphocyte alloantigens to the A blood group system, the ELA locus was defined (Bailey et al., 1979). By then it was clear that all of these scientists were investigating a single, very Table 7.1. Class I and class II genes of the equine MHC. The loci that have been identified, the effective test methods and number of alleles and loci are indicated. Locus ELA-A ELA-B ELA-C ESCI ELA-D DRA DRB DQA DQB 5DQA

MHC class Test methods I I I I II II II II II II

Serological Serological Serological Western blotting Serological/MLR Sequence/SSCP Sequence/SSCP Sequence/RFLP/SSCP Sequence/SSCP Sequence/SSCP

No. of alleles Comments 22+ 1+ 1+ 2 5+ 6 11+ 23+ 13+ 1+

Significant effort Limited investigation Limited investigation Significant effort One locus, unique polymorphism Two or three loci transcribed One locus One locus DQA exon 2 sequence on ECA5

Sequence indicates DNA sequencing; SSCP indicates the detection of single strand conformational polymorphism; RFLP indicates detection by restriction fragment length polymorphism; serological testing indicates the use of lymphocytotoxicity testing; MLR indicates testing for mixed lymphocyte response.

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important system that would be useful in research on the immune response and to uncover disease associations. To standardize the nomenclature and to collaborate in further research, a series of five workshop meetings were held during the 1980s, under the auspices of the Dorothy Russell Havemeyer Foundation (Bull, 1983; Bailey et al., 1984; Antczak et al., 1986; Bernoco et al., 1987a; Lazary et al., 1988). Based on the workshop effort and independent studies by workshop participants, powerful testing systems were devised to investigate the genetics, biology and significance of the MHC to health and welfare in horses. Lymphocyte typing was also useful simply to characterize genetic variation within and between breeds of horses (Bailey, 1983b; Varewyck et al., 1985; Antczak et al., 1986; Halldorsdottir et al., 1991; Horin et al., 1998). These workshops led the way to subsequent epidemiological, immunological and molecular studies on the MHC during the 1990s. Table 7.1 summarizes the results of serological and molecular studies to detect equine MHC variation as described below.

Characterization of MHC Class I Genes Serological studies MHC class I genes were characterized biochemically in humans and mice as 42–44 kDa transmembrane glycoproteins associated with the 12 kDa β2-microglobulin at the surface of all tissues (Fig. 7.1). The initial methods to study the MHC were based on serological tests of lymphocyte alloantigens. Lymphocytes were isolated from horse blood samples and tested with antisera for the presence and absence of antigens in microcytotoxicity tests. The antisera were derived from the serum of parous mares and the anti-lymphocyte antibodies appeared to be the product of stimulation during pregnancy. The early genetic studies on horses characterized a single genetic system designated the ELA system (Bull, 1983). This system was closely linked to the A blood group system, exhibiting only 1.6% recombination (Bailey et al., 1979; Bailey, 1983a; Guérin et al., 1988). A standardized typing method was adopted by the workshop participants to facilitate sharing of reagents (Bailey et al., 1984). Based on extensive studies in families and different horse populations, most of these antigens appeared to be the products of a single locus; specifically, no horse was ever found with more than two of the antigens, and therefore they were designated as products of the A locus of the ELA system or, more properly, the ELA-A locus (Antczak et al., 1986; Bernoco et al., 1987a). By the conclusion of the last workshop, the ELA-A locus was shown to include 17 alleles which were designated A1–A10, A14, A15, A19 and provisionally W16, W17, W18 and W20 (Bernoco et al., 1987a; Lazary et al., 1988). The ELA-A locus clearly encoded MHC class I molecules based on biochemical studies demonstrating that their size corresponded to that of MHC class I antigens in humans and mice (Bernoco et al., 1987b; Donaldson et al., 1988). At the same time, evidence was found for a second ELA class I locus,

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designated ELA-B, based on serological, family and biochemical studies (Bernoco et al., 1987a). Additional evidence for a second locus was provided by biochemical studies (Donaldson et al., 1988), but these studies did not result in workshop assignment of specificity or locus designation. However, Hirni and co-workers (1988) conducted biochemical and family studies on a specificity previously adopted by the workshop, W21, and demonstrated that it was a class I gene product which segregated in families in coupling with genes for ELA-A and ELA-B. Therefore, W21 was the product of a third ELA class I locus, presumably ELA-C. These studies also suggested that the presumptive ELA-B and ELA-C loci were minimally polymorphic. After 1988, the focus of ELA work shifted to molecular studies and the workshop verification of new serological specificities was discontinued. Nevertheless, other class I antigens continued to be discovered, including Be-22, -24, -25, -26 and -108 (reviewed in Marti et al., 1996). At the end of these studies, three MHC class I loci were described for the horse, but only one showed extensive polymorphism. This was a distinct contrast to serological studies of the human HLA system that identified three highly polymorphic class I loci (A, B and C) and the murine H-2 system that exhibited two polymorphic class I loci (K and D) (Fig. 7.3). Whether the limited polymorphism of class I antigens in the horse reflected a significant biological difference or was simply an artefact of the limited horse research remains undetermined. However, molecular studies demonstrated that variation of MHC class I genes is greater than that detected serologically.

Molecular studies: Southern blotting and RFLPs Vaiman and co-workers (1986) were the first to use Southern blotting of DNA to identify the MHC genes in horses and investigate restriction fragment length polymorphisms (RFLP). They isolated horse DNA, digested it with restriction enzymes and separated the resulting fragments on agarose gels. The DNA was blotted on to a nitrocellulose membrane, then DNA from cloned human class I, class II (DRB and DQA) and class III (complement C4) genes was hybridized to the nitrocellulose to determine whether the horse genes showed homology to the human genes and also to determine whether they could detect genetic variation between horses. All four genes hybridized to the horse and showed genetic variation, including a complex pattern for MHC class I genes. Alexander and co-workers (1987) used mouse clones for class I and class II (Aa, Ea, Ab and Eb) genes and likewise observed hybridization and polymorphism. They compared the RFLP patterns for 14 Standardbred horses known to be homozygous for ELA alleles A1, A3, A4, A5, A6 and A10. Each horse possessed 12–14 non-polymorphic fragments and 10–19 fragments which were polymorphic. No two horses had the same RFLP pattern; however, horses sharing the same ELA types were more similar than horses with different ELA types.

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This report was significant because, up to this time, only one class I MHC locus had been described for the horse using serological studies. These results suggested that, like humans and mice, horses may possess as many as 30 class I loci. However, as for humans and mice, many of those genes may not be expressed and many may not be polymorphic. If the genes are not expressed or if they are not polymorphic, then serological studies will not detect them. No locus-specific MHC class I clones were found and, as a result, RFLP patterns were highly complex and of almost no use for the development of a typing system because of the large number of class I loci. Guérin and coworkers (1987, 1988) did use Southern blotting techniques to type several horse families and demonstrated co-segregation of the patterns with inheritance of ELA-A genes. They also described a recombinant family for the A blood group and ELA-A locus in which the A blood group system was shown to fall outside the region encompassed by the class I and class II MHC genes.

Equine soluble class I substance (ESCI) Normally, MHC class I molecules are expressed on the cell surface. However, in the mouse, a soluble class I molecule was discovered and designated Q10 (Lew et al., 1986a). Investigations on a wide range of species demonstrated that most members of the family Perissodactyla also possessed a soluble class I molecule, while none was found for members of any other families, including closely related Artiodactyla species (Lew et al., 1986b). In the horse, the molecule was slightly smaller than the membrane-bound form (36 kDa as opposed to 44 kDa) and present in serum in concentrations of 30 µg ml−1. The molecule was designated equine soluble class I substance (ESCI). ESCI was polymorphic in horses in that ESCI was not detected in some domestic horses nor in four of four Przewalski’s horses tested. The genetic variation found among horses led to the demonstration that ESCI was a product of the equine MHC by way of linkage analysis (Lew et al., 1986c). A function for ESCI and Q10 is unknown; however, its absence in some horses and mice indicates that it is not an essential component of the immune system.

Molecular studies: DNA sequencing Although Southern blotting studies indicated that many class I loci existed for the horse, all of these loci may not be expressed. Only those genes transcribed into RNA can be expressed as proteins, so a first step to address this question is to look at transcription. Barbis and co-workers (1994a) isolated and sequenced class I complementary DNA (cDNA) clones from a library derived from a horse known to be MHC homozygous by descent. Only two different sequences were revealed, suggesting expression of only two loci. However, Ellis and co-workers (1995) screened a cDNA library from an ELA heterozygous individual and uncovered seven different MHC class I transcripts,

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suggesting expression of at least four class I loci. However, it was not possible to identify locus-specific DNA sequences in order to assign the genes to specific loci or to determine the number of loci that might be polymorphic. Holmes and Ellis (1999) compared cDNA sequences for MHC class I exons 4–8 for Perissodactyla species including the domestic horse (E. caballus) Przewalski’s horse (E.f. przewalskii), domestic donkey (E. asinus), Grevy’s zebra (E. grevyi), Persian onager (E. hemionus onager), two rhinoceros species (Rhinocerus unicornus, Dicerorhinus sumatrensis) and a Malayan tapir (Tapirus indicus). The resulting sequences fell into six phyletic groups, designated, A–F. Group F contained the sequences associated with the rhinoceros and the tapir. The sequences for the equids were distributed among groups A–E. Group B was the largest and contained sequences from all the equid species. Since some individuals were found to contain more than two sequences from group B, this group probably represents at least two loci. Together, these results indicate that evolutionary expansion of the MHC class I loci occurred among Equidae after divergence from the other Perissodactyla but before speciation of Equidae. Developing a molecular approach to MHC class I typing would be advantageous. Serological typing of MHC class I genes requires fresh cells and the reagents are difficult to produce. Unfortunately, a systematic approach has not yet been developed for molecular typing of MHC class I genes for the horse due to the costs involved and the complexity of class I genes. With many class I genes encoded in the DNA but few loci being expressed, it will be difficult to sort out the expression of these genes using the molecular approaches at hand.

Characterization of MHC Class II genes Structure of class II genes MHC class II molecules bind soluble molecules specifically and present them to the helper T lymphocytes that augment the immune response. The entire MHC molecule is composed of two membrane-bound glycoproteins, a 33 kDa α-chain and a 28 kDa β-chain, which combine to form an antigen-binding site (Fig. 7.2). Antigen binding occurs in the N-terminal domain of the molecule. This region appears to be responsible for most of the genetic variation detected between class II gene products and reflects different binding capacities of each class II allele. Every species possesses a series of class II loci. Strong homology has been observed for some loci across species boundaries when comparing DNA and protein sequences. For example, the class II E loci of the mouse (α and β) are homologous to the DR loci (α and β) of humans; likewise the A loci of the mouse correspond to the DQ loci of humans (Fig. 7.3). (For human class II gene products, HLA scientists adopted the practice of representing ‘α-chain’ gene products with an ‘A’ and ‘β-chain’ gene products with a ‘B’ rather than

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using the Greek symbols. Research in other species, including the horse, has followed this practice.)

Mixed lymphocyte culture and serological tests Most of the MHC and disease associations discovered for people were associations with class II genes. Initially, these genes were characterized using a technique called mixed lymphocyte culture (MLC). For this test, lymphocytes from two individuals were cultured together. If the individuals had different class II genes, then the lymphocytes responded to the differences by dividing rapidly. If the individuals were the same, then no stimulation or cell division occurred. Lazary and co-workers (1980b) demonstrated that responsiveness to MLC in families co-segregated with their ELA-A type. However, this association was not apparent among randomly selected horses sharing class I alleles in the population. This study was the first demonstration of class II genes in the horse and effectively the first demonstration that the ELA system was the MHC of the horse since it included both class I and class II genes that were tightly linked. Developing serological tests for class II gene products became a major priority for the last two ELA workshops (Bernoco et al., 1987a; Lazary et al., 1988). This work proved to be more difficult than expected. In humans and mice, class II antigens are expressed primarily on the nylon wool-adherent cells that make up approximately 20% of the lymphocyte population. These cells include B lymphocytes and monocytes. Initially, the ELA scientists looked for antigens that would be expressed only in such a subset of lymphocytes. However, Crepaldi and co-workers (1986) demonstrated that class II antigens are expressed on all lymphocytes in the horse, as was subsequently found for dogs (Doveren et al., 1986) and sheep (Dutia et al., 1993). Therefore, testing class II in horses did not require development of the complex nylon wool adherence cells separation procedures. However, pregnant mares did not readily produce antisera to class II antigens, so the reagents needed to be produced by matching horses for class I specificities then immunizing them to produce antisera to class II antigens. Following the work of Lazary and co-workers in Switzerland and corroboration in other laboratories, three antigens were identified as MHC class II gene products and designated ELA-W13, -W22 and -W23 by the workshop (Lazary et al., 1988). Lazary and co-workers (1986) demonstrated that cells homozygous for ELA-W13, -W22 or -W23 behaved as ‘homozygous typing cells’ in MLC. Immunoprecipitation studies demonstrated that these antigens had a molecular weight characteristic of MHC class II antigens (Hesford et al., 1989). Other class II antigens described by Lazary and co-workers but not included in the workshop report were BeVIII and Be200 (Marti et al., 1996). Most work investigated the presence or absence of these antigens on the cell surface. However, Barbis and co-workers (1994b) used monoclonal antibodies and flow cytometry to compare the expression of MHC class II

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antigens on the cell surface. They discovered that the W13 antigen, referred to as D3 in that manuscript, was expressed at lower levels than other antigens. The consequence of this variation in expression is unknown but worthy of future study, especially since the studies described below reported that this antigen was associated with susceptibility for several diseases. In a previous review, a nomenclature convention was adopted to signify homology to the human MHC class II genes, specifically, introduction of a ‘D’ prefix to the allele or antigen designation (Marti et al., 1996). Henceforth, we shall refer to these alleles as ELA-Dw13, Dw22, Dw23, DBeVII and DBe200. However, the relationship of the serological determinants to the DR and DQ loci remains unknown and is signified by the ‘w’ or ‘Be’ following the prefix.

Molecular studies: Southern blotting and RFLPs Class II MHC genes in the horse were investigated using Southern blotting and RFLP techniques in the same way as for class I genes. While serological studies identified limited genetic variation of class II, Southern blotting and RFLP studies demonstrated extensive variation of class II in horses using a variety of DNA probes from mice and humans (Vaiman et al., 1986; Alexander et al., 1987; Hänni et al., 1988; Guérin et al., 1987, 1988). The levels of variation were comparable with that observed in human populations, suggesting a similar genetic structure involving multiple polymorphic loci. While this approach seemed more promising for the detection of MHC class II variation compared with serological approaches, the cost of the tests, the cross-hybridization of DNA probes between loci and the complexity of the system made Southern blotting an unattractive method for routine typing class II MHC. Fortunately, Southern blotting techniques were soon replaced by methods which relied on the use of polymerase chain reaction (PCR) and DNA sequencing.

Molecular studies: DNA sequencing DRA The DRA locus homologue was the first equine MHC gene sequenced (Albright et al., 1991). In other species there is one DRA locus with no or limited variation. Indeed, the early Southern blotting studies of the horse indicated limited variation (Alexander et al., 1987; Hänni et al., 1988). Studies of the second exon using allele-specific PCR demonstrated genetic variation, with three common alleles found among horses (Bailey, 1994). Sequence studies and the use of single-strand conformation polymorphism (SSCP) demonstrated that most of the base changes resulted in amino acid changes in the putative antigen-binding site of this molecule (Albright-Fraser et al., 1996). Eight species of the genus Equus were tested, including horse, donkey, onager and zebra species, resulting in the discovery of five alleles at a single locus. A sixth DRA allele was found in donkeys by DNA sequencing (E. Marti, unpublished

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observations). Since the DRA locus has little or no variation in other species, this discovery was remarkable. The variation occurs within the antigen-binding site and may result in differences in the ability of horses to respond to different antigenic stimuli. DRB In humans, nine loci were discovered for DRB, four of which encoded functional class II β molecules and five which proved to be pseudogenes. At the same time, variation in the number of loci was found, with individual haplotypes possessing two to five loci. Only one or two loci are expressed within a given haplotype, but those loci are highly polymorphic, resulting in high DRB variability across the population (Trowsdale, 1995). Gustavsson and Andersson (1994) used PCR to amplify the second exon of DRB in the horse and sequenced it to investigate genetic variation. They uncovered three alleles, designated DRB*1, DRB*2 and DRB*3. Fraser and Bailey (1996) used this approach to uncover an additional eight alleles, designated DRB*4–DRB*11. SSCP and family studies were used to demonstrate that one horse family possessed three DRB loci. However, comparison of the second exon sequences did not reveal locus-specific sequences. Amplification of these three alleles from a cDNA library indicated that the message is transcribed and may be expressed for all three loci. This locus appears to be highly polymorphic based on SSCP studies, and many additional alleles remain to be described for the DRB loci. Hedrick and co-workers (1999) investigated variation in the second exon of DRB among 14 Przewalski’s horses. They identified six new sequences, designated Eqpr-DRB*1–*6. Since this locus shows a greater level of polymorphism in other species, the authors suggested that the Przewalski’s horse may have reduced variation as a result of a reduced founder population in the early 1900s. A phylogenetic comparison of those sequences with those reported for the horse suggested that some allelic variation of DRB was ancestral to divergence of the two species. Family and population studies indicated that the Przewalski’s horse has only two loci for DRB. Fraser and Bailey (1996) reported three loci for the horse, although evidence was reported suggesting only two loci in some horses. DQA A full-length cDNA clone for DQA was sequenced and found to be of the expected size and homologous to DQA genes in other species (Szalai et al., 1994a). However, investigations of exon 2 sequences from six horses revealed limited genetic variation, with only two alleles. This was in distinct contrast to studies of DQA homologues in other species that were found to be highly polymorphic. Using the sequence information from this study plus sequence information from other species, DNA oligonucleotide primers were designed to amplify the second exon of DQA in the horse, and genetic variation was investigated using SSCP and DNA sequencing (Fraser and Bailey, 1998). Sequence studies in

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horses, onagers and donkeys led to the description of 16 additional alleles, of which 13 alleles were found in horses (DQA*0201–1501), one sequence was unique to the onager (DQA*1601) and two sequences were unique to donkeys (DQA*1701–1801). The allele represented by each sequence could be distinguished readily using SSCP, and population and family studies were conducted readily. Family studies indicated that most alleles belonged to a single locus. An additional six DQA alleles (DQA*1901–2401) were found in further family studies among a variety of domestic horse species. The most remarkable discovery of this study was the occurrence of a second DQA locus homologue on a separate chromosome. While the equine MHC is located on horse chromosome 20, DQA*1301 clearly belonged to a separate locus on horse chromosome 5 (Fraser et al., 1998). This was remarkable because it represented the first time that MHC genes have been found distributed on more than one chromosome. To distinguish the locus on chromosome 5 from the MHC-linked locus on chromosome 20, it was designated 5DQA. It remains to be seen whether an entire class II gene is present on chromosome 5 and whether or not it is expressed and plays a role in regulating the immune system. DQB PCR and DNA sequencing were used to investigate sequence variation for the second exon of the equine homologue of DQB with primers shown effective in a wide range of species (Gyllensten et al., 1990; Szalai et al., 1993). A single locus with 13 alleles was described (Szalai et al., 1993). Fourteen horses were lymphocyte typed for four MHC class II specificities (ELA-Dw23, Dw13, Dw22 and Dbe200), and the serological results correlated exactly with the amino acids in the antigen-binding site (positions 55–60). This suggested that the ELA serological determinants were a product of the DQB locus. A full-length cDNA clone was sequenced and found to be longer than the DQB gene in most other species (Szalai et al., 1994b).

Characterization of MHC Class III genes The unifying characteristic of class III genes is that they occur within the MHC region and have been reported within the MHC of most species. They are not members of the MHC/immunoglobulin gene family or any other known gene family. Although the genes and their products are important for a variety of functions, significant polymorphism has not been found which influences health or the immune response in natural populations. Consequently, little work has been conducted in developing this aspect of the equine MHC. As shown in Fig. 7.3, both CYP21 and C4 have been mapped to the equine MHC using Southern blotting studies (Kay et al., 1987). TNFA has been sequenced (Su et al., 1991) and synteny demonstrated with the ELA system (Bailey et al., 1995).

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Characterization of Non-MHC Lymphocyte Alloantigens During the 1970s and 1980s, thousands of antisera to lymphocyte alloantigens were investigated to determine the genetics of the determinants they detected. Almost all were specific for MHC gene products. However, two polymorphic non-MHC systems were uncovered. ELY1 was described by Lazary et al. (1982) based on discovery of the ELY-1.1 antigen. This system is expressed on lymphocytes but not on platelets or erythrocytes. A second antigen was found and this allele, ELY1.2, closed the system, i.e. no other alleles are know to exist (Byrns et al., 1987). The function of this system and possible homology to systems in other species is unknown. Antczak (1984) described a second non-MHC system, designated ELY2. This system had a single antigen, ELY2.1, which was detectable on lymphocytes, platelets and erythrocytes by absorption studies. Population and family studies demonstrated that ELY2 was not homologous to any of the known blood group systems for the horse (Bailey and Henney, 1984). Only a single gene product has been detected for this system and its function and possible homology to systems in other species is also unknown.

MHC and Bone Marrow Transplantation MHC typing is used to match organ recipients with organ donors in human medicine. The ability to exchange tissue grafts was one of the first characteristics identified for the MHC and is why they were described as ‘histocompatibility’ genes. The control of tissue graft rejection is not part of the interest in MHC genetics for the horse. However, there was one instance in which ELA typing was used for tissue grafts in a horse. Bue et al. (1980) used ELA typing to match donor and recipient horses for a successful bone marrow transplantation in an Arabian horse suffering from severe combined immunodeficiency (SCID). SCID-affected horses, like the human counterpart, lack both T and B lymphocytes. They are unable to make immune responses and die within 2 months of birth without treatment. In this set of experiments, the ELA-matched bone marrow stem cells were engrafted successfully from a normal donor animal into the SCID recipient. As a treatment, this may be too expensive for routine application to horses. Fortunately, the question of developing this treatment should be moot in the future since the gene defect responsible for equine SCID was identified and a test for detection of carriers was developed (Shin et al., 1997; see Chapter 8).

MHC and Reproduction From the beginning, it was apparent to the ELA serologists that foaling mares commonly produced antibodies directed against paternally inherited

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alloantigens expressed on lymphocytes from the foal in MHC-incompatible pregnancies. Antczak and co-workers (1984) performed a thorough investigation and discovered that over 90% of the primiparous mares produce such antibodies and that these antibodies appear as early as day 60 of gestation. This contrasted strongly with data for human pregnancies in which such antibodies were observed only 15% of the time in multiparous women. These antibodies do not lead to abortions and may be beneficial to pregnancy. If so, this would be a powerful tool to maintain MHC polymorphism. (See Chapter 12 for more details.) Bailey (1986) investigated the inheritance of ELA antigens in Standardbred pregnancies and found no evidence for a selection against or for histoincompatible pregnancies. However, transmission ratio distortion was discovered in one extended family. The effect was a small but statistically significant segregation distortion of the ELA-A10 allele. Heterozygous stallions transmitted to their offspring the A10 allele more often than their other ELA allele (57% of the time; P < 0.005). This distortion was not seen among mares from the same family nor in unrelated Standardbred families carrying the ELA-A10 allele. Therefore, the effect was probably determined by other genes occurring on this MHC haplotype and not the A10 allele itself. Initially, the effect was thought to be analogous to a set of genes found in the mouse, called the T-complex, which influence spermatogenesis, among other things. However, subsequent studies demonstrated that T-complex genes are not linked to the MHC in the horse (Langemeier et al., 1993). MacCluer et al. (1988) found no effect of ELA on gestation length. However, their study found some evidence for influence of the ELA types of the dam, the sire and of sire–dam ELA incompatibility on fertility rate. The effects did not reach statistical significance. In a subsequent study to investigate the association of ELA types and results from uterine biopsy, a trend but not statistical significance was observed for pathological status and ELA types (Weitkamp et al., 1991). The authors regarded the results as inconclusive and suggestive for future work.

Association of MHC with Antibody Production Genetic studies in mice (McDevitt et al., 1972), guinea pigs (Levine et al., 1963), chickens (Koch and Simonsen, 1977) and cattle (Lie et al., 1986) have shown strong associations of the MHC with the capacity to produce low or high antibody titres in response to immunization. However, such associations were only observed when vaccination antigens of limited structural heterogeneity and with a small number of antigenic determinants, such as synthetic polypeptides, were used. An influence of the MHC on the immune response has also been observed with more complex antigenic molecules when they were applied in small quantities (Vaiman et al., 1978; Pevzner et al., 1979; Longo and Paul, 1982). The immune response to complex antigens is probably under polygenic control; therefore, the influence of the MHC is less, if at all,

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demonstrable. Selection for high or low antibody production against complex antigens over generations was carried out in mice (Biozzi et al., 1975, 1979), chickens (Van der Zijpp and Nieuwland, 1989) and goats (Eide et al., 1990), and resulted in descendant groups producing generally low or high antibody responses. In these experiments, differences in the distribution of MHC haplotypes were observed between high and low antibody titre-producing descendant groups. In the horse, Lazary et al. (1978) found a relationship between the immune response to tetanus toxoid in unrelated adult horses and the presence of specific lymphocyte alloantigens, which were detected with antisera not defined for ELA specificities. In a more recent study (Bodo et al., 1994), antibody titres were measured after the first vaccination and after a boosting injection in ELA-typed foals (n = 184) belonging to various breeds and living in different stud farms. The vaccine (Resequin®) contained multivalent viral antigens such as equine herpes virus type 1 (EHV-1), influenza A/equi 1 and 2, and traces of ovalbumin. The age of the foals, the specific environment of the stud farm and, for some vaccine antigens, the sex of the animal influenced the immune response significantly. When all foals were taken into account, evidence for a weak influence (0.1 > P > 0.01) of certain ELA antigens was demonstrated by analysis of variance. Further analysis showed that Thoroughbreds carrying ELA-A10 displayed lower antibody titres against EHV-1 than foals lacking this ELA specificity (P < 0.01). Among warmblood foals, an association with the same level of significance was found between an ELA class II specificity and higher antibody titres against influenza A equi 2 virus haemagglutination antigen. This study does not really prove a genetic linkage or a true association of the immune response with MHC alleles. However, it leads one to suspect that an actual effect might be present but that the complexity of the immunogens used and the great heterogenicity of the material (five different breeds and eight different studs!) do not allow the detection of stronger associations.

MHC and Disease Associations The identification of genes or markers associated with disease predisposition is one important aim of equine genetics research. A wide variety of diseases have shown associations with MHC genes in humans (Tiawara and Terasaki, 1985) and in other species (Schook and Lamont, 1996). Therefore, a series of studies were conducted between 1985 and 1992 to investigate associations between MHC and diseases in horses. Table 7.2 summarizes these results. The experiments uncovered a strong association between ELA genes and equine sarcoid tumours as well as, to some extent, susceptibility to insect bite dermal hypersensitivity (sweet itch). For laryngeal hemiplegia (Poncet et al., 1989) and chronic bronchitis (Marti et al., 1991), a non-MHC-based genetic predisposition was shown. Meredith et al. (1986) studied laminitis among Standardbreds and Thoroughbreds, but failed to demonstrate increased frequency of any ELA antigens in diseased animals versus controls.

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chronic bronchitis laminitis hemiplegia laryngis

sweet itch

Swiss warmblood Swiss warmblood Irish warmblood French warmblood Freiberger Swiss warmblood Thoroughbred Swedish warmblood Icelandic ponies Swiss warmblood Swiss warmblood Thoroughbred/Standardbred Swiss warmblood

Thoroughbred

EHV1 vaccine

Occurrence of: sarcoid tumours

Breed

ELA-A5, Dw13 (P < 0.01) ELA-Dw13 (P < 0.001) ELA-Dw13 (P < 0.01) ELA-A3 (P < 0.01), Dw13 (P < 0.0001) ELA-DBe108 (class I) (P < 0.01) Co-segregation with ELA haplotypes (P < 0.0001) ELA-A3, Dw13 (P < 0.0001) Co-segregation with ELA-A3, Dw13 (P < 0.01) ELA-Dw22 (P < 0.01) Co-segregation with ELA-Dw23 (P < 0.01) None None None

Low response with ELA-A10 (P < 0.01)

Association

Summary of investigations of MHC influences on horse diseases (adapted from Marti et al., 1996).

Condition

Table 7.2.

Lazary et al. (1985) Lazary et al. (1994) Lazary et al. (1994) Lazary et al. (1994) Lazary et al. (1994) Gerber et al. (1988) Meredith et al. (1986) Broström et al. (1988) Halldorsdottir et al. (1991) Marti et al. (1992), Lazary et al. (1994) Marti et al. (1991) Meredith et al. (1986) Poncet et al. (1989)

Bodo et al. (1994)

Reference

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Equine sarcoid tumours The equine sarcoid is the most common skin tumour in the horse. Although it does not metastasize, it often recurs after surgical removal and, when located in the saddle or girth region, impairs the use of the horse. These fibroblastic, wart-like lesions are caused by a virus closely related or identical to the bovine papilloma virus (BPV) type 1 or 2 (Angelos et al., 1991; Reid and Smith, 1991; Otten et al., 1993). The association between ELA class I or class II antigens and susceptibility to sarcoids has been described in the USA (Meredith et al., 1986), in Sweden (Broström et al., 1988) and in Switzerland (Lazary et al., 1985, 1994), for different breeds. In French, Swiss, Irish and Swedish warmbloods as well as in Thoroughbreds, the class II antigen Dw13 occurs with increased frequency in sarcoid-affected horses compared with controls (P, corrected for multiple comparisons, < 0.01). ELA-Dw13 shows linkage disequilibrium (association in populations) to the class I alleles A3, A5 and A15. Therefore, depending on the investigated subpopulation, these class I alleles can also display weak associations with susceptibility to sarcoid. In breeds where the class II allele Dw13 does not occur, sarcoids are sometimes very rare, e.g. among Standardbreds. Among Freibergers (Swiss draught horse), the class I antigen encoded by Be108 is associated with the occurrence of sarcoid tumours and a linked class II antigen is not yet identified. Despite the strong association of Dw13 with sarcoid susceptibility among warmblood horses, the aetiological fraction varies between 0.28 and 0.72 within warmblood breeds; in other words, some of the affected horses do not carry the ELA-Dw13 and some of the horses with ELA-Dw13 do not have sarcoid tumours. The possible segregation of a specific haplotype among diseased animals was investigated in half-sibling groups sired by ELA heterozygous stallions (Gerber et al., 1988). Within such half-sibling groups, nearly all sarcoid-affected offspring had inherited one particular paternal ELA haplotype. However, the haplotypes co-segregating with sarcoid affection in families are not necessarily those found associated with the disease in the population studies. Moreover, as we have already seen above, sarcoid susceptibility is associated with different class I or class II antigens depending on the breed or the family studied. These facts suggest that the class I or class II genes might only act as genetic markers for the disease and that a putative ‘sarcoid-susceptibility’ gene might be located between these loci. Between 1991 and 1994, similar associations between class II MHC genes and papilloma virus-induced tumours were described in other species. Wank and Thomssen (1991) have shown that women carrying the HLA-QW3 class II MHC allele have an increased risk of developing papilloma virus-induced squamous cell carcinoma of the cervix. In a more recent study, Apple et al. (1994) found a papilloma virus type-specific association between HLA DR-DQ haplotypes and the same disease. In rabbits, class II MHC genes are also associated with the development of tumours induced by Shope papilloma virus (Han et al., 1992). These comparable findings in three different species

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suggest similar virus–host interactions in oncogenic transformation which have a papilloma virus aetiology and are influenced by MHC class II or closely linked genes.

Insect bite dermal hypersensitivity Insect bite dermal hypersensitivity, also called sweet itch, summer eczema or summer dermatitis, is a seasonal pruritic dermatitis of horses identified throughout the world in places where midges and other biting flies are present. It is caused, in most cases, by an immediate-type allergic skin reaction to bites from insects of the genera Culicoides and Simulium (Fadok and Greiner, 1990). It can occur in any breed, and a hereditary predisposition to this disease has been described by many authors (Riek, 1953a,b; Ishihara and Ueno, 1957; McCaig, 1975; Strothman, 1982; Marti et al., 1992). An association between ELA and susceptibility to summer dermatitis has been described in two different breeds. Halldorsdottir et al. (1991) studied the distribution of MHC antigens in 303 Icelandic horses; 136 were affected with summer dermatitis. The class II antigen Dw22 (former Be8) occurred more often in affected animals than in controls (corrected P < 0.01), with a relative risk of 2.53. The aetiological fraction was low (0.186) because the frequency of the antigen in the population is low. In Swiss warmblood horses, an association between the MHC class II antigen Dw23 was found in two half-sibling groups from two different stallions (Marti et al., 1992; Lazary et al., 1994). ELA typing of four Swiss warmblood multiple-case families descended from the same healthy sire gave the following results: eight of the nine affected offspring carried the paternal haplotype A15, DW23; on the other hand, only three of the 15 healthy offspring displayed this paternal haplotype (P < 0.005). The distribution of the ELA haplotypes in offspring from the second sire showed that 11 of 12 offspring affected with summer dermatitis inherited the paternal haplotype with the class II antigen Dw23. It still remains to be investigated whether association between MHC and summer dermatitis will also be found in population studies within warmblood breeds.

Immunoglobulin Genetics Immunoglobulins, also called antibodies, are molecules that circulate in blood and through tissues, binding foreign substances, representing one of the major aspects of the immune system. Our understanding of the genetics and function of immunoglobulins is derived largely from studies of mice and humans. Most immunoglobulins are proteins composed of two heavy chains (~446 amino acids) and two light chains (~214 amino acids) covalently linked by disulphide bridges (Fig. 7.2). The light chain and heavy chain align with each other to form an antigen-binding site. Each immunoglobulin chain is the product of multiple genes defining a variable region (V), a joining region (J) and a

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constant region (C). The constant region defines the antibody isotype while the variable region confers the antigenic specificity. Each of the immunoglobulin isotypes and subisotypes has different immunological characteristics and they play diverse and important roles in resistance to disease and response to vaccines. The genetic organization and regulation of immunoglobulins is among the most complex systems yet known. Although genetic studies of horse immunoglobulins are relatively recent, immunological studies of these molecules date from early in the 20th century. Hyperimmune horse serum was once used commonly as a prophylaxis and therapeutic treatment for human disease. Horses responded well to antigenic challenge and large amounts of serum could be collected from horses. The active components of hyperimmune serum were immunoglobulins. IgG and IgM were distinguished in 1939 followed by the discovery of a hyperimmune T-protein (reviewed in Roberts, 1975). Serological and immunochemical studies of immunoglobulins revealed the existence of at least four subisotypes of horse immunoglobulin, IgG (a, b, c and T), IgA, IgM and IgE (Rockey, 1967; Zolla and Goodman, 1968; McGuire and Crawford, 1972; McGuire et al., 1973; Montgomery, 1973; Suter and Fey, 1983). The immunology of horse immunoglobulins was reviewed recently by Lunn et al. (1998). Since immunoglobulins could be studied so readily using immunological and immunochemical techniques, genetic approaches to the study of horse immunoglobulins have been neglected until the advent of molecular research approaches. Molecular approaches to immunogobulin genetics allow us to identify the genomic organization and understand better the regulation of immunglobulin expression.

Organization of immunoglobulin heavy chain loci The heavy chain loci encode the immunoglobulin isotypes M, G, A and E. These genes are found within a single chromosome region in most species and are coordinately regulated. Wagner and co-workers (1997) created a restriction map for the chromosome region encoding the constant chains of IgE and IgA. Their work demonstrated that the horse has one locus for IgE and for IgA. In addition, four immunoglobulin haplotypes were discovered based on RFLPs. Navarro and co-workers (1995) cloned and sequenced the equine IgE heavy chain, demonstrating homology to IgE sequence in other species. Schrenzel and co-workers (1997) analysed 15 IgM clones and identified a single constant region for the M chain, at least five genes encoding joining segments (Igh-J) and seven variable region sequences associated with strong representation of one of the four previously described variable region ‘clans’ for the horse. Other studies with heterohybridomas suggested that the order of loci beginning from the 5 ′ end were IgE, IgA, IgM and IgG constant region, similar to the order found in other species. Investigations of the IgG constant region genes demonstrated the existence of five, and possibly six, loci (Overesch et al., 1998). This is the largest number of IgG genes described for any species investigated, so far.

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Immunoglobulin light chains Immunoglobulin light chains are regulated and encoded by genes in a separate chromosome region in other species. Their genomic locations have not yet been mapped in the horse. All species possess two classes of light chains, λ and κ. The ratio of the two classes varies between species, with λ chains in excess among horses and κ chains in excess among mice. Up to 90% of horse immunoglobulins contain λ chains. Home and co-workers (1992) determined that the λ chain is encoded by three, and possibly four, loci for the constant region of the molecule and between 20 and 30 loci for the antigen-binding, variable region. The repertoire of variable region genes was limited compared with other species. This observation suggested that the predominance of the λ chains over the κ chains was not due to a large number of germline λ genes, but rather due to an aspect of genetic regulation. This conclusion was reinforced when Ford et al. (1994) determined that the horse possesses a single locus for the constant region of the κ chain and approximately 20 loci for the variable region. The organization of the κ genes was similar to that found in the mouse.

T-cell Receptor Genetics T-cell receptors are responsible for antigen recognition by T cells (T lymphocytes). Two lineages of T cells exist and are distinguished by the presence of either α/β-chain receptors or γ/δ-chain receptors. T cells with the α/β-chains are essential for humoral and cellular aquired immune responses. The function of the α/β receptor is associated with simultaneous recognition of self-MHC and is refereed to as MHC restriction. The γ/δ receptor also recognizes antigen but, unlike the α/β receptor, does not require a dual recognition of self-MHC and antigen. As with immunoglobulins, T-cell receptors are composed of a variable region gene responsible for binding, a joining region and a constant region that defines the chain as α, β, γ or δ (Fig. 7.1). Schrenzel and co-workers (1994) cloned and sequenced 33 T-cell receptor β-chain (TDRB) cDNA clones, finding 16 unique variable segments (TCRBV), 14 joining genes (TCRBJ) and two constant region genes (TCRBC). RFLP studies demonstrated genetic variation of the TCRBC genes among horses of different breeds. Schrenzel and Ferrick (1995) also characterized T-cell receptor cDNA for α (TCRA), γ (TCRG) and δ (TCRD). They demonstrated five genes for the TCRA variable region and joining region and a single gene for the TCRA constant region, based on sequencing 12 clones. Eight TCRG genes were sequenced and two distinct constant regions found, but no variable or joining genes. Fifteen TCRD clones were characterized and eight variable genes, four joining genes and one gene for the constant region were identified. The authors investigated the distribution of transcripts for these genes in different

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tissues, finding evidence for presence of α-β T-lymphocytes and δ-γ lymphocytes in all tissues except the bone marrow, where only α-β transcripts were found (Schrenzel and Ferrick, 1995).

Severe Combined Immunodeficiency Disease Several immunodeficiencies have been established as clear clinical entities for the horse, including selective IgM deficiency and transient hypogammaglobulinaemias and agammaglobulinaemias (reviewed in Lunn et al., 1998). However, a heredity role has not been established for these conditions. In contrast, an autosomal, recessive genetic defect is clearly responsible for SCID of Arabian foals (McGuire and Poppie, 1973). This disease results in absence of both T and B lymphocytes and death due to opportunistic infections of the immunocompromised foal. Several SCID candidate gene defects were considered based on the homology to diseases in humans, but the corresponding enzymes were found to be normal in SCID-affected foals (reviewed in Magnuson et al., 1985). However, Wiler and co-workers (1995) demonstrated a defect in V(D)J recombination for horse immunoglobulins and T-cell receptors that was homologous to the defect associated with murine SCID. In mice, the defect was the consequence of a genetic mutation affecting DNA-dependent protein kinase catalytic subunit (DNA-PKcs), essential for the production of immunoglobulin and T-cell receptor molecules. Shin and co-workers (1997) sequenced a cDNA clone of the horse DNA-PKcs homologue and discovered a deletion of five nucleotides. While this mutation was different from the one observed in mice, it did explain the absence of DNA-PKcs activity in SCIDaffected horses and why no immunoglobulin or T-cell receptor could be produced. Based on these results, a DNA-based test could be developed to detect carriers of the allele causing SCID.

Conclusions Agricultural and veterinary research usually focuses on application. Major health problems continue to demand research in horses including developmental bone diseases, muscle diseases, poor responses to vaccines, allergic diseases and metabolic diseases. Applied research has provided many effective therapies and vaccines for some health problems, but many problems remain. Clearly, we cannot solve these problems using our current base of knowledge. Basic immunogenetics research is one of the keys to addressing these problems. Basic research in mice demonstrated MHC-restricted cytotoxicity, specifically the observation that cytotoxic T lymphocytes kill virus-infected cells only when they share the same MHC antigens. Allen and co-workers (1995) and McGuire and co-workers (1997) developed in vitro cytotoxic T-cell assays

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using the ELA typing reagents in order to characterize the cellular immune response to equine herpes viruses and equine infectious anaemia viruses, respectively. The goal of this work is to develop better vaccines. This work would not have been possible without the development of the ELA typing system. Likewise, Marti and co-workers (1997) needed to develop better tools to investigate allergic diseases. They cloned the horse gene for IgE in an expression vector and used the expressed molecule to generate antibodies to this molecule. This application of basic research allowed them to investigate allergic diseases such as chronic obstructive pulmonary disease and insect bite dermal hypersensitivity. This gene family is among the most well studied genetic systems in all species. As a consequence, we know that the genetic structure is largely conserved in all vertebrate species. Similarities allow us to use the lessons learned with other species. Differences give us clues regarding those aspects of health, physiology and disease that are unique to the horse. Since the late 1970s, scientists have actively pursued genetic studies to characterize this gene family for the horse. These studies have not led to complete understanding of the MHC in the horse. We still do not know the number of MHC class I, class II and class III genes nor their pattern of expression. The discovery that Dw13 has a lower level of expression (Barbis et al., 1994b) than other class II molecules coupled with the association of Dw13 with several diseases offers fruitful ground for developing new, testable hypotheses involving MHC gene regulation. We know little about the organization of genes for immunoglobulin and T-cell receptors for the horse. Genetic variation has been found for all of these genes, indicating strong selective pressure, perhaps by infectious pathogens. This chapter documents the transition of early, basic MHC research to the more recent reports in which MHC research was applied to understanding health problems. Many groups bridged the gap between the basic research scientist who develops new tools and understanding, and the clinician, who studies diseases. Transfer of basic research knowledge to applied experimentation will help construct better experiments and find solutions for diseases that have resisted traditional veterinary medical approaches.

Acknowledgements This review is dedicated to Professor Jack Bryans and the late Professor Heinz Gerber both of whom strongly encouraged the authors to conduct research on immunogenetics. This report was supported by the Swiss National Science Foundation grant No. 31–49618.96 and in connection with a project of the University of Kentucky Agricultural Experiment Station and published as paper number 99–14–70.

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References Albright, D., Bailey, E. and Woodward, J.G. (1991) Nucleotide sequence of a cDNA clone of the horse (Equus caballus) DRA gene. Immunogenetics 34, 136–138. Albright-Fraser, D.G., Reid, R. Gerber, V. and Bailey, E. (1996) Polymorphism of DRA among equids. Immunogenetics 43, 315–317. (Erratum published in Immunogenetics 44, 487.) Alexander, A.J., Bailey, E. and Woodward, J.G. (1987) Analysis of the equine lymphocyte antigen system by Southern blot hybridization. Immunogenetics 25, 47–54. Allen, G., Yeargan, M., Costa, L.R.R. and Cross, R. (1995) Major histocompatibility complex class I restricted cytotoxic T-lymphocyte responses in horses infected with equine herpes virus 1. Journal of Virology 69, 606–612. Angelos, J.A., Marti, E., Lazary, S. and Carmichael, L.E. (1991) Characterization of BPVlike DNA in equine sarcoids. Archives of Virology 119, 95–109. Ansari, H.A., Hediger, R., Fries, R. and Stranzinger, G. (1988) Chromosomal localization of the MHC of the horse by in situ hybridization. Immunogenetics 28, 362–364. Antczak, D.F. (1984) Lymphocyte alloantigens of the horse. III ELY-2.1. A lymphocyte antigen not coded by the MHC. Animal Blood Groups and Biochemical Genetics 15, 103–115. Antczak, D.F. (1991) The major histocompatibility complex of the horse. In: Plowright, W., Rossdale, P.D and Wade, J.F (eds), Equine Infectious Diseases VI. R&W Publishing, Newmarket, UK, pp. 99–112. Antczak, D.F., Bright, S.M., Remick, L.H. and Bauman, B.E. (1982) Lymphocyte alloantigens of the horse. I. Serologic and genetic studies. Tissue Antigen 20, 172–187. Antczak, D.F., Miller, J.M. and Remick, L.H. (1984) Lymphocyte alloantigens of the horse II. Antibodies to ELA antigens produced during pregnancy. Journal of Reproductive Immunology 6, 283–297. Antczak, D.F., Bailey E., Barger, B., Bell, K., Bernoco, D., Bull, R.W., Byrns, G., Guérin, G., Lazary, S., McClure, J.J., Mottironi, V., Templeton, J. and Varewyck, H. (1986) Joint Report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, held April, 1984. Animal Genetics 17, 363–373. Apple, R.J., Erlich, H.A., Klitz, W., Manos, M.M., Becker, T.M., and Wheeler, C.M. (1994) HLA DR–DQ associations with cervical carcinoma show papillomavirus-type specificity. Nature Genetics 6, 157–162. Bailey, E. (1980) Identification and genetics of lymphocyte antigens in the horse. Immunogenetics 11, 499–506. Bailey, E. (1983a) Linkage disequilibrium between the ELA and A blood group systems in Standardbred horses. Animal Blood Groups and Biochemical Genetics 14, 37–43. Bailey, E. (1983b) Population studies on the ELA system in American Standardbred and Thoroughbred horses. Animal Blood Groups and Biochemical Genetics 14, 201–211. Bailey, E. (1986) Segregation distortion within the equine MHC; analogy to a murine T/t complex trait. Immunogenetics 24, 225–229. Bailey, E. (1994) Variation within the antigen binding site of the major histocompatibility complex DRA gene of domestic horses. In: Nakajima, H. and Plowright, W. (eds), Equine Infectious Diseases VII. R&W Publications, Newmarket, UK, pp.123–126. Bailey, E. and Henney, P.J. (1984) Comparison of ELY-2.1 with blood group and ELY-1 markers in the horse. Animal Blood Groups and Biochemical Genetics 15, 117–122.

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Genetic Aspects of Disease in Horses 1 and Marianne Rasmuson2 E. Collinder 8Genetic Eje Aspects and Collinder of M.Disease Rasmuson 1Laboratory of Medical Microbial Ecology, Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden; 2Institute of Genetics, Umeå University, S-901 87 Umeå, Sweden

Introduction Causes of Disease Identifying Genes Complex Diseases Search for Genetic Risk Factors Conclusions References

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Introduction Horse breeding differs from other attempts to improve livestock performance and production in that the primary goal is not to enhance breed averages (except in the diminishing category of workhorses) but to produce individual animals that can conquer their fellow competitors in racing and other sports. Since training and breeding horses usually requires large investments, people involved in these trading and breeding transactions need assurance of the health and good constitution of the animal in question. Official registration for use as a breeding animal can provide some assurance for this. Registration events are clearly the time and place to exert influence over the genetic condition of the breed and to define the future goals for desired characteristics of a breed. In many breeds, the demands for stallions are much more stringent than for mares, which is perhaps a necessary consequence of their greater number of offspring. In judgement for approval as a breeding animal, much weight is placed on physical appearance and on the absence of discernible defects and infectious diseases, but, if available, the horse’s ancestry and information about performance in offspring are also considered. Since it is the competitive performance of the horse that usually decides its value for the owners, results ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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of performance tests and prize money earned are also important factors in the popularity of an individual horse for use in breeding.

Causes of Disease Sex chromosome abnormalities occur in several breeds. They are found mostly in small mares with gonadal dysgenesis, i.e. chronic infertility, very small gonads, lack of follicular activity and a flaccid uterus and cervix. Most cases are analogous to the human condition called Turner’s syndrome, and have only one X chromosome or a pair where one of the Xs is only half of the normal length. However, several cases have been found in different breeds where mares have a normal male karyotype with one X and one Y chromosome. This example of chromosomal abnormality has a familial occurrence and seems to follow a dominant inheritance. It may be due to translocation of a segment of the Y chromosome to an autosome (Trommershausen Bowling et al., 1987). There are many ailments that reduce the performance capacity of horses. They may have genetic causes, or be mainly of environmental origin, such as inadequate feeding, wear and overloading, or the result of infections and parasites. However, many diseases with apparently environmental causes can still have an appreciable genetic component. It has been shown in other domestic animals that selection of certain breed characteristics, or blood lines within a breed can be effective in increasing the resistance to various infections. In horses, however, more emphasis appears to have been placed on keeping a high standard of hygiene, diet and management in general, rather than focusing on general genetic improvement of disease resistance. Defects in young horses such as deformities, severe locomotor and neurological problems, etc. are always unacceptable and a common cause for culling. If there is regular inheritance, the defective gene can be spotted through pedigrees, and carriers of such genes removed from further breeding. However, due to the long generation time and small number of offspring per mare it is not always easy to prove the heredity behind equine diseases, even when over time they prove to be of genetic origin. Some diseases assumed to be inherited regularly are listed in Table 8.1. Since the dawn of genetics, the rules for monogenic traits in pedigrees have been known and used to ascertain the mode of inheritance, i.e. autosomal or sex-linked, dominant or recessive. This is still the preferred method where possible. Dominant genes with lethal effects in homozygotes are most easily determined. Instances of this are two coat colour genes, dominant white and roan, which cause lethality when inherited from both parents (Pulos and Hutt, 1969; Hintz and Van Vleck, 1979). However, even in this well-defined case, the inheritance pattern is not absolute, as the roan-like coat colour may sometimes be caused by other genes or be difficult to recognize. In such cases, a molecular genetic diagnosis would be of value, and a search for such tests is under way (Marklund, 1997; Marklund et al., 1999). Recently, this has succeeded for the Overo lethal white syndrome (OLWS) in American Paint

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Genetic Aspects of Disease Table 8.1.

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Some inherited monogenic diseases in horses.

Disease

Signs and genetics

Deformed extremity bones

Misdeveloped ulnae and fibulae causing lameness and abnormal movements. Probably autosomal recessive

Dominant white Hyperkalaemic periodic paralysis (HYPP) Multiple exostosis (ME) Overo lethal white syndrome (OLWS) Patellar (sub)luxation in Shetland ponies Roan Severe combined immunodeficiency (SCID)

Prevalence

Reported among Shetland ponies in the UK and other countries. Estimated gene frequency in Sweden 18% (Philipsson et al., 1998) Heterozygotes have pink skin, white coats, Exclusive ‘blancos’ with unknown spread. Small economic impact (Pulos mane, tail and hooves. Homozygosity and Hutt, 1969) (WW) causes prenatal lethality Widespread in Quarter Horses causing Muscle weakness, fasciculation and great economic losses. Molecular tests tremors. Codominant autosomal (HYPP). are available (Bowling et al., 1996) Similar to human HYPP Unknown spread. Important economic Numerous bony outgrowths on the ribs, disadvantage (Gardner et al., 1975) long bones and vertebra, tumours. Autosomal dominant gene (Me). Similar to human HME Mostly in Paint horses (Santschi et al., Popular colour pattern in heterozygotes, overo spotted. Homozygosity causes early 1998) death with severe intestinal abnormalities. In humans, Hirschsprung’s disease In The Netherlands, one of the most An abnormality located in the frequent undesired conditions (Hermans femoropatellar articulation of the stifle et al., 1987) joint. Recessive autosomal Autosomal dominant coat characteristics, Moderately spread in several breeds. Molecular tests are under way roan. Homozygosity (RnRn) causes (Marklund et al., 1999) lethality in utero Spread in Arabian horses. Carriers Born healthy, early clinical signs, death around 10% (USA). Significant within 3 months. Recessive autosomal economic wastage. Diagnostic DNA test (ir) available (Bernoco and Bailey, 1998)

horses, where a DNA variant has been shown to be associated with the disease. Similar conditions occur in humans (Hirschsprung’s disease) and rodents, where mutations in the gene for endothelin receptor B (EDNRB) are responsible. An amino acid substitution in the product of the analogous equine gene has now been shown to be homozygous in all OLWS foals (Santschi et al., 1998), and to occur in heterozygous form in both parents, of which all but one were coloured overo spotted. This is an example of the possibilities for molecular genetic tests, and may also add to our understanding of coat colour inheritance in horses. The characteristic inheritance of genes located on the X chromosome, i.e. from healthy carrier mother to sons, which exhibit the disease, has only been demonstrated for one disease – haemophilia A – in the horse. Haemophilia A has a sex-linked inheritance in both horses and man, and in both species it causes a deficit of the coagulation factor VIII: C in the blood, leading to spontaneous haematomas and other bleeding disorders (Henninger, 1988).

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Even if the clinical signs indicate the same defect, the underlying causes may vary. For example, a Swedish warmblood stallion, with the roaring defect (laryngeal hemiplegia), had sired many offspring, among which an appreciable number (8%) were found to develop the same defect. This stallion accordingly was rejected from use in further breeding. Another stallion of the same breed was taken out of breeding when he developed roaring, but was later re-approved when it was found that none of his many offspring had inherited the defect (Stenlund, 1994; Philipsson et al., 1998). This implies a different genetic background in the two cases, or that the defect in the second case was due to some environmental cause. Until specific biochemical or DNA-based tests for specific disease(s) can be found, there will always remain possibilities for heterogenous causes, even when clinical signs of a disease are quite uniform. The heterogeneity may involve environmentally induced phenocopies, different genes blocking the same metabolic chain and leading to the same end result, or different molecular defects within the same gene, which may confound attempts at diagnosis through DNA sequencing.

Identifying Genes For diseases with a clear Mendelian inheritance, further aims are to map and identify the disease-causing gene. The major advances in identifying human genetic diseases during the last 10 years show the possibilities. They have been based on the mapping of the human genome, involving the technologies for gene identification that have emerged from the Human Genome Project (Collins, 1995). The primary efforts include linkage and association studies using marker genes spaced over the entire genome. Having obtained a crude localization, further studies may involve candidate genes, i.e. genes with known functions, which have been identified in earlier studies and are situated within the indicated chromosome section. The many markers in the human genome, which have made localization of disease genes so successful in human genetics, are not yet available in the horse. The first markers were polymorphic variants in animal form and colour, with simple inheritance and good penetrance. They are rather common in mice but only a few are available in the horse. During the 1950s, several polymorphic blood group factors (red cell alloantigenes) were identified by immunological procedures involving hemagglutination and haemolysis. The inheritance of some ten such systems has been analysed and the number of known variants in a system, identified with test sera, varies from two up to 20. To these markers have been added several blood protein variants (allozymes) which can be identified with electrophoresis, either on starch or polyacrylamide gel, and subsequent specific protein staining. The inheritance in such systems is usually codominant so that heterozygotes produce both gene products and the genotypes thus can be identified easily. A number of equine blood group systems and several systems of protein polymorphism are now internationally recognized (Trommershausen Bowling and Clark, 1985;

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Ouragh et al., 1994), and routinely used in many countries (Chapter 5). Several horse breeds have an obligatory registration of blood type for each animal, for subsequent identification and paternity testing. Recently, the number of available markers has increased by use of DNA analyses, involving restriction fragment length polymorphisms (RFLPs) and microsatellite loci (see Chapter 6). The latter are highly polymorphic and therefore especially useful. They are sequences of variable number of di- and trinucleotide repeats, such as CTCTCT . . . or CGACGA . . ., which can be analysed by the polymerase chain reaction (PCR) technique that magnifies specific segments of DNA. The equine genetic map accordingly has improved considerably. Markers have been assigned to most of the 32 equine chromosomes and approximately 30 coding genes have been mapped (Lindgren et al., 1998). When clinical features reflect genetic or biochemical homology among different species, genomic analysis can help to identify equine disease genes. Comparative genomic analysis recently has become a workable way to obtain information about monogenic diseases. It is well known that the main part of the genome is common to many organisms. This implies that defects in homologous genes may cause phenotypic effects of the same kind in different species. Indeed, it has been found that several genetic diseases in the horse have counterparts in other mammals, including man (Chapter 4). Mutation-related diseases in the horse have only been revealed in a few instances, and they have all been found by means of genomic comparison with other species (Raudsepp, 1999). The OLWS foals are one example. Two other well-analysed instances of disease homology involving horse and man are severe combined immune deficiency (SCID) and hyperkalaemic periodic paralysis (HYPP). SCID is not a common disease in domestic animals, but has been found as an autosomal recessive trait in Arabian horses (Studdert, 1978; Perryman and Torbeck, 1980; Bernoco and Bailey, 1998), as well as in mice (Shin et al., 1997). The disease is also recognized in humans, where several syndromes of this type, with varying molecular causes, are described. HYPP, called Gamstorp’s disease in man, is a defect in the skeletal muscle sodium channel gene, which causes a potassium-induced paralysis. By means of molecular methods, a mutation causing an amino acid change from Phe to Leu has been identified, and shown to be responsible for the disease in Quarter Horses. The DNA sequencing of the HYPP gene in humans identified two different mutations in the same transmembrane part of the sodium channel gene as in the affected horses, but with different amino acid changes (Rudolph et al., 1992). Here molecular analysis has not only revealed true animal analogues of human genetic defects, but also has made accurate diagnosis by DNA tests possible in cases where clinical clues are non-diagnostic (Bowling et al., 1996).

Complex Diseases Many traits that run in families do not show a regular pattern of inheritance. Gene–environment interaction may influence the expression of a gene,

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sometimes making it so slight as to escape discovery. More refined and exact methods of diagnoses may help to identify clinically normal carriers of disease genes. Often, however, the appearance of a certain defect depends on many interacting genetic and environmental factors. Examples of less well characterized diseases, which are found in many mammalian species, and may have genetic links, are congenital malformations (Hermans et al., 1987), allergies (Marti et al., 1991) and neurological diseases (Beech and Haskins, 1987; Poncet et al., 1989) (Table 8.2). The aetiological factors for such diseases are multifactorial and thus vary between cases. As yet, no genes involved with these diseases have been identified in the horse. Irregular appearance of a disease in families and an increased incidence among close relatives to affected individuals are suggestive of a genetic basis to the disease. Both in humans and horses, these complex diseases pose a difficult problem. They occur as a combined result of a genetic predisposition and specific (often unknown) environmental factors, involving management, diet and training. In human genetics, much effort recently has been directed towards identifying genes behind some common diseases, such as heart failure, diabetes and cancer. Such diseases can be seen as threshold characters on an underlying liability scale. Only when the combined effects of many factors exceed a presumed threshold will the trait diverge from normality and a Table 8.2.

Some polygenic complex diseases in horses.

Disease

Signs and genetics

Prevalence

Spread in several breeds, most in horses older than 4 years (Marti et al., 1991) Occurs in all domestic breeds. Less Cryptorchidism than 5% of all foals, frequently associated with intersex cases. Small economic impact (Leipold et al., 1986) Common in Quarter Horses and other Intermittent stiffness and muscular Exertional breeds. Prevalence 5% in Thoroughbred cramping. The genetic causes are rhabdomyolysis racehorses. Major obstacle in horse uncertain. Genetic heterogeneity (Rha) racing indicated Neuraxonal dystrophy Pelvic limb dysmetria and uncoordinated Spread in the Morgan breed (Beech and Haskins, 1987) movements. Familial inheritance (NAD) Disturbance of endochondral ossification. Common skeletal disorder. Prevalence Osteochondrosis 11% in warmbloods and Standardbreds. Radiographic diagnosis is used. (OC) Usually little effect on performance Heritability 0.1–0.3 (Philipsson et al., 1998) Polysaccharide storage Glycogen storage disorder. Clinical signs Thoroughbreds, Quarter Horses and other breeds (Valberg, 1997) as in Rha, but early onset myopathy (PSSM) Increased respiratory sound and irregular Low prevalence, 2/10,000 year−1 in Roaring, laryngeal vocal cord causing impaired performance. Sweden. Clinical treatment gives good hemiplegia Uncertain genetic basis, heterogeneous results (Stenlund, 1994) background Chronic bronchitis (CB)

Expiratory dyspnoea and cough, with or without destructive emphysema. Genetic factors in the pathogenesis uncertain Failure of one or both testes to descend in their normal position in scrotum. Genetic basis uncertain

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disease become manifest. One of the aims of investigating genetic contribution to these diseases is to identify genes (quantitative trait loci, QTL) with appreciable though moderate influence on the disease in question. Another aim is to find cases where genes and environment interact in a non-additive manner, for instance genes which affect individual sensitivity to the environment. There are several difficulties in this enterprise. There is usually no one-toone relationship between disease and genotype, and different genes may contribute proportionally more or less in different breeds. Genetic heterogeneity is a common cause of inability to replicate positive findings. A gene variant that is of importance for the manifestation of a disease in one population may be irrelevant in another, either because it is missing or because other factors have a greater influence on manifestation of the disease. Exertional rhabdomyolysis (Table 8.2) is an example of a clinical sign with many underlying causes. One of these is the polysaccharide storage myopathy (PSSM) in Quarter Horses (Valberg, 1997), where the disease is apparent already in foals. Other studies have indicated recessive as well as dominant autosomal inheritance in different pedigrees and breeds (Valberg et al., 1996; MacLeay et al., 1999). Quantitative genetics can provide a first guide towards the importance of genetics for complex diseases. The estimate of the total genetic part of the phenotypic variation (the heritability) can be obtained from incidence in sib groups, in progeny from affected animals or from sire–offspring regression, based on the frequency of the trait in progenies from stallions with, and without, the trait. Since diseases are all-or-none traits, and the heritability refers to the assumed liability scale; the confidence limits are wide. The estimates of heritability for osteochondrosis (a disturbance of ossification of disputed clinical significance) vary from 23 to 52% in Scandinavian populations of Standardbred trotters (Grøndahl and Dolvik, 1993; Philipsson et al., 1993). Diseases with a multifactorial background may sometimes occur more often in one sex than in the other. Males are more likely to show skeletal defects, such as lesions in the limbs, as well as neuromuscular problems such as roaring (Stenlund, 1994), locomotor weakness and ataxias (Falco et al., 1976). A sex predisposition in females has been found for rhabdomyolysis syndrome (Rha) in Swedish Standardbred trotters (Collinder et al., 1997; MacLeay et al., 1999). Since the triggering factors in these cases are unknown, it is not possible to identify their nature. They may be found in management, training and racing habits, but may also be due to differences in the hormonal physiology of the sexes.

Search for Genetic Risk Factors Another important epidemiological parameter in disease causation in relation to genetics is the relative risk of an individual acquiring a disease, defined as the risk for a relative of an affected individual as compared with the risk in the general population. Further, beyond merely demonstrating the importance of genes for complex diseases are attempts to characterize affected individuals as

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a specific group with a gene content that deviates from that of the general population. Comparing gene frequencies for a set of markers in the affected group and in the population at large, using χ2 homogeneity analysis, can test this hypothesis. The total results for all markers give an estimate of the genetic distance between the groups. Marker alleles, which differ significantly in frequency from the control group, are said to be associated with the disease and may be used to identify a risk group. However, association between a marker and a disease can occur from two different causes. Either the marker itself is a QTL, where alleles in some way differently influence the risk of obtaining the disease, or there is linkage disequilibrium between a marker allele and a nearby QTL for the disease. Disequilibrium is to be expected for ancestral mutations in an isolated population, which have been established relatively recently and where recombination has not had time to break the linkage and balance the disequilibrium. The closer the linkage, the longer the time needed to obtain equilibrium. In breeds of different heritage, linkage may therefore be due to different alleles in a nearby marker locus, and an established risk group cannot be declared valid in other breeds. In all these attempts, one has to be aware of false-positive results, which are to be expected when a large number of independent results are tested simultaneously. Therefore, the significance level in these methods has to be stringent. A search for genetic factors behind a complex disease in the horse has been undertaken with exertional rhabdomyolysis (Rha), also called ‘tying up’, in Swedish Standardbreds (Stbs). This ailment has been reported in many breeds of domestic horses, including Thoroughbreds, Quarter Horses, Standardbreds, warmbloods and pony breeds. It is manifest by nervousness, recurrent attacks of muscle stiffness and sweating during and after exercise, accompanied by acute muscle damage. Sometimes the signs are mild and may go unnoticed. The syndrome can also be diagnosed by finding abnormally high values of AST (asparate aminotransferase) and CK (creatine kinase) post-exercise in blood serum. In some pedigrees, the occurrence of a main gene has been indicated (Valberg et al., 1996; MacLeay et al., 1999), but in the Swedish Standardbreds no such findings have been made. With the aim of searching for association between genetic markers and Rha in this breed, the gene frequencies in a randomly selected sample of 663 Stbs with a diagnosis of Rha were compared with those in the total population of the breed. They were compared with respect to six blood types and nine protein polymorphic systems from serum and red blood cells. None of the loci for these markers are closely linked. Since all Stbs are typed and registered for these 15 blood group systems, the register in Sweden comprises over 100,000 horses. Gene frequencies have been calculated over the years and have shown no tendency to change since the start of registration (1970). In the calculations shown below, a random sample of 1000 horses born during the same period as those in the Rha sample was used.

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A significant heterogeneity was established between the Rha sample and the population, making an estimate of genetic distance meaningful. Many indices based on frequencies have been suggested for genetic distances between pairs of populations. For example, Nei’s measure of genetic distance is based on an evaluated mean number of codon differences (Nei, 1975). Nei’s genetic distance is based on an evaluation of genetic identity (I), i.e. the probability of identity between two randomly chosen alleles at a certain locus. Genetic diversity (D) is defined as 1–I. Since many codon differences cannot be detected with immunological reactions or electrophoresis techniques, the distance may be underestimated. However, this holds for all estimates not based on DNA sequencing. Another way to estimate a genetic distance uses angular transformation of gene frequencies. This gives the distance for each locus as a chordal or an arch distance in a Euclidian superspace where the two populations are placed on the surface of a hypersphere (Cavalli-Sforza and Edwards, 1967). For both measures, the distance is unity (= 1) when the two populations have no common allele. Distances from different loci can be pooled into a total distance by taking the square root of the mean of the squared distances for all loci (Wright, 1978). The pooled distance, based on differences in allele frequencies of blood group and blood protein systems, between the Rha group and the total population of Stbs, as estimated in Dchordal and Darch (Cavalli-Sforza and Edwards, 1967), turned out to be about one-tenth of the comparable distance between the two different Swedish breeds (Stbs) and North-Swedish trotters (NSt). Nei’s distance (Dstandard), on the other hand, being a squared distance between the Rha group and the total population, was 80 times smaller than between the breeds (Table 8.3).

Table 8.3. Three different measures of genetic distance and discriminant score between a group of Swedish Standardbreds with one of the diseases rhabdomyolysis (Rha), palmar/plantar osteochondral fragments (POF) or polycythemia (Pc), and the total population of Swedish Standardbreds (Stbs). Methods Genetic distance Dstandard (Nei’s) Dchordal Darch Discriminant score, D2 Variance ratio, F P for H0

Rha–Stbs

POF–Stbs

Pc–Stbs

Stbs–NSt

< 0.0008 < 0.024 < 0.022 < 0.180 < 50.07 < 0.001

< 0.0021 < 0.045 < 0.040 < 0.080 < 1.82 > 0.05

< 0.0026 < 0.047 < 0.042 < 0.473 < 11.28 < 0.001

< 0.063 < 0.231 < 0.210 < 3.57 < 303.32 < 0.001

From Håkansson (1995) and Collinder et al. (1997). For comparison, corresponding scores between Stbs and the total population of North-Swedish trotters (NSt) are given. For the discriminant score, the variance ratio and probability for H0 are also given. The five most distinguishing genetic markers were used, including sex in Rha and Pc.

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Whereas the genetic distance is a way to judge the importance of genetic differences between two groups, the establishment of risk criteria from available information is of more practical importance. For this purpose, a linear discriminant function can be estimated, in which the difference between the group of individuals with a liability for the disease and the population at large is maximized. Such methods have been used for population characterization and for classifying individuals as belonging to a certain group by means of characters, which, taken singly, do not give a reliable classification (Sneath and Sokal, 1973). The discriminating characters in this case are genotypes or phenotypes at the marker loci. One arbitrarily chosen homozygous genotype, or the dominant phenotype, is given the value of 1, heterozygotes when discernible 0.5, all others have 0 values. The differences in arithmetic mean values between the two groups and the mean variance within the groups for every character form the basis for the discriminant score. Covariances are assumed to be zero for independent characters such as the phenotype frequencies in unlinked loci. Additions over loci give the discriminant score but, since the characters are of variable importance, the score can be made more effective by including only the really important characters. The significance is tested by a variance ratio. The score for an unclassified individual is calculated from its value for the included marker phenotypes, but it can also comprise sex and other criteria, if these are found to be important for the disease. The individual will be assigned to the risk group if its score falls above the limit value midway between the scores of the disease group and the population. The final score for discriminating the Rha group included four marker loci and sex; mares being more affected than males. The score obtained was: X = 1 A −2.48 Hb −4.59 PGM + 2.86 Tf 0 + 6.64 ‘

In order to confirm this result, the calculations were repeated with another group of 521 Stbs with Rha. A significant score was obtained with the same markers as before, but an exchange of the Hb system for that of K improved the discrimination. Thus, it is possible to identify a risk group for Rha within Stbs using four or five marker genes in blood group systems and/or systems of blood protein variants (Collinder et al., 1997). Whether the score can be used in other breeds for this disease is as yet uncertain, since the association may be due mainly to spurious linkage disequilibrium. Evaluation of the same diagnostic parameters, including heterogeneity among marker phenotype or allele frequencies, genetic distance and discriminant score, has also been applied to two other equine diseases with a complex background (Table 8.3), in the same breed. The heritability of palmar/plantar osteochondral fragments (POF), has been estimated to be 0.17–0.23 in Stbs (Philipsson et al., 1993). In a study of POF in 124 horses, there was heterogeneity for two markers among 14, and a total heterogeneity, which was significant only at the 0.05 level (Håkansson, 1995; Roneus et al., 1998). For the other disease, polycythemia, which is an increase in the red blood

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cell mass and reduced maximum performance (overtraining), no heritability estimate is available, but a familial disposition is presumed. For 131 horses diagnosed as having polycythemia (Persson, 1967; Persson, 1997), three loci showed heterogeneity at the 0.001 level of significance, as did the combined homogeneity test (Håkansson, 1995). The genetic distances from both the POF and polycythemia groups to the population at large were about double the distance between the Rha group and the population (Table 8.3). No reliable discriminant score was obtained for POF, but the polycythemia sample gave a discriminant score that could be reduced to four markers. Mares were shown to be less liable to this disease. The discriminant markers were D02, Es, Al and Hb (Håkansson, 1995). Only the Hb marker was also found to be discriminating for the Rha group. In order to evaluate the importance of the risk groups, genetic distance and a comparable discriminant function were also calculated between Stbs and NSt, using the same marker systems (Table 8.3). The best score was highly significant and included five loci, Tf, Hb, P, Ap and Q. It is thus obvious that the important markers vary between comparisons, which is to be expected since they can be seen as randomly sampled loci from the total genome. If no association is obtained, the genes involved with the disease either are not close enough to the chosen markers or are of less importance than other, mainly environmental factors. Since the available markers only cover a small part of the equine genome, the possibilities are great that genetic factors have gone unnoticed in these attempts.

Conclusions New insights into the genetic mechanisms behind equine diseases are under way. Can they lead to reduced disease risks and a better state of health? A breed organization can set the lower limit for approval, but the decision to use a horse for breeding purposes is then a choice for the owner, who has mainly his own interest in view. There is little opportunity for investments to improve the breed as such. Judgements on the suitability for breeding can be improved, if easily performed and reliable test methods become available to detect carriers of defect genes in animals with no clinically detectable abnormalities. This is especially important for stallions which otherwise can spread the gene to numerous offspring. The way in which to obtain such test methods is long and laborious. Data indicating the mode of inheritance, search for linkage to chromosomal markers and genomic comparisons between species are steps on the way to genetic identification. Development of diagnostic DNA tests, such as those for SCID and HYPP, are examples of specific genetic identification. Since there are hundreds of different genetic diseases, each usually restricted to one or a few blood lines, it is clear that the availability of such methods will remain scarce for a long time yet.

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A better strategy may be to direct efforts towards investigating the genetic role in the more common diseases, which, however, have complicated and multifactorial backgrounds. This may be done by estimating individual risk profiles, which, based on knowledge of early environment and presence in the genotype of genetic markers, have been found to be associated with the disease. For this, a specific evaluation of each risk situation is necessary. Only if there are strong indications and the risk is high can such methods be used as a cause for restriction in approval for breeding. At present, there is little to indicate any immediate reduction in the genetic disease load of the horse.

References Beech, J. and Haskins, M. (1987) Genetic studies of neuraxonal dystrophy in the Morgan. American Journal of Veterinary Reseach 48, 109–113. Bernoco, D. and Bailey E. (1998) Frequency of the SCID gene among Arabian horses in the USA. Animal Genetics 29, 41–42. Bowling, A.T., Byrns, G. and Spier, S. (1996) Evidence for a single pedigree source of the hyperkalaemic periodic paralysis in Quarter Horses. Animal Genetics 27, 279–281. Cavalli-Sforza, L.L. and Edwards, A.W.F. (1967) Phylogenetic analysis: models and estimation procedures. American Journal of Human Genetics 19, 233–257. Collinder, E., Lindholm, A. and Rasmuson, M. (1997) Genetic markers in Standardbred trotters susceptible to the rhabdomyolysis syndrome. Equine Veterinary Journal 29, 117–120. Collins, F.S. (1995) Positional cloning moves from perditional to traditional. Nature Genetics 9, 347–350. Falco, M.J., Whitwell, K. and Palmer, A.C. (1976) An investigation into the genetics of ‘wobbler disease’ in Thoroughbred horses in Britain. Equine Veterinary Journal 8, 165–169. Gardner, E.J., Shupe, J.L., Leone, N.C. and Olson, A.E. (1975) Hereditary multiple exostosis. Journal of Heredity 66, 318–322. Grøndahl, A.M. and Dolvik, N.I. (1993) Heritability estimations of osteochondrosis in the tibiotarsal joint and of bony fragments in the palmar/plantar portion of the metacarpo- and metatarsophalangeal joints of horses. Journal of the American Veterinary Medical Association 203, 101–104. Henninger, R.W. (1988) Hemophilia A in two related Quarter Horse colts. Journal of the American Veterinary Medical Association 193, 91–94. Hermans, W.A., Kersjes, A.W., van der Mey, G.J. and Dik, K.J. (1987) Investigation into the heredity of congenital lateral patellar (sub)luxation in the Shetland pony. Veterinary Quarterly 9, 1–8. Hintz, H.F. and Van Vleck, L.D. (1979) Lethal dominant roan in horses. Journal of Heredity 70, 145–146. Håkansson, E. (1995) Search for genetic Risk Factors in Two Diseases of Standardbred Trotters. Report from the Institute of Genetics, Umeå University, Sweden. Leipold, H.W., DeBowes, R.M., Bennett, S., Cox, J.H. and Clem, M.F. (1986) Cryptorchidism in the horse: genetic implications. In: Milne, F.J. (ed.) Proceedings of the 31st Annual Convention of the American Association of Equine Practitioners,

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1985, Toronto, Canada. American Association of Equine Practitioners, pp. 579–589. Lindgren, G., Sandberg, K., Persson, H., Marklund, S., Breen, M., Sandberg, B., Carlstén, J. and Ellegren, H. (1998) A primary male autosomal linkage map of the horse genome. Genome Research 8, 951–966. MacLeay, J.M., Valberg, S.J., Sorum, S.A., Sorum, M.D., Kassube, T., Santschi, E.M., Mickelson, J.R. and Geyer, C.J. (1999) Heritability of recurrent exertional rhabdomyolysis in Thoroughbred racehorses. American Journal of Veterinary Research 60, 250–256. Marklund, S. (1997) Applied molecular genetics in domestic animals with particular focus on the horse. Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden. Marklund, S., Moller, M., Sandberg, K. and Andersson, L. (1999) Close association between sequence polymorphism in the KIT gene and the roan coat colour in horses. Mammalian Genome 10, 283–288. Marti, E., Gerber, H., Essich, G., Oulehla, J. and Lazary, S. (1991) The genetic basis of equine allergic diseases 1. Chronic hypersensitivity bronchitis. Equine Veterinary Journal 23, 457–460. Nei, M. (1975) Molecular Population Genetics and Evolution. Elsevier, North Holland Publishing Co., Amsterdam, pp. 128–209. Ouragh, L., Mériaux, J.C. and Braun, J.P. (1994) Genetic blood markers in arabian, barb and arab-barb horses in Morocco. Animal Genetics 25, 45–47. Perryman, L.E. and Torbeck, R.L. (1980) Combined immunodeficiency of Arabian horses: confirmation of autosomal recessive mode of inheritance. Journal of the American Veterinary Medical Association 176, 1250–1251. Persson, S.G.B. (1967) On blood volume and working capacity in horses. Acta Veterinaria Scandinavica 19 (Suppl.), 1–189. Persson, S.G.B. (1997) Heart rate and blood lactate responses to submaximal treadmill exercise in the normally performing Standardbred trotters – age and sex variations and predictability from the total red blood cell volume. Journal of Veterinary Medicine A 44, 125–132. Philipsson, J., Andrèasson, E., Sandgren, B., Dalin, G. and Carlsten, J. (1993) Osteochondrosis in the tarsocrural joint and osteochondral fragments in the fetlock joints in Standardbred trotters. II. Heritability. Equine Veterinary Journal (Suppl.) 16, 38–41. Philipsson, J., Brendov, E., Dalin, G. and Wallin, L. (1998) Genetic aspects of diseases and lesions in horses. In: Proceedings of the 6th World Congress on Genetics Applied to Livestock Production, Vol. 24, pp. 408–415. Poncet, P.A., Montavon, S., Gaillard, C., Barrelet, F., Straub, R. and Gerber, H. (1989) A preliminary report on the possible genetic basis of laryngeal hemiplegia. Equine Veterinary Journal 21, 137–138. Pulos, W.L. and Hutt, F.B. (1969) Lethal dominant white in horses. Journal of Heredity 60, 59–63. Raudsepp, T. (1999) Comparative genome analysis in the horse. Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden. Roneus, B., Arnason, T., Collinder, E. and Rasmuson, M. (1998) Arthroscopic removal of palmar/plantar osteochondral fragments (POF) in the metacarpo- and metatarsophalangeal joints of Standardbred trotters – outcome and possible genetic background to POF. Acta Veterinaria Scandinavica 39, 15–24.

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E. Collinder and M. Rasmuson Rudolph, J.A., Spier, S.J., Byrns, G., Rojas, C.V., Bernoco, D. and Hoffman, E.P. (1992) Periodic paralysis in Quarter Horses: a sodium channel mutation disseminated by selective breeding. Nature Genetics 2, 144–147. Santschi, E.M., Purdy, A.K., Valberg, S.J., Vrotsos, P.D., Kaese, H. and Mickelson, J.R. (1998) Endothelin receptor B polymorphism associated with lethal white foal syndrome in horses. Mammalian Genome 9, 306–309. Shin, E.K., Perryman, L.E. and Meek, K. (1997) A kinase-negative mutation of DNA-PK(CS) in equine SCID results in defective coding and signal joint formation. Journal of Immunology 158, 3565–3569. Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy. Freeman, San Francisco, pp. 400–408. Stenlund, E. (1994) Roaring and Horse Breeding – An Offspring Survey and Clinical Records Study. Report from Department of Anatomy and Histology, Swedish University of Agricultural Sciences, Sweden. Studdert, M.J. (1978) Primary, severe, combined immunodeficiency disease of arabian foals. Australian Veterinary Journal 54, 411–417. Trommershausen Bowling, A. and Clark, R.S. (1985) Blood group and protein polymorphism gene frequencies for seven breeds of horses, in the United States. Animal Blood Groups and Biochemical Genetics 16, 93–108. Trommershausen Bowling, A., Millon, L. and Hughes, J.P. (1987) An update of chromosome abnormalities in mares. Journals of Reproduction and Fertility, 35 (Suppl.), 149–155. Valberg, S.J. (1997) Polysaccharide storage myopathy (PSSM) In: Proceedings of the 15th Annual Veterinary Medical Forum. American College of Veterinary Medicine, Colorado. Valberg, S.J., Geyer, C., Sorum, S.A. and Cardinet, G.H., III (1996) Familial basis of exertional rhabdomyolysis in quarter horse-related breeds. American Journal of Veterinary Research 57, 286–290. Wright, S. (1978) Variability within and among natural populations. In: Evolution and Genetics of Populations, Vol. 4. University of Chicago Press, Chicago, Illinois.

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Cytogenetics and Physical Gene Maps B.P. Chowdhary 9Cytogenetics Bhanu andand P. Physical Chowdhary T. Raudsepp Gene Mapsand Terje Raudsepp Divison of Animal Genetics, The Royal Veterinary and Agricultural University, Grønnegårdsvej 3, 1870 Frederiksberg C, Denmark Introduction Cytogenetics Horse chromosomes – a historical background The horse karyotype Meiotic chromosomes Chromosome aberrations Gene Mapping in Horses – Physical Gene Maps Historical background Synteny mapping Radiation hybrid mapping In situ hybridization – radioactive and non-radioactive Flow sorting of chromosomes Chromosome microdissection Chromosome painting Comparative chromosome painting Future Prospects References

171 172 172 172 178 180 191 191 192 196 197 209 209 211 211 220 223

Introduction Since their inception, physical gene maps are portrayed routinely in a cytogenetic background because, in general, the mapping data talk ‘cytogenetic language’. The map primarily indicates the location of a marker on a chromosome. The location is shown either by a chromosome number assigned through a standard karyotype, or more precisely by cytogenetically defined bands determined through a nomenclature system. Hence this chapter will first address equine chromosomes by providing a brief overview of the status of cytogenetics in the horse. Differences between normal and abnormal karyotypes will be summarized mainly to show how the latter affects viability and fertility in the horse. This will be followed by a detailed survey of various ©CAB International 2000. The Genetics of the Horse (eds A.T. Bowling and A. Ruvinsky)

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physical gene mapping techniques hitherto applied in this species, which provide a broad outlook of the current equine physical gene map.

Cytogenetics Horse chromosomes – a historical background Preliminary studies to visualize chromosomes of several domestic species and to assess their modal number were carried out mainly during the early 1900s. Among the first documented reports, the haploid chromosome number varied between ten and 19 while the diploid number was between 20 and 37 (Kirillow, 1912; Masui, 1919; Painter 1924). Although initial studies suggested that the horse has an XO sex chromosome configuration (Wodsedalek, 1914), it was later evident that, like other animal species, the horse also has an XY sex system (Makino, 1942). However, it took another 17 years before the correct chromosome number in the horse was established. Using cell cultures from kidney tissue, Rothfels et al. (1959) studied five horses and showed unambiguously that the chromosome constitution in horse was 64,XY. The results were later confirmed by independent groups using cultures from peripheral blood lymphocytes (PBLs; Moorhead et al., 1960; Makino et al., 1963). The success with enumeration of horse chromosomes sparked a series of cytogenetic studies in several related equid species. Today, information on chromosome number in ten of the 12 extant Equus species is available. A comparative summary of these data is presented in Table 9.1. It needs to be mentioned that two of the species – E. quagga and the Eurasian wild horse, the tarpan – became extinct during the 19th century (Short, 1975). The E. przewalskii species has just emerged from the verge of extinction, and is still listed as a highly endangered species. Of interest in the present context is the variation in chromosome number among the existing Equus species. In spite of being closely related, the diploid number within this genus varies considerably from 2n = 32 in Hartmann’s mountain zebra to 2n = 66 in Przewalski’s horse (see Table 9.1 for details). Similarly, the nombre fondamental (NF) in the genus ranges from 62 in Hartmann’s mountain zebra to 102 in the domestic ass and the Mongolian and Transcaspian wild asses. These variations will be of significance while briefly discussing hybrids between different Equus species in a later section of this chapter.

The horse karyotype The early arrangement and the present-day karyotype Ever since the correct chromosome number in horse (2n = 64) was determined (Rothfels et al., 1959), equine chromosomes have been presented in several ways. Among the first known karyotypic arrangements are those by Trujillo et al. (1962) and Benirschke et al. (1962). However, it was not until the correct

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number of meta- and submetacentric (13 pairs of autosomes + the sex chromosomes) and acrocentric (18 pairs of autosomes) chromosomes was ascertained (Benirschke et al., 1965; Hsu and Benirschke, 1967) that a stable karyotype pattern began emerging. The first ‘standard karyotype’ (Ford et al., 1980) provided a reasonably good basis for various equine cytogeneticists to converge on common grounds. We will therefore present only a summary of developments following this ‘standard’. The Second Standardization Conference (Richer et al., 1990) accepted a rearranged, improved and compact version of the equine karyotype. In this version, nine of the first 13 chromosomes from the first standard were rearranged. The new karyotype had only six rows of chromosomes as compared with the eight presented earlier. The first three rows comprised 13 meta- and submetacentric autosomes arranged according to their relative length (see Richer et al., 1990, for details). The sex chromosomes were placed at the end of the third row. The 18 pairs of acrocentric autosomes were arranged in the next three rows (each with six pairs), with the same decreasing size order as followed in the first standard karyotype (Ford et al., 1980). In spite of the changes and presentation of both G- and R-banded karyotypes, the new standard fell short of an enumerated nomenclature of chromosome bands, which became increasingly important in light of developments in the field of gene mapping. This need was fulfilled when ISCNH (1997) provided a detailed description of both G- and R-banded chromosomes along with ideograms showing landmarks and band numbers for each of the banding approaches (Fig. 9.1). No changes in chromosome numbering as compared with the second standard (Richer et al., 1990) were made. To cater to the needs of both cytogeneticists and the gene mapping community in correctly identifying equine chromosomes, three resolutions ranging from moderate to reasonably elongated chromosomes were presented.

Application of different banding techniques A wide range of banding techniques has been applied to study the horse chromosomes. The early studies concentrated primarily on the morphology, size and count of the chromosomes, and hence used, for example, orcein (Benirschke et al., 1962), Giemsa or basic fuchsin (Trujillo et al., 1962) for visualizing the chromosomes. These applications, however, did not allow unequivocal identification of all homologous pairs. Though basic techniques to identify homologues among metaphase chromosomes from a cell were reported during the early 1970s (Q-banding, Caspersson et al., 1970; Gbanding, Seabright 1971; both techniques highlight AT-rich DNA as positive bands), their application to horse chromosomes was first reported only a few years later by Biemont and Laurent (1974), Short et al. (1974) and Hageltorn and Gustavsson (1974). A couple of years later, Buckland et al. (1976) provided for the first time a detailed description of individual G-banded equine chromosomes, thus adding a new phase to chromosome study in the horse. The latter made a significant contribution towards preparing a basis for

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B.P. Chowdhary and T. Raudsepp Table 9.1. A summary of the present status of cytogenetics in the genus Equus. Species name supplemented with common name, diploid chromosome number, ‘nombre fondamental’ (NF), karyotypic characteristics and references for individual species within the genus are presented. Species

2n

NF

E.f. przewalskii Przewalski’s wild horse

66

92

NK

—

64

92

62

102

62/63

96

E.f. ferus Tarpan, Eurasian wild horse E. caballus Domestic horse E. asinus Domestic ass, donkey

E. africanus somaliensis Somalian wild ass

E q u u s

E. hemionus hemionus Mongolian wild ass E. hemionus onager Persian wild ass

NK

—

55/56

102

E. heminonus kulan Transcaspian wild ass

54/55

102

NK

—

51/52

92

E. grevyi Grevy’s zebra, Imperial zebra

46

80

E. burchelli Grant’s zebra, common zebra

44/45

80

E. zebra Hartmann’s mountain zebra

32

62

E. quagga Quagga

NK

—

E. hemionus khur Indian wild ass E. kiang Tibetan wild ass

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Continued.

Comments

References

Thirteen pairs of meta-/submetacentrics, 18 pairs of acrocentrics + sex chromosomes; karyotype similar to domestic horse except for an extra pair of acrocentric chromosomes

Benirschke et al. (1965), Ryder et al. (1978)

Extinct during the 19th century

Short (1975)

Most probably similar to wild horse except for a metacentric which corresponds to two acrocentrics in the wild horse Nineteen pairs of autosomes meta-/submetacentric while remaining acrocentric; except for some similarities, karyotype is different from horse; Robertsonian polymorphism involving chromosome 3

Benirschke et al. (1962), Trujillo et al. (1962), Ryder et al. (1978), Bowling and Millon (1988)

Banding homology with domestic ass; 19/21 centric fusion/ fission polymorphism

Benirschke and Malouf (1967), Benirschke and Ryder(1985), Houck et al. (1998)

—

—

Twenty two pairs of meta-/submetacentrics and five pairs of acrocentric autosomes; centric fusion/fission polymorphism of chromosomes 23/24

Benirschke et al. (1965), Ryder et al. (1978)

Twenty two pairs of meta-/submetacentric and five pairs of acrocentric autosomes; 13/24 centric fusion/fission polymorphism

Ryder et al. (1978)

—

—

Polymorphism involves same elements participating in Robertsonian rearrangements in E. hemionus kulan and E. hemionus onager (chromosomes 22/23 in E. kiang)

Ryder and Chemnick (1990)

Sixteen pairs of meta-/submetacentric and six pairs of acrocentric autosomes; probable ancestral equid karyotype

Mutton et al. (1964), King et al. (1965, 1966), Benirschke and Malouf (1967), Ryder et al. (1978)

Seventeen pairs of meta-/submetacentric and four pairs of acrocentric autosomes; polymorphism in chromosome 5. Karyotype similar to Grevy’s zebra

Benirschke et al. (1963), Benirschke et al. (1964a), Hansen (1975), Ryder et al. (1978), Power (1984), Whitehouse et al. (1984)

Fourteen pairs of meta-/submetacentric and one pair of acrocentric autosomes; lowest chromosome number in genus Equus

Benirschke et al. (1964b), King et al. (1966), Benirschke and Malouf (1967)

Extinct during 19th century

Short (1975)

NK denotes not known. A slash between diploid chromosome number indicates polymorphism.

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B.P. Chowdhary and T. Raudsepp 18 17 15 14.3 14.1 13 12.2 11 11 13 14 15 16 17.1 17.2 17.3 21.1 21.2 21.3 22 23 24

18 17 16 15.3 15.1 14 13 11 11 12 13 14.1 14.3 21.1 21.2 21.3 22 24 25

26 28

1 15 14 13 12 11 11 12 13 21.1 21.2 21.3 22 23 24

2 16 15 14.3 14.1 13 11 11 12 13 14 15 16 17 18 19

6 14 13 12.3 12.1 11 11 13 14.1 14.3 15 16

16 15 14 13 12 11 11 12 13 14.1 14.3 21 22.1 22.3 23 25 26

14 15 16 17

4

16 17 18

15 14 12 11 11 12 13 14 15

13

5 15 14 13 12 11 11 12 13 14 15 17 21 22 23

15 14 13 12 11 11 13 14 15

8

12

11 11 12

24 26 27

17 16 15 14 12 11 11 12 13 14 16 21.1 21.3 22

14 13 12 11 11 12 13 14

18 17 16 15 14 13

21.1 21.3 22

3

7

11

16 14 12 11 11 12 13 14

9 25 23 21 15 14 13 11 11 13 15 17 21 22 24 26 27 28 29

10

12 11 12 13 14 15

Y

X 11 12 13 14

11 12 13 14 15 16 21.1 21.2 21.3 22.1 22.3 23.1 23.3 24

11 12 13 14 21.1 21.3 22 23 24 25 26 27

15 16 21 22.1 22.3 23 24 25 26 27

14 11 12 13 14 15 16

15 11 12 13 14 15 16 17

21.1 21.2 21.3 22 24

20 11 12 13 14

21 11 12 13 14 15 16 17 18

15 16 17

26

15 16 17 18 19

22 23 24

21

17

15 16.2 16.3

23

24 11 12 13 14

25 11 13 14 15 16 17

15

29

19 11 12 13 14 15 16 17 18 19

14

11 12 13 14 15 16

28

18 11 12 13

18 19

22

22 24 25

25 26

15 16

11 12 13 14 15 16 17 18

27

22 23 24 25 26 27

11 12 13 14 15 16 17 18 21

14 15

11 12 13 14

14

19.2

11 12

16 11 12 13

18

11 12 13 15 21.1 21.3

30

31

Fig. 9.1. Schematic drawing of G-banded equine chromosomes as described in the latest standard horse karyotype (ISCNH, 1997).

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describing G-banded horse chromosomes in the first standard karyotype of the horse (Ford et al., 1980). Application of the R-banding technique (positive bands are the reverse of the G- and Q-positive bands), which primarily highlights GC-rich DNA, was first reported during the early 1980s (Murer-Orlando et al., 1982). Equine cytogeneticists used both the fluorescent- (RBG; Molteni et al., 1982) and the heat treatment–Giemsa staining-based approaches (RHG; e.g. Murer-Orlando et al., 1982; Power, 1987a; Romagnano et al., 1987a), leading to distinct R-bands (Marki and Osterhoff, 1983; Maciulis et al., 1984; Richer and Romagnano, 1985; Romagnano and Richer, 1984, 1985; Romagnano et al., 1987a; Rønne et al., 1993). In the later reports, enhanced banding quality on elongated chromosomes was produced by the use of cell cycle synchronization and incorporation of bromodeoxyuridine (BrdU) into chromosomal DNA (e.g. see Romagnano et al., 1983; Marki and Osterhoff, 1983; Power, 1987a, 1990). Using this approach, a detailed description of R-banded chromosomes with corresponding ideograms was presented by Rønne et al. (1993). Besides banding techniques that allow chromosome identification, there are methods which specifically highlight constitutive heterochromatin. This banding technique, also referred to as C-banding, and which can be produced by acidic and/or alkaline and heat treatment, was in fact the first differential staining technique applied to horse chromosomes (Comings and Mattoccia, 1972). Later, Melchior and Höhn (1976), Buckland et al. (1976) and others used it routinely for various purposes. Among the autosomes, all horse (ECA) chromosomes, except ECA11, show darkly stained pericentromeric heterochromatic blocks. Interstitial heterochromatin has been reported on ECA1pter, 12q and Xq, while the Y chromosome is known to be almost entirely heterochromatic (Buckland et al., 1976; Ryder et al., 1978; see Power, 1990). Size polymorphism of centromeric C-bands has been shown for ECA1, 8, 12 and 13 (Buckland et al., 1976; Ryder et al., 1978; Haynes and Reisner, 1982; see Power, 1990), and a broad range of heterochromatin polymorphism has been observed also using the chromomycin A3/distamycin A–4 ′,6-diamidino-2phenylindole (DAPI) technique (Kopp et al., 1988). T-banding, which highlights telomeric repeat sequences, has been applied only rarely in the horse as compared with other farm animals. Horse telomeric repeats were first studied using molecular cytogenetic approaches (de La Seña et al., 1995). Except for their usual location, no intercalary telomeric repeats were observed. Recently, the same approach was used to study telomeres of equine sperm cells (Zalensky et al., 1997). Chromosomal regions containing transcriptionally active rRNA genes (rDNA or nucleolus-organizing regions, NORs) can be visualized by NOR-banding. The technique usually involves staining with silver nitrate (Goodpasture and Bloom, 1975; Bloom and Goodpasture, 1976) but can now be conducted using molecular cytogenetic approaches. The presence of three pairs of NOR chromosomes in the horse karyotype was first shown by Sysa et al. (1977). However, assignment of NORs to specific chromosomes could be carried out only after the application of a combination of NOR and

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chromosome identification techniques (G- or R-banding) (Wockl et al., 1980; Kopp et al., 1981; Cribiu, 1981; Gadi and Ryder, 1983; Power, 1987a). These studies suggested ECA1, 28 and 31 to be the NOR-bearing chromosomes. Electron microscopy showed that the NORs reside outside the terminal secondary constriction on the terminal portion of ECA1p, beside the proximal region of ECA28 and outside the secondary constriction on the proximal region of ECA31 (Romagnano et al., 1987b). As in other species, variation in the expression of rDNA genes has also been observed within and between horses (Kopp et al., 1988). Recently, horse rDNA was studied using a cluster of human 18S–5.8S–28S rDNA as a probe (Deryusheva et al., 1997). In this study an additional NOR-bearing chromosome, ECA27, was reported. In addition to these banding techniques, attempts have also been made to produce bands on equine chromosomes specific for electron microscopy (Messier et al., 1989; Richer et al., 1989). A monoclonal anti-BrdU antibody and a protein A–gold complex were used to detect discontinuous incorporation of BrdU in the chromosomes, resulting in the depiction of well-defined band-like patterns. The patterns were similar to those observed by light microscopy and readily facilitated identification of homologues and, consequently, construction of a karyotype. Chromosome measurements and size polymorphism The relative length of horse chromosomes is reported in only a few studies (Melchior and Höhn, 1976; Stranzinger, 1980; Molteni et al., 1982; Hansen, 1984a). The results vary slightly between authors depending on whether G(Melchior and Höhn, 1976), R- (Molteni et al., 1982) or Q-banding (Hansen, 1984a) was used for chromosome identification. Q-banding followed by Giemsa staining is proposed to be the most suitable approach for the measurements because it does not disturb or remove parts of the chromosomes (Hansen, 1984a). It is agreed that the X chromosome is the second largest element in the karyotype and forms about 5% of the genome (Stranzinger, 1980; Hansen, 1984a), while the Y chromosome is one of the smallest. However, it is known that the Y chromosome shows a broad range of size variation (more than twice as compared with the smallest known size). This polymorphism is not considered to be breed dependent or connected to any clinical abnormalities (Hansen, 1984b; Power, 1988).

Meiotic chromosomes As in other species, the study of meiotic chromosomes in the horse has helped in analysing the basis of chromosome aberrations. It is well known that nondisjunction during male/female meiosis, in the majority of cases, is the source of sex chromosome or autosome aneuploidy. Hence information on chiasma frequency at diakinesis and estimating the frequency of non-disjunction and irregular segregation at meiotic metaphase II is of significance (Scott and Long, 1980; King et al., 1990). Meiotic studies in hybrids between different equid

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species have contributed to developing an understanding about the likely causes of hybrid sterility. Different aspects of meiotic studies in the horse are briefly discussed below. Male meiosis All the early chromosome studies in the horse were conducted using meiotic preparations from spermatocytes or spermatogonia. However, the main aim of these works was not to investigate specifically the meiotic chromosomes, but to find out the normal chromosome number of the horse. Later, normal male meiosis was studied cytogenetically in order to compare pachytene spermatocytes between fertile stallions and the mule and hinny (Chandley et al., 1974). Until now, the only detailed study of normal male meiosis in the horse is presented by Scott and Long (1980). Chromosome analysis of testicular material from eight normal stallions at diakinesis and meiotic metaphase II showed that the overall mean chiasma number in horses is 54.4 and the non-disjunction frequency is 3.4%. These values closely resemble the corresponding figures reported for other domestic species (Scott and Long, 1980). The study also showed that at diakinesis, the Y chromosome is associated with the short arm of the X chromosome. Female meiosis As far as is known, a very limited number of studies concerning equine female meiosis have been carried out to date. The primary aim of these investigations was to detect the time of ovulation (King et al., 1987, 1990) and to study the fine structure of the follicular oocytes of a mare (Vogelsang et al., 1987). The findings showed that, as in other mammals, equine oocytes ovulate after completion of the first meiotic division (King et al., 1987, 1990) and that nondisjunction at this stage leads to hypo- or hyperhaploid cells in metaphase II. It was also proposed that fertilization of such oocytes causes aneuploid zygotes (King et al., 1990). Chromosome studies of in vitro matured oocytes have also been carried out (Sosnowski et al., 1997). It was observed that as compared with cattle, sheep and goats, the maturation time of the oocytes was longer in the horse. This information is of value for in vitro fertilization and embryo transfer in the horse (Sosnowski et al., 1997). Synaptonemal complex analysis Synaptonemal complex analysis (SCA) involves electron microscopic investigation of the pairing pattern of homologous chromosomes during meiotic prophase I. The study provides vital information concerning normal/abnormal pairing of the chromosomes during this stage. SCA of cases with an aberrant chromosomal constitution helps in understanding the basis of fertility disturbances at the meiotic level (Safronova and Pimenova, 1988; Switonski and Stranzinger, 1998). As yet, only one study involving SCA analysis has been carried out in the horse (Power et al., 1992). The horse, a carrier of ECA28 trisomy, demonstrated a trivalent (synapsis involving the two homologues together with the extra ECA28) or a bivalent (a normal synapsis between the

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two homologues) plus a univalent (the third homologue which could not pair anywhere and sometimes formed a ring) in primary spermatocytes. Azoospermy observed in this trisomic horse was attributed to degeneration observed in the germinal epithelium, which in turn affects the spermatocyte and spermatid levels in the semen. Meiotic studies of interspecific hybrids A considerable amount of data regarding analysis of meiotic cells of hybrids between horse and donkey, zebra and horse, zebra and donkey, and domestic horse and Przewalski’s horse is available (Wodsedalek, 1916; King et al., 1966; Trujillo et al., 1969; Taylor and Short, 1973; Chandley et al., 1974, 1975a; Short et al., 1974). In the majority of the studies, male meiosis was investigated. However, in one of the works, the development of germ cells in the ovary of the mule and hinny was also analysed (Taylor and Short, 1973). It is interesting to note that in horse ×donkey hybrids, testicular meiosis shows a ‘block’ at the primary spermatocyte stage. This is caused by incompatibility of synaptal pairing between paternal and maternal chromosomes, resulting in a total arrest of spermatogenesis (Chandley et al., 1975a). However, it is noteworthy that spermatozoa occasionally have been observed in ejaculates from male (Trujillo et al., 1969; Chandley et al., 1974), and oocytes in ovaries of female mules and hinnies (Taylor and Short, 1973). The only totally fertile equid hybrid hitherto known is that between E. caballus and E. przewalskii. Meiotic studies of these hybrids show formation of a trivalent which segregates into two classes of genetically balanced germ cells (Short et al., 1974; Chandley et al., 1975a).

Chromosome aberrations Like some of other farm animal species, chromosome aberrations were reported in the horse even prior to the introduction/application of banding techniques (e.g. Payne et al., 1968; Gluhovschi et al., 1970; Basrur et al., 1970; Marx et al., 1973). During the pinnacle years of horse cytogenetics, which most likely were between 1970 and 1990, a vast number of abnormal karyotypes were reported. Although the cumulated data are not as diverse in numbers as those catalogued in, for example, pigs and cattle, the vast array of reports on chromosomal abnormalities do indicate that equine chromosomes have been studied extensively. Because the majority of these aberrations are discussed at length in earlier published reviews (e.g. Chandley et al., 1975b; McFeely, 1975; Halnan, 1985; Bowling et al., 1987; Long, 1988; Chandley, 1997) and chapters (Eldridge, 1985; Bowling, 1992; Power, 1990; Bowling, 1996), and no significant additions have been made since then, we shall provide only a brief overview of the chromosomal abnormalities discussed therein. Therefore, for an extended description of these aberrations, we would refer readers to the aforementioned reviews/chapters. However, data published thereafter will be discussed at length.

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A summary of all chromosomal aberrations as yet reported in the horse is presented in Table 9.2. For the sake of convenience, chromosomal aberrations are classified into two major categories, i.e. (i) autosomal (includes autosome–sex chromosome and vice versa translocations) and (ii) those involving only the sex chromosomes. Each of the categories is classified further into two subcategories: structural and numerical. A large number of mosaic/chimaeric karyotypes involving the sex chromosomes, as well as cases of sex reversal, are listed separately from the structural and numerical aberrations. Chromosome aberrations involving autosomes After four decades of cytogenetic analysis in the horse, it might appear unusual that only six structural and seven numerical autosomal aberrations have been detected as yet in this species. Compared with the vast amount of information available in human and mouse, where the studies have been very comprehensive, the equine information seems limited. However, how the equine data relate to that available in farm animal species such as cattle, pigs, sheep, etc., can be of interest. None the less, before making any comparisons, it must be noted that, over the years, not more than 1400–1500 horses (predominantly mares) have been examined, resulting in the detection of about 300 cases with chromosome aberrations (note that cases of sex reversal which amount to ~110 are not included in this count; see explanation later in the section below). One must also bear in mind that in many instances the cases were hand-picked, due either to phenotypic deviations or to fertility problems. As far as known, there are only 18 reports where, within a study, more than ten individuals were analysed cytogenetically, and only nine where the number was over 40. Thus no large-scale organized cytogenetic studies were ever conducted in the horse. This itself shows the limitations of the cytogenetic data in this species compared with those available in pigs and cattle, where more organized, and in some cases mandatory karyotype analyses, were carried out. However, one cannot rule out two other possibilities regarding autosomal chromosome aberrations in the horse: that they (i) are less common in this species – which probably is highly unlikely; and (ii) animals with these abnormalities do not survive to full term and might result in early embryonic death – which might be more rational. However, due to lack of comprehensive karyotype analysis of the equine population, the plausibility of these two scenarios needs verification. Of the six structural aberrations, three are deletions (loss of part of chromosome) and three are translocations (both balanced and unbalanced). Among the latter, two involve only autosomes while the remaining one involves the X chromosome (see Table 9.2 for references). Except for an extra chromosomal fragment (unspecified) reported in a few of the cells of a mare with reproductive disorders (Chandley et al., 1975b) and a loss of a small metacentric chromosome in a chimaeric Standardbred mare (Klunder et al., 1990), all numerical aberrations in the horse are trisomies of different

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Table 9.2.

An overview of different chromosome aberrations found in the horse.

Abnormality I Autosomes Structural aberrations 64,XX, del(12q) 64,XY, del(13qter) 64,XX, del(4p) 64,XX, t(1q;3q) 63,XY, t(1;30) 64,X,-X+der(X),t(X;15q) Numerical aberrations 65,XY,+23 65,XX,+26 centric fusion type trisomy 65,XX,+27 65,XY,+28 65,XX,+30 65,XY,+31 64,XX/65,XX,+fragment 63,XX(-small metacentric)/64XX/64,XY II Sex chromosomes Structural aberrations 64,X,-X+der(X),t(X;15q) 64,X,del(Xp) 64,XX, del(Xp) 63,X/64,X-Y,+t(Yq;Yq) 64,X,i(Xq) 64,XY/64,XY,del(Xq) Numerical aberrations 63,X0

65,XXX

65,XXY 66,XXXY

References

Trommershausen-Smith et al. (1979) Halnan et al. (1982) Rønne (1992) Power (1991) Long (1996) Power (1987) Klunder et al. (1989) Bowling and Millon (1990) Buoen et al. (1997) Power (1987) Bowling and Millon (1990) Lear et al. (1999) Chandley et al. (1975b) Klunder et al. (1990)

Power (1987) Trommershausen-Smith et al. (1979), Bowling et al. (1987), Rønne (1992) Rønne (1992) Herzog et al. (1989) Mäkelä et al. (1994) Halnan (1985) Payne et al. (1968), Chandley et al. (1975b), Hughes et al. (1975a, b), Taylor and Trommershausen-Smith (1975), Hughes and Trommershausen-Smith (1977), Blue et al. (1978), Bruère et al. (1978), Metenier et al. (1979), Miyake et al. (1979), Trommershausen-Smith et al. (1979), Walker and Bruère (1979), Metenier and Cribiu (1980), Cribiu and Losfeld (1982), Buoen et al. (1983a), Halnan (1985), Mäkinen et al. (1986), Bowling et al. (1987), Long (1988), Klunder and McFeeley (1989), Long (1989), Klunder et al. (1990), Buoen et al. (1993), Breen et al. (1997a) Chandley et al. (1975), Buoen et al. (1983b), Bowling et al. (1987), Power (1987a), Stewart-Scott (1988), Klunder and McFeeley (1989), Moreno-Milan et al. (1989), Klunder et al. (1990), Mäkinen et al. (1999) Kubien et al. (1993) Gluhovschi et al. (1970, 1975)

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Continued.

Abnormality Mosaicism/chimaerism 63,XO/64,XX

63,XO/65,XXX 64,XX/65,XXX 63,XO/64,XX/65,XXX 64,XX/64,XY 63,XO/64,XY 64,XX/65,XXY 63,XO/65,XYY 64,XX/65,XXX/65,XXY 63,XO/64,XX/64,XY 63,XX/64,XX/64,XY 63,XO/64,XY/65,XXY 63,XO/64,XX/65,XXY 64,XX/64,XY/65,XXY 64,XX/96,XXY 63,XO/64,XX/64,XY/65,XXY 63,XO/64,XX/65,XXX/65,XXY 63,XO/64,XX/65,XXX/65,XXY/ 66,XXYY/66,XXXY III Sex reversal syndromes 64, XY (female)

64,XX (male; intersex)

References Chandley et al. (1975b), Hughes et al. (1975a, b),Hughes and Trommershausen-Smith (1977), Bruère et al. (1978), Trommershausen-Smith et al. (1979), Walker and Bruère (1979), Cribiu and Losfeld (1982), Halnan et al. (1982), Buoen et al. (1983b), Cribiu (1984), Halnan (1985), Kent et al. (1986), Bowling et al. (1987), Long (1988), Stewart-Scott (1988), Klunder and McFeeley (1989), Long (1989), Klunder et al. (1990) Breen et al. (1997a) Gill et al. (1988) Long (1989), Klunder et al. (1990) McIlwraith et al. (1976), Dunn et al. (1981), Buoen et al. (1983b), Halnan (1985), Klunder and McFeeley (1989), Klunder et al. (1990) Hughes and Trommershausen-Smith (1977), Trommershausen-Smith et al. (1979), Dunn et al. (1981), Halnan et al. (1982), Kent et al. (1986), Bowling et al. (1987) Bouters et al. (1975), Gluhovschi et al. (1975), Bielanski et al. (1980), Halnan et al. (1982), Bowling et al. (1987) Höhn et al. (1980), Herzog et al. (1989) Walker and Bruère (1979) Forster and Braun (1984), Stewart-Scott (1988), Klunder et al. (1990) Klunder et al. (1990) Halnan et al. (1982), Klunder and McFeeley (1989) Fretz and Hare (1976) Dunn et al. (1974) Power and Leadon (1990) Basrur et al. (1970) Klunder and McFeeley (1989) Klunder et al. (1990)

Chandley et al. (1975b), Kieffer et al. (1976), Hughes and Trommershausen-Smith (1977), Sharp et al. (1980), Buoen et al. (1983b), Cribiu (1984), Power (1986), Kent et al. (1986), Bowling et al. (1987), Long (1988), Stewart-Scott (1988), Klunder and McFeeley (1989), Long (1989), Klunder et al. (1990), Pailhoux et al. (1995), Mäkinen et al. (1999) Bornstein (1967), Gerneke and Coubrough (1970), Miyake et al. (1982), Constant et al. (1994), Kent-First et al. (1995), Milliken et al. (1995), Meyers-Wallen et al. (1997)

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autosomes. Interestingly, all of them involve the small acrocentric chromosomes (nos 23–31; see Table 9.2 for details). This is in agreement with similar data in other species where viable trisomies generally involve smaller chromosomes. For example, in humans, the most common survivors are trisomies of the HSA13, 18, 21 and 22 (see Chandley, 1997), while in cattle a viable trisomy 22 has been reported (Mayr et al., 1985, 1987). It is possible that trisomies involving larger chromosomes might have a drastic effect on early embryonic survival or later neonatal development. As in other species, the majority of equine trisomies are associated with increased maternal age, causing more errors during the first maternal meiotic division at oogenesis (Buoen et al., 1997; Lear et al., 1999a). Some of the salient features of viable individuals with autosomal aberrations (both structural and numerical) are: reduced to completely impaired fertility, minor to moderate anatomical malformations primarily affecting gait or orientation, atypical poor confirmation and moderate to notably smaller height compared with the breed/parents average. Although, as in other species, production of offspring to full term by individuals bearing balanced reciprocal translocations and tandem fusions has been reported in the horse (see Power, 1991; Long, 1996), the most noticeable is the foaling of a ECA26 trisomy carrier to produce a karyotypically normal colt (Bowling and Millon, 1990). A similar incidence has been reported for a cow carrying an autosomal trisomy (Mayr et al., 1987). Survival of trisomic individuals to full term, or even after birth, is exceptional in humans. Those who do survive suffer from various forms of hereditary malformations. Cognate observations have been made for autosomal trisomies observed in pigs (Bösch et al., 1985), cattle (Coates et al., 1988; Kulikova et al., 1991; Agerholm and Christensen, 1993), chickens (Bloom, 1970) and cats (Benirschke et al., 1974). In the majority of the cases, neonatal death is reported. Of the five viable trisomic individuals hitherto described in the horse, that with the ECA26 trisomy (Bowling and Millow, 1990; see Power, 1990) is of particular interest. Recent comparative studies showed that this equine chromosome corresponds completely to HSA21 (Raudsepp et al., 1996; see Fig. 9.5 and Table 9.6). Mental retardation has been shown to be associated with trisomy of HSA21 (Zuhlke et al., 1994). Compared with this, the ECA26 female appeared mentally dull, showed atypical poor confirmation, angular deformities, lack of vigour, and was small for her breed. However, at 5 years of age, she foaled a karyotypically normal colt and showed normal maternal behaviour. This aspect is of particular interest because it corresponds to similar reproductive status observed in some human Down syndrome females (Masterson et al., 1970). Overall, the equine example provides reasonable parallels between chromosomal abnormality and consequent phenotypic/ physiological effect observed in two evolutionarily distantly related species. an almost similar analogy has been shown for HSA21 and mouse chromosome 16 (MMU16) (see Kola and Hertzog, 1998). However, considerable care must be taken before drawing conclusions about a correlation between chromosome aberration and phenotypic effects, both within and across species.

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Chromosome aberrations involving sex chromosomes The vast majority of chromosomal abnormalities reported in the horse involve the sex chromosomes (predominantly the X). Of the approximately 300 abnormal karyotypes reported to date, about 94.5% involve the sex chromosomes (about 93% involving only the sex chromosomes and 1.5% involving the sex chromosome–autosome). Structural aberrations Five types of structural chromosome abnormalities involving the sex chromosomes are known at present in the horse (see Table 9.2 for details). With the aberrations ranging from translocations and deletions to isochromosomes, it is very difficult to generalize their effects on individuals. However, as is usual with most of the chromosome aberrations, an adverse effect on fertily is a common factor. The mare carrying the unbalanced X;15 translocation (Power, 1987b) was infertile, which is in agreement with similar observations for X–autosome unbalanced translocations in humans (Keitges and Palmer, 1986). The significant part of this investigation was the observation that the derivative X chromosome (which lost the short arm and instead had a large middle to distal segment of ECA15, in addition to two normal ECA15), predominantly showed inactivation. Interestingly, the latter extended also into the attached autosomal segment. It, however, needs to be mentioned that Power (1987b), with an excellent R-banded karyotype, reported the translocated chromosome to be ECA15, with banding patterns similar to HSA13. There appears to be some ‘switch over’ for the equine chromosome because in all standard karyotypes (Ford et al., 1980; Richer et al., 1990; ISCNH, 1997), the chromosome with this banding pattern is listed as ECA17. This has been reiterated further by Rønne (1992) who compared ECA17 and HSA13 with the help of R-banding patterns. Among the mares showing deletion of the short arm of the X chromosome (Bowling et al., 1987), one was infertile while the other, surprisingly, produced two filly foals – one with a karyotype similar to the mare, and the other was normal (Bowling, 1992). This is among the rare examples where a mare with gonadal dysgenesis and chromosome abnormality was fertile. The phenotype of these mares resembled those with monosomy of the X chromosome. In humans, a similar pattern has been reported for patients with Xp deletion. Some of the fertile females either produced karyotypically normal offspring or those bearing the same deletion as themselves (Fryns et al., 1981, 1982). Further, in the male horse with isochromosome Y (Herzog et al., 1989), pseudo-hermaphroditism was found. Lastly, the filly with isochromosome Xq had the short arm of one of the X chromosomes replaced by the long arm. The animal showed retarded growth, impaired sight, small inactive ovaries, and was considered mentally ‘dull’ (Mäkelä et al., 1994). In humans, the general phenotypic manifestations of i(Xq) females is not very different from those with monosomy of the X chromosome (Therman et al., 1980). Large-scale analysis of human patients with Xp and Xq deletions has shown that 65 and 93%, respectively, suffer from ovarian dysgenesis and that Xq deletions do not induce specific symptoms different from those caused by Xp deletions (Therman and Susman, 1990). Whether the same holds true for horses needs to

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be examined carefully. The only two reported cases of Xp deletions in mares with some reproduction problems (Rønne, 1992) give insufficient information for broader conclusions to be drawn. Numerical aberrations A wide range of numerical aberrations of the sex chromosomes are known in the horse. Monosomy (lack) of the X chromosome (karyotype constitution 63,XO) is amongst the most common and comprises around 47% of all reported chromosome abnormalities and 57% of the sex chromosome abnormalities. All XO mares studied to date are reported to be infertile. However, Power (1987a) is of the view that an occasional fertile 63,X mare would not be surprising, especially if attempts are made to cover the mare at an early age. Other characteristics of XO mares include shorter than normal stature, poor body confirmation, small gonads lacking follicular development, and an irregular or absent oestrous cycle (see Power, 1990). Although no concrete evidence has been found as yet in a horse to link the condition with increased embryonic/prenatal mortality, in the absence of an organized investigation, a possible connection between the two cannot be ruled out completely. Monosomy of the X chromosome has also been reported in a number of other mammalian species. In humans, the condition is known as Turner’s syndrome and, as for horses, is by far the most common chromosome abnormality. The effects of this condition in humans are strikingly similar to those observed in the horse (see Walker and Bruère, 1979). Unlike equine XO individuals, there are abundant data to indicate that it invariably (99% of conceptions having the condition) results in early abortion in humans. Sporadic cases of fertile XO human females have also been reported (see Lippe, 1991). Compared with this, in general, XO mice are known to be fertile (e.g. see Burgoyne and Baker, 1981), though reports suggest that these mice reproduce only for a short time compared with normal female mice. Pure trisomy of the X chromosome (65,XXX) is not frequent in horses. To date, a total of only 11 cases of trisomy X have been reported. Although all mares carrying this abnormality were phenotypically normal, they were found to be infertile. In one instance, the trisomy was discovered to be associated with male pseudo-hermaphroditism (Moreno-Millan et al., 1989). In cattle, the abnormality usually results in phenotypically normal females ranging from those with a varying range of ovarian function (Swartz and Vogt, 1983) to those with impaired reproductive physiology (see Moreno-Millan et al., 1989). X trisomy in the dog was in one case found to be associated with gonadal dysgenesis (Johnston et al., 1985). Of two X trisomies reported in buffaloes, one was fertile (Yadav and Balakrishnan, 1982), while the other was sterile (Prakash et al., 1994). In humans, trisomic X females are observed more frequently than in the horse. The majority of these females are subfertile, with varying degrees of gonadal malfunction. However, normal fertile females producing karyotypically normal to trisomy carrier offspring have also been reported in humans. In the light of this evidence, Power (1990) suggested that XXX mares might be present in the general population but, due to lack of

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large-scale karyotype analysis in the horse, might so far have escaped detection. In additon to these numerical aberrations, cases of pure 65,XXY (Kubien et al., 1993) and 66,XXXY (Gluhovschi et al., 1970, 1975) have also been reported. In both cases, most of the clinical symptoms observed in the horse resembled those of Kleinfelter’s syndrome in humans, and analogous condition in bulls and rams (Kubien et al., 1993). Mosaicism/chimaerism Numerous categories of mosaics/chimaeras involving the sex chromosomes (primarily the X) have hitherto been reported in the horse. In the following discussion, a brief overview of the prominent cases of these categories will be presented. Mosaicism of cells having normal female sex chromosome (XX) with those monosomic for the X chromosome, in the same individual – a condition expressed as 63,X/64XX, is the second most common abnormality involving the sex chromosomes in the horse (excluding sex reversal). It represents about 20% of all the chromosome aberrations reported in the horse, and almost a quarter of all sex chromosome-related aberrations. Although phenotypically these mares appear normal, their clinical manifestation closely resembles that of 63,X mares. The animals are known to have normal external genitalia associated with gonadal dysgenesis. Except for the report of Halnan (1985) showing one foaling in six of the ten X/XX mares, there is overwhelming evidence that this mosaicism is associated with sterility. Both Halnan (1985) and Bowling (1992) are of the view that the mosaic individuals can best be described as subfertile – a statement which needs further verification. In humans, X/XX females can range from a prototype of a Turner’s syndrome individual to those having normal offspring (Kohn et al., 1980). It has also been reported that in humans, the X/XX embryos have a significantly higher rate of survival to term than the XO types (Hook and Warburton, 1983). Among other mixoploids, the 63,X/64,XY are worth mentioning because, like humans, they may range from normal looking females with gonadal dysgenesis (Dunn et al., 1981; Halnan et al., 1982) to those apparently intersex (pseudo-hermaphrodites with varying degrees of gonadal dysgenesis; Hughes and Trommerhausen-Smith, 1977; Trommerhausen-Smith et al., 1979). A contrast between whole body chimaeras and blood chimaeras are worth mentioning at this stage. The former, with XX/XY constitution in all cells, may result from the fusion of male and female embryos and are reported as true hermaphrodites in the horse (McIlwraith et al., 1976; Dunn et al., 1981). The latter, with XX/XY constitution only in blood cells (in varying percentages depending upon the sex of the individual), are a result of heterosexual twins. Unlike in cattle and sheep (e.g. see Greene et al., 1977; Power, 1986), the female co-twin in the horse does not suffer from reduced/impaired fertility. A very unusual diploid–triploid chimaerism (64,XX/96XXY) was reported in a Thoroughbred foal presumed to be a female at birth (Power and Leadon, 1990). This type of mixoploid with 2n/3n cells, the first of its kind reported in the horse, showed a perfectly normal karyotype picture through lymphocyte

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culture. However, the fibroblast culture showed a chimaeric cell population with approximately 85% of the cells of 96,XXY type. Clinical examination classified the foal as intersex. As a yearling, it was described as an attractive, healthy and aggressive individual, manageable only in the presence of geldings. Cases of diploid–triploid chimaeras are also known in early embryos, neonates with malformation or in intersexes of other species such as humans (increased viability compared with pure triploids; Boué et al., 1975), cattle, pigs, cats, mink and chickens (see Power and Leadon, 1990 for details). Another type of unique chimaerism is that reported in an 8-year-old Arabian mare with a history of fertility problems, and later diagnosed as having gonadal dysgenesis (Klunder et al., 1990). Fibroblast cultures from this otherwise physically normal mare showed XX and XXX cell lines in a ratio of 1 : 3. However, the picture from leucocyte culture was amazing, exhibiting six different cell types (63,XO/64,XX/65,XXX/65,XXY/66,XXXY/66,XXYY), with a clear predominance (93%) of the XX and XXX types. The presence of a Y chromosome in three of the cell types is intriguing. It could not be determined whether the mare was born a co-twin to a colt. The birth and viability of such an individual is interesting and worth further investigation. A number of other mosaic/chimaeric conditions are presented in the last section of Table 9.2. For details on these, we refer readers to the reviews and chapters listed at the beginning of the section. However, a general word about these mixoploids may be appropriate to define the effects that they may have on carrier individuals. Most of the reported chimaeric/mosaic cases are registered as females with a wide range of clinically detectable reproductive system deviations. The animals show varying degree of virilization of the external genitalia. In cases where initially cryptorchidism is reported, a wide range of deficiencies of testicular development are observed (Power, 1990). Sex reversal It is difficult to ascribe sex reversal to a true chromosomal abnormality because, as is evident from the karyotype of the individual, it is neither structural nor numerical in nature. Without prior knowledge of clinical manifestations of sex reversal, a cytogeneticist will categorize the examined animal as karyotypically normal. However, sex reversal can be described most appropriately as a disagreement between the karyotypic sex and the phenotypic/ anatomical sex. Hence, considering that the condition still involves the chromosomes, and is a deviation from normal, it is included within the broad heading of chromosome aberration. However, because of its nature, it is described separately here. As far as is known, over 120 such cases have been reported to date in the horse – a number which is sizeable when compared with the approximately 300 other chromosome abnormalities observed in the horse. Normally, individuals with an XY sex chromosome constitution are expected to be males (64,XY). However, in cases where they appear more like females, with varying degrees of male-like characteristics, the condition is called sex reversal. The terminology holds good even if the karyotype indicates the animal to be a female (64,XX), but phenotype and clinical

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examination suggest a preponderance of male-like characteristics. In the following discussion, we briefly exemplify both situations, with an extended summary of latest results/developments. 64,XY sex reversal The degree of significance of this condition is evident from the fact that it is the second most common anomaly found among infertile mares (Bowling et al., 1987). A number of the affected individuals may have normal female external genitalia, along with intra-abdominal testes (see Power, 1986, 1990 for details). In some cases, rudimentary (rarely greatly; see Kent et al., 1986) male-like external sex characteristics may be noticed. There can be others with varied degrees of testicular feminization (e.g. Kieffer et al., 1976) and gonadal dysgenesis (e.g. Trommerhausen-Smith et al., 1979; Sharp et al., 1980). In exceptional cases, reduced normal fertility may also be observed (Sharp et al., 1980; Kent et al., 1986), resulting in phenotypically and karyotypically normal male/female as well as XY female offspring. In several instances, the condition most closely resembles monosomy of the X chromosome. On the basis of different characteristics, Kent et al. (1988) classified the XY sex reversal mares into four categories: (i) phenotypically normal females with a normal reproductive tract (some are even fertile); (ii) females with small and non-functional ovaries; (iii) intersexes with fibrous streak gonads, infantile curpus uteri and an enlarged clitoris; and (iv) virilized intersexes with testes or ovotestes and a hypoplastic uterus and cervix. Analysis of six pedigrees with 64,XY sex reversal mares (Kent et al., 1986) led to the conclusion that the condition could be congenital and attributed to a single gene or the combined effect of a group of genes. An X-linked recessive or an autosomal sex-limited dominant locus was considered responsible for XY females arising through maternal lineages, with an autosomal sex-limited dominant locus for those arising through paternal lineage (see Kent et al., 1986 for details). It was also indicated that in each of the pedigrees with XY females, the sex ratio among the progeny deviated significantly from normal. Since the identification of a zinc finger protein gene on the Y chromosome (ZFY; Page et al., 1987) and sex-determining region on the Y chromosome (SRY; Palmer et al., 1989) as candidates for testis determining factor (TDF) in humans and other mammals, the possibility of loss of Y chromosome material (or specifically either or both of these genes) during paternal meiosis has also been proposed as a likely cause of XY sex reversals. Recently, use of SRY and ZFY probes on DNA from an XY mare with gonadal dysgenesis showed deletion of the SRY gene (Pailhoux et al., 1995). The latter was, therefore, assigned as the reason for the sex reversal phenotype. It must, however, be noted that the second candidate, ZFY, which according to Palmer et al. (1989) is not the testis-determining factor, was present. 64,XX sex reversal Horses with this type of sex reversal have captured the attention of cytogeneticists ever since analysis of equine chromosome became prevalent (Bornstein, 1967; Gerneke and Coubrough, 1970). The condition was further described as intersexuality in later reports and reviews (Hare and Singh,

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1979; Miyake et al., 1982, Long, 1988; Power, 1990; Bowling, 1996). As far as is known, no more than ten cases of this type of sex reversal have been reported to date in horses. The affected individuals have female sex chromosomes, but show the presence of both ovarian and testicular tissue. Phenotypically, the animals are reported as males (or by some owners as females; e.g. see MeyersWallen et al., 1997), with an overall masculine confirmation and moderate to aggressive stallion-like behaviour. Some external female characteristics are not uncommon, and may range from mild, e.g. increased amount of mammary tissue, enlarged nipples, slight udder development (Constant et al., 1994; Milliken et al., 1995), to moderate but abnormal, e.g. normal vulval lips, phalluslike clitoris, blind-ending vagina, etc. Of the three recently studied XX sex reversal cases, two showed retention of small soft testes in the inguinal region/canal (Constant et al., 1994; Milliken et al., 1995), and the horses were referred to as cryptorchid and pseudo-hermaphrodite, respectively. In the most recent case (Meyers-Wallen et al., 1997), the animal was referred to as a true hermaphrodite. The two small, flat, soft gonads detected on rectal palpation turned out to be ovotestes, with 80% of the gonad composed of testicular-like tissue. In horses, cytogenetic and phenotypic examinations of XX sex reversal individuals has also been complemented with endocrine testing. In such individuals, the human chorionic gonadotrophin (HCG) stimulation test normally increases serum testosterone levels by threefold, within 30 min of administration. This test provided only a slight increase in testosterone levels in animals studied by Constant et al. (1994) and Milliken et al. (1995), while a significant increase (3–5 times the pre-test value after 7–23 h) was found in the study of Meyers-Wallen et al. (1997). Further, assay of follicle-stimulating hormone (FSH), oestrogen, and luteinizing hormone (LH) concentrations, which normally are used to assess fertility in stallions, indicated deviant levels for the latter two hormones (Constant et al., 1994). Serum oestrone sulphate concentration in the horse studied by Milliken et al. (1995) clearly diagnosed the animal as having retained testes. A combination of these tests together with phenotypic and karyotype examination of animals can thus prove useful in arriving at a definitive conclusion regarding XX sex reversal cases. The majority (~90%) of human XX male syndrome patients carry Y chromosomal DNA sequences, including the transposition of the SRY gene on one of the X chromosomes (Boucekkine et al., 1994). Recently, Meyers-Wallen et al. (1997) used equine-specific PCR primers for the SRY gene on DNA from a ‘filly’ with ovotestes and some external male-like characteristics. In contrast to a normal male horse, no PCR product of the expected size was obtained from this individual, indicating that this is a case of SRY-negative sex reversal. The authors claim that, as far as is known, this is the first such case in a horse. SRY-negative XX sex reversal cases have also been reported in humans (Berkovitz et al., 1992; McElreavey et al., 1992), dogs (Meyers-Wallen et al., 1995a, b) and pigs (Thomsen and Poulsen, 1993). However, in the latter two species (pig, Sittman et al., 1980; dog, Meyers-Wallen and Patterson, 1988), and in the goat (Soller et al., 1969), XX sex reversal is also shown to be inherited as an autosomal recessive trait. Although XX sex reversal cases have been

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found in a number of other mammalian species (for details, see Meyers-Wallen et al., 1997), it is still unclear whether it is due to mutation in the same gene or in different genes. In the recent past, mutations in the testicular 17-βhydroxysteroid dehydrogenase 3 (HSD17B3) gene located on HSA9q22 have been found to be one of the causes of male pseudo-hermaphroditism (Geissler et al., 1994). Fragile sites Fragile sites cannot be defined as a purely aberrant condition of chromosomes. Rather they represent non-randomly distributed points along the chromosome length, at which chromosomes tend to break under special conditions, e.g. thymidine starvation (Sutherland and Hecht, 1985) and/or exposure to various chemicals. Therefore, it has been recommended that fragile sites be always designated with the source of their production. Following fluorouracil/BrdU synchronization and subsequent fluorochrome photolysis-Giemsa (FPG) staining, Rønne (1992) observed several breaks and gaps in 15 phenotypically normal horses, of which eight mares displayed lower than average foaling rates. Eight of the equine chromosomes (ECA1, ECA4, ECA8, ECA11, ECA16, ECA17, ECA23 and ECAX) showed a relatively higher frequency of fragility at one or more sites, especially in the latter group of mares. Two of the mares also displayed deletions distal to the fragile site on ECA4p and ECAXp. Although the author concluded that chromosomes of the control group (normal stallions/mares) showed a significantly low frequency of fragility as compared with mares with fertility problems, the data are insufficient to deduce firm conclusions because of the low number of animals examined, the techniques employed and the obvious overlap of inducing agents. It is noteworthy that chromosomes from the same 15 animals showed no breakage after thymidine/cytidine synchronization and subsequent GWL-banding. Further, comparison of chromosome banding patterns and the location of fragile sites between horse and human revealed three evolutionarily conserved breakage points on ECA17q21, ECAXq22 and ECAXq27 versus proposed corresponding band locations on HSA13q21, HSAXq22.1 and HSAXq27.3, respectively. Chromosome studies in humans have correlated fragile sites with cancer-related breakpoints, likely targets for mutagens and carcinogens (Yunis et al., 1987), probable recombinational hotspots (Glover and Stein, 1988) and potential sites with evolutionary breakpoints in chromosome evolution (Miro et al., 1987). In the horse, a considerable amount of work is needed before any such inferences can be drawn.

Gene Mapping in Horses – Physical Gene Maps Historical background Among farm animals, the horse enjoys a unique privilege as the only domestic animal in which humans started closely monitoring inheritance of various

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phenotypic characters ever since domestication began over 6000 years ago (reviewed by Chowdhary and Gustavsson, 1992; Bailey, 1998; Bailey and Binns, 1999). Although it is thought that initial genetic studies in horses were carried out already during the 18th century (e.g. analysis of coat colour), it was not before the 1930s and 1940s, that a congenital basis for some of the defects was proposed (see Jones, 1982). With the advent of biochemical genetics in the 1950s, a number of equine blood group and blood protein polymorphisms were detected (for details see Chapter 5). The first gene mapping data for horses came from the mid-1960s, when G6PD was assigned to the X chromosome (Trujillo et al., 1965; Mathai et al., 1966). However, it took 10 more years before the first autosomal linkage group (EAK-PGD) was detected (Sandberg, 1974; for details, see Chapter 5). This work did not provide any major impetus to equine genome analysis, though some small but significant contributions were made during the early 1980s (Andersson and Sandberg, 1984; Sandberg and Andersson, 1984). Chromosome studies, although initiated in the horse, had not progressed sufficiently to assign any of the linkage groups physically to specific chromosomes, except the X chromosome. It was during the late 1980s when in situ hybridization was first utilized on equine chromosomes (Ansari et al., 1988; Mäkinen et al., 1989). This can be termed as the true beginning of physical gene mapping in the horse. The following years showed a slow but steady progress. However, the major stimulus came 3–4 years ago when organized efforts to develop a gene map (both physical and genetic) in the horse began at a global level (First International Workshop for Equine Genome Analysis, Lexington, Kentucky, USA, October 1996). This was possible because both the breeders and the horse geneticists clearly identified that a framework map is necessary to study the underlying genetics of numerous congenital disorders known in horses and to find means to control them. In the following section, we restrict our discussion to the physical mapping techniques applied in horses and will provide details of the current status of the equine physical gene map.

Synteny mapping Synteny means on the same chromosome, and a synteny map simply represents a list of loci known to reside on the same chromosome in a particular species. The basic method for building synteny maps is through the construction of a somatic cell hybrid (SCH) panel by fusing cell lines of two species (Gross and Harris, 1975), one of which is the species in which the map is to be made. Analysis of pairs of genes in an SCH panel reveals concordance or discordance of their retention, thus showing their synteny or asynteny, respectively. The main methods for analysing SCH panels are enzyme electrophoresis, Southern blotting and PCR amplification with species-specific primers (see Kao, 1983; Dionne and Jaye, 1993). The latter nowadays is the most extensively used approach. The precision of synteny mapping depends on how well the hybrid clones are characterized cytogenetically as well as

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through mapping of a sufficient number of markers. Once the chromosome segments are characterized accurately, it is possible to assign markers even to segments/bands of individual chromosomes. The main drawback of this method, however, is that it shows synteny but not gene order or distances between loci. Nevertheless, the possibility of mapping any kind of DNA sequences, including type I or non-polymorphic markers, is an advantage of the method. The first syntenic group in the horse can be traced back to indirect analysis carried out in a mule ×mouse hybrid cell panel (Deys, 1972). Three genes (G6PD, HGPRT and PGK) were then assigned to the X chromosome. However, the first direct synteny study was based on biochemical detection methods using an SCH panel obtained from horse ×mouse heterohybridoma cells (Williams et al., 1993). The analysis resulted in identification of three syntenic groups with eight enzyme genes (Table 9.3). Later, three more mouse ×horse SCH panels were constructed (Bailey et al., 1995; Raney et al., 1998; Shiue et al., 1999). These panels have hitherto not been characterized for the equine chromosomes/segments they contain. However, attempts have been made to see numerically how many whole chromosomes or parts are present in individual clones of some of the panels (Lear et al., 1992; Bailey et al., 1995; Shiue et al., 1999). The authors also report on the preferential loss of large submetacentric equine chromosomes from the hybrids. This makes the panels unsuitable for excluding syntenic groups. Using the panel described by Lear et al. (1992), six syntenic groups including two genes and 15 microsatellites were established after PCR analysis (Bailey et al., 1995). At that time, none of the syntenic groups were assigned to specific chromosomes. Recently, 33 syntenic groups with 182 microsatellites and 58 random amplified polymorphic DNA (RAPD) markers were synteny mapped (Shiue et al., 1999). Based on fluorescence in situ hybridization (FISH) mapping data for some of the markers, 23 syntenic groups were chromosomally located. Further, using a trisomic individual, two microsatellite markers were assigned to ECA30 and it was also possible to reveal the maternal origin of the extra chromosome (Bowling et al., 1997). At present, including the unpublished data, over 500 markers have been mapped using the above-mentioned SCH panels (Bowling et al., 1999; Caetano et al., 1999a,b). Thus the overall status of the equine syntenic map looks very encouraging. Despite this significant progress, the available syntenic map in the horse does not enable adequate comparative analysis with the genomes/chromosomes of other species because the number of mapped type I markers is very low. Although attempts are being made to map specific genes within the available panels, alternative approaches such as the use of comparative anchor-tagged sequence (CATS) primers (Lyons et al., 1997) is also in progress. Preliminary results showed that of the 52 CATS primer sets used, 34 gave a single PCR product, making them potentially useful for mapping in the panel. Recently, eight CATS primer pairs (from HSA5) were mapped into two equine syntenic groups, which were later assigned indirectly to ECA14 and 21 (Caetano et al., 1999a; Table 9.3). The latter are known to be HSA5

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Name

Adenosine deaminase Complement component 3 Complement component 9 Calcium/calmodulin-dependent protein kinase Colipase Colony-stimulating factor 1 receptor Colony-stimulating factor 2

Major histocompatibility complex Oestrogen receptor Fibroblast growth factor receptor 3 Glucose-6-phosphate dehydrogenase Growth hormone Growth hormone receptor Growth hormone-releasing hormone Granzyme A Hexosaminidase Heart fatty acid-binding protein-like Hypoxanthine phosphoribosyltransferase 1 Hyperkalaemic periodic paralysis

ADA C3 C9 CAMK4 CLPS CSF1R CSF2

ELA-DRA ESR FGFR3 G6PD GH GHR GHRH GZMA (CTLA3) HEXB HFABP-L HGPRT HYPP

Method E P P P P P P P P P E, P P P P P P P E P

Location 6? 7pter 21 14 20 14 14 20 31q15–q17 3 X 11 21 22 21 14 2 X 11

Williams et al. (1993) Caetano et al. (1999b) Caetano et al. (1999a) Caetano et al. (1999a) Caetano et al. (1999b) Caetano et al. (1999a) Caetano et al. (1999a)

20q12–q13 19p13.3 5p14–p12 5q21–q23 6pter–p21 5q33.3–q34 5q31.1 5q23–q31 6p21.3 6q25.1 4p16.3 Xq28 17q22–q24 5p14–p12 20q11.2 5q11–q12 5q13 — Xq26.1 17q23

194

Bailey et al. (1995), Caetano et al. (1999) Caetano et al. (1999b) Caetano et al. (1999b) Deys (1972) Caetano et al. (1999b) Caetano et al. (1999a,b) Caetano et al. (1999b) Caetano et al. (1999a) Caetano et al. (1999a) Caetano et al. (1999b) Deys (1972) Caetano et al. (1999b)

Reference

Human location

A list of genes synteny mapped in the horse using somatic hybrid cell panels. Detection methods: E, enzymatic; P, PCR.

Gene

Table 9.3.

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IDH2 IGF1 IGF1R IGF2 IL7R LDHB LEP MPI MSTN NP PEPB PEPC PGK PLG PRKDC/ DNA-PK SPARC TNFA TUB

Isocitrate dehydrogenase 2 Insulin-like growth factor 1 Insulin-like growth factor 1 receptor Insulin-like growth factor 2 Interleukin 7 receptor Lactate dehydrogenase B Leptin (murine obese) Mannose phosphate isomerase Myostatin Nucleoside phosphorylase Peptidase B Peptidase C Phosphoglycerate kinase 1 Plasminogen DNA protein kinase catalytic subunit, candidate gene for SCID Secreted protein, acidic, cysteine rich Tumour necrosis factor-α Tubby

Williams et al. (1993) Williams et al. (1993); Caetano et al. (1999b) Caetano et al. (1999b) Caetano et al. (1999b) Caetano et al. (1999a) Williams et al. (1993) Caetano et al. (1999b) Williams et al. (1993) Caetano et al. (1999b) Williams et al. (1993) Williams et al. (1993) Williams et al. (1993) Deys (1972) Caetano et al. (1999b) Caetano et al. (1999b) Caetano et al. (1999a) Bailey et al. (1995) Caetano et al. (1999b)

15q26.1 12q22–q23 15q25–q26 11p15.5 5p13 12p12.1 7q31.3 15q22–qter 2q12–q22 14q11.2–q13.1 12q21 1q25 Xq13.3 6q26 8q11 5q31–q33 6p21.3 11p15

E P P P P E P E P E E E E P P P P P

1 6, 8, 28? 1 12q14 21 6, 8, 28? 4 1 18 1 6, 8, 28? 2, 5, 30 X 31 9p12 14 20 7

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homologues (Raudsepp et al., 1996; Chaudhary et al., 1998). Further, PCR mapping of 57 type I markers using universal mammalian sequence-tagged sites is in progress (Terry et al., 1999). A summary of synteny-mapped genes in horses is presented in Table 9.3, while data for synteny/FISH-mapped microsatellites and anonymous DNA sequences are listed in Table 9.4.

Radiation hybrid mapping Radiation hybrid mapping is basically an SCH technique with the difference that before fusion of cell lines, the whole or partial genome of the species of interest is exposed to high doses of X-ray irradiation that cause fragmentation of chromosomes (e.g. see Cox et al., 1990). RH mapping shows not only synteny between loci, but also helps to analyse physical distance between them. The farther apart two markers are on a chromosome, the greater are the chances that they will be separated by X-ray treatment, and vice versa. The range of resolution of RH maps is dependent on the irradiation dosage applied. RH mapping enables the integration of linkage maps based on polymorphic markers with non-polymorphic type I markers. The technique has proved to be a powerful tool for high-resolution mapping in human and mouse (McCarthy, 1996; McCarthy et al., 1997; Stewart et al., 1997; Flaherty and Herron, 1998). Among farm animals, whole-genome RH panels recently have been constructed for cattle (Womack et al., 1997) and pigs (Alexander et al., 1998; Yerle et al., 1998), with over 1000 markers mapped in each of the panels. Rigorous expressed sequence tag (EST) mapping studies at present are underway in both species. Because the resolution of RH maps exceeds that of linkage and cytogenetic maps, it provides a new perspective for constructing high-resolution ordered comparative maps between species. The very recent comparison between HSA17 and BTA19 using RH maps is one of the best examples of the power of parallel RH mapping for comparative purposes (Yang and Womack, 1998; Yang et al., 1998). In horses, as far as is known, two radiation hybrid panels currently are being established (B.P. Chowdhary, personal communication; M. Binns, personal communication). The Copenhagen–Texas panel (COPTEX-panel) prepared by the Copenhagen group in collaboration with Texas A&M University, USA, was generated by the irradiation of a horse fibroblast cell line with a cobalt-60 source delivering 185 rad min−1, for a total of 5000 rad. Following this, the cells were fused in the presence of polyethyleneglycol (PEG) with the Chinese hamster TK− fibroblast line A23. The fusion has yielded over 110 hybrid clones which have been propagated for DNA extraction. Analysis of the panel is in progress with the help of different primer pairs. The Cambridge panel (prepared by the Department of Genetics, Cambridge University, Cambridge, in collaboration with Research Genetics, Cambridge, UK) is also being propagated, and analysis of the panel currently is in progress to generate a

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framework map in the horse (Binns, 1999, Plant and Animal Genome VII Conference, San Diego, USA, oral presentation).

In situ hybridization – radioactive and non-radioactive In situ hybridization is a technique which is widely used in several branches of biology. However, with reference to gene mapping, the technique allows direct visualization of the location of specific genes or anonymous DNA segments on the chromosomes. The location is thus a reflection of the molecular constitution of the chromosome at that site. There are two major components of in situ hybridization, i.e. chromosomes, which are the targets, and probes, which are DNA segments of various lengths. Usually, the target is either metaphase or prometaphase chromosomes but, in cases where high-resolution physical mapping is conducted, the chromatin fibre could either be from interphase cells or from mechanically stretched cellular DNA. The probes, however, vary considerably in size as well as origin. The size can range from a few base pairs (bp), e.g. the telomeric or centromeric repeat oligonucleotide sequences, to several hundred kilobases (kb) cloned in a yeast artificial chromosome (YAC) vector. Depending on the type of in situ hybridization, the DNA probe can be labelled radioactively or non-radioactively. Radioactive in situ hybridization, which has now almost become a technique of the past, used radioactively tagged nucleotides as labels, of which tritium [3H] was most extensively used (see Chowdhary, 1998a). However, from the 1980s onwards, the nonradioactive approach has progressed significantly (Pinkel et al., 1986; Lawrence et al., 1988; Lichter et al., 1991; Trask, 1991a, b), and during recent years has become the method of choice. In this approach, the DNA is labelled with nucleotides tagged with biotin, digoxigenin (DIG), di-/trinitrophenol or with other labelling molecules. The hybridization is then detected with a variety of reporter molecules which have affinity for the labels. The detection can either be carried out enzymatically or with the help of fluorochrome conjugates. The latter approach, which is also referred to as FISH, is the most widely used. Direct physical assignment of loci to horse chromosomes by in situ hybridization started a decade ago using the radioactive approach. Due to lack of horse-specific probes, human and pig genomic or cDNA clones were used. The first assignments were for the equine major histocompatibility complex (ELA) to ECA20q14–q22 (Ansari et al., 1988; Mäkinen et al., 1989) and glucose phosphate isomerase (GPI) to ECA10pter (Harbitz et al., 1990). During the initial 5 years, not more than 6–8 genes were mapped in the horse. Later, when the trend shifted to the application of the FISH technique, Oakenfull et al. (1993) mapped haemoglobin-α (HBA) to ECA13q. From here onwards, and with the beginning of international collaboration for developing a gene map in the horse, the number of FISH-mapped loci has soared. During recent years, significant progress has been made in constructing equine genomic libraries and isolating genes and microsatellites from large (Cont’d on p. 205)

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Table 9.4. A list of all microsatellites and anonymous DNA markers mapped in the horse by fluorescence in situ hybridization (FISH) and somatic hybrid cell panel analysis (SCH). Name

Location

Reference

A3 A4 A5 A-14 A-17 AHT2 AHT4 AHT5

17q23 1p14 26q12–q13 2q14–q21 26q13–q14 15 24q14 8*

ASB1 ASB2 ASB3 ASB4 ASB5 ASB6 ASB7

13 15q21–q23 4p12–p13 9q16–q18 9q16–q18 10p13 19q14–q16*

ASB8 ASB9 ASB10 ASB11

1q16–q17 10q21–q23 Centromeric 19q21–q22*

ASB12 ASB13

1q12–q13 2q14.3–q21.1*

ASB14

8*

ASB15 ASB16 ASB17 ASB18

15q21 13q13–q15 2p14–p15 2p17–p18*

ASB19 ASB21 ASB22 ASB23 ASB25 ASB37 ASB38

15q21–q23 Multiple 4q21 3q22.1–q22.3 19q21–q23 13q11–q12 27*

ASB39 ASB40 ASB41 ASB42 ASB43

23q15 7q18–q19 1p14 16q21 29q16

Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Marti et al. 1998) (F) Marti et al. 1998) (F) Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Shiue et al. (1999) (S), International Equine Gene Mapping Workshop (1999) (S) Breen et al. (1997b) (F), Shiue et al. (1999) (S) Breen et al. (1997b) (F), Shiue et al. (1999) (S) Breen et al. (1997b) (F) Breen et al. (1997b) (F), Shiue et al. (1999) (S) Breen et al. (1997b) (F) Breen et al. (1997b) (F), Shiue et al. (1999) (S) Breen et al. (1997b) (F), Shiue et al. (1999) (S), International Equine Gene Mapping Workshop (1999) (F) (S) Breen et al. (1997b) (F), Shiue et al. (1999) (S) Breen et al. (1997b) (F), Shiue et al. (1999) (S) Breen et al. (1997b) (F) Breen et al. (1997b) (F), International Equine Gene Mapping Workshop (1999) (F) (S) Breen et al. (1997b) (F) Breen et al. (1997b) (F), International Equine Gene Mapping Workshop (1999) (F) (S) Breen et al. (1997b) (F), Shiue et al. (1999) (S), International Equine Gene Mapping Workshop (1999) (S) Breen et al. (1997b) (F), Shiue et al. (1999) (S) Breen et al. (1997b) (F) Breen et al. (1997b) (F), Shiue et al. (1999) (S) Breen et al. (1997b) (F), International Equine Gene Mapping Workshop (1999) (F) (S) Breen et al. (1997b) (F) Lear et al. (1999c) (F) Breen et al. (1997b) (F) Lear et al. (1999c) (F) Lear et al. (1999c) (F) Lear et al. (1999c) (F) Lear et al. (1999c) (F), International Equine Gene Mapping Workshop (1999) (S) Lear et al. (1999c) (F) Lear et al. (1999c) (F) Lear et al. (1999c) (F) Lear et al. (1999c) (F) Lear et al. (1999c) (F)

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Name

Location

Reference

B4 B7 B8 B-8 B9 BAC1 BAC2 BAC3 BAC4 BAC6 BAC8 BAC9 BAC11 BAC12 C10 C11 COR001 COR002 COR003 COR004 COR005 COR006 COR007 COR008 COR009 COR010 COR011 COR012 COR013 COR014 COR015 COR016 COR017 COR018 COR019 COR020 COR021 COR022 COR023 COR024 COR025 COR026 COR027 COR028 COR029 COR030

13q14–q15 8q14–q15 3p16–p15 15q14–q21 5p16–p15/14q23–q25 Xq27–q28 24q12–q13 23q24 22q12–q13 22q18–q19 1q24 13q12–q13 1q25 6p12 16q12–q13 18q12–q14 22 14 6/3 4 3 1 17 9 12 2 16 8 9 15 10 22 UCD-A/ 27? 25 6 10 29 22 5 24 24 2 29 3 20 12

Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Marti et al. 1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) Skow et al. (1998) (F) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S)

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200 Table 9.4.

B.P. Chowdhary and T. Raudsepp Continued.

Name

Location

Reference

COR031 COR032 COR033 COR037 COR038 COR040 COR041 COR042 COR043 COR044 COR045 COR046 COR047 COR048 COR050 COR052 COR053 COR054 COR055 COR056 COR057 COR058 COR059 COR060 D-8 EB2E8 ECA7 ECA14 ES200 HLM3 HMS1 HMS2 HMS3 HMS5 HMS6 HMS7 HMS8 HMS9 HMS15 HMS16 HMS18 HMS19 HMS20 HMS22 HMS23 HMS25

UCD-A/27? 17 3 2 31 UCD-A/27? 2 15 2 16 10 1 4 10 20 16 1 1 23 6 19 12 1 23 11p12–p13 26 18q26 9p11 (9p12) Centromeric 18 15 10 9 5 4 1 19 4q21.3 1q21–q23 6 30 4q21 16 4 10 17

International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Marti et al. (1998) (F) Shiue et al. (1999) (S) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Sakagami et al. (1994) (biotin/avidin/BCIP/NBT) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1998) (F), Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Godard et al. (1998) (F), Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S)

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Name

Location

Reference

HMS41 HMS42 HMS45 HMS46 HMS47 HP13 HP7.2 H27A1 HTG3 HTG4 HTG5 HTG6 HTG7 HTG8 HTG9 HTG10 HTG11 HTG12 HTG13 HTG14 HTG15 I-12 I-18 LEX02 LEX03 LEX04 LEX05 LEX06 LEX07 LEX08 LEX09 LEX10 LEX11 LEX14 LEX15 LEX16 LEX17 LEX19 LEX20 LEX22 LEX23

17q21.3–q22 20q24 UCD-A/27? 18 22q19 24q15–q16 16q24–q25 1p16–p15 16q13 9 20 15q26–q27 4 9 4q21.3 21 14 1 16 22 5 19q12–q14 16q23–q25 22 X 5 UCD-A/27? 2 3 10 10 X 20 5 7 18 10 9 1 X 8*

LEX24 LEX25 LEX26 LEX27

X 30 X X

Shiue et al. (1999) (S), Godard et al. (1999a) (F) Godard et al. (1998) (F), Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Godard et al. (1998) (F), Shiue et al. (1999) (S) Lear et al. (1998d) (F) Lear et al. (1998d) (F) Lear et al. (1998d) (F) Shiue et al. (1999) (S), Godard et al. (1999a) (F) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Godard et al. (1998) (F), Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Godard et al. (1998) (F), Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Marti et al. (1998) (F) Marti et al. (1998) (F) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S), International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Bowling et al. (1997) (S), Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S)

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B.P. Chowdhary and T. Raudsepp Continued.

Name

Location

Reference

LEX28 LEX29 LEX30 LEX32 LEX33 LEX34 LEX35 LEX36 LEX37 LEX38 LEX39 LEX40 LEX41 LEX42 LEX43 LEX44 LEX45 LEX46 LEX47 LEX48 LEX50 LEX51 LEX52 LEX53 LEX54 LEX55 LEX56 LEX57 LEX58 LEX59 LEX60 LEX61 LEX62 LEX63 LEX65 LEX66 LEX68 LEX70 MPZ003 SGCV01

X 8 1 24 4 5 19 19 21 7 1 19 13 24 14 26 7 15 14 16 4 15 20 23 18 17 16 3 1 16 21 4 10 23 6 10 11 9 19 22*

SGCV02 SGCV03 SGCV04 SGCV06 SGCV07

1q14 13q12 23q19 15q24 18q21

Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S), International Equine Gene Mapping Workshop (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S)

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Name

Location

Reference

SGCV08

12*

SGCV10 SGCV13 SGCV14 SGCV16 SGCV17

12p13 11q12 21q13 21q13 10p*

SGCV18 SGCV19 SGCV20

3p13–p14 22q19 10p13–p14*

SGCV21 SGCV22 SGCV23 SGCV24 SGCV25 SGCV28 SGCV30 SGCV31 SGCV32

15q23 11p14 4q27 11p12 1q14 7 10qter X 8*

SGCV33

3p12*

SGCV35 TKY01 (ECA1) TKY02 (ECA2) TKY3 (ECA3)

19 1q24–q25 1q17.2–q17.3 2p15.1–p15.3

TKY04 (ECA4) TKY05 (ECA5) TKY07 (ECA6) TKY08 (ECA8) TKY09 (ECA9) TKY10 (ECA10) TKY12 (ECA12) TKY13 (ECA13) TKY14 TKY15 TKY16 TKY17 TKY18 TKY19 TKY20 (#4–6) TKY21 TKY22

19q21 7p12 1p12.1 20q21.1 19q23–q24 11p12.1 7p14.3 1q12 1q12 1q12 18q26 18q26 28q14 18q13 Xq29 20q13 20q21.1

Godard et al. (1997) (F), International Equine Gene Mapping Workshop (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S), International Equine Gene Mapping Workshop (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F) Godard et al. (1997) (F), International Equine Gene Mapping Workshop (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Godard et al. (1997) (F) Godard et al. (1997) (F), Shiue et al. (1999) (S) Shiue et al. (1999) (S) Godard et al. (1997) (F), Shiue et al. (1999) (S) Shiue et al. (1999) (S) Godard et al. (1997) (F), International Equine Gene Mapping Workshop (1999) (S) Godard et al. (1997) (F), International Equine Gene Mapping Workshop (1999) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Sakagami et al. (1995) (F), Shiue et al. (1999) (S) Tozaki et al. (1995) (F), Kakoi et al. (1998) (F), Shiue et al. (1999) (S) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Kakoi et al. (1998) (F) Kakoi et al. (1998) (F) Kakoi et al. (1999) (F) Kakoi et al. (1998) (F) Kakoi et al. (1998) (F) Kakoi et al. (1999) (F) Hirota et al. (1997) (F) Kakoi et al. (1999) (F) Kakoi et al. (1998) (F)

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B.P. Chowdhary and T. Raudsepp Continued.

Name

Location

Reference

TKY23 TKY24 TKY25 TKY26 TKY27 TKY28 TKY29 TKY30 TKY31 (#2–33) UCDEQ002 UCDEQ004 UCDEQ005 UCDEQ006 UCDEQ014 UCDEQ046

9p16 2p15.1–p15.3 10q21 10q21 10q21 10q21 10q21 10q14 13p15 26 22 UCD-A/27? 4 17 6*

UCDEQ062 UCDEQ136 UCDEQ304 UCDEQ380 UCDEQ387 UCDEQ405 UCDEQ411 UCDEQ412 UCDEQ425 UCDEQ428 UCDEQ437 UCDEQ439 UCDEQ440 UCDEQ455 UCDEQ457 UCDEQ464 UCDEQ465 UCDEQ467 UCDEQ482 UCDEQ487 UCDEQ493 UCDEQ497 UCDEQ502 UCDEQ505 UM001 UM002 UM003 UM004 UM005 UM007

11 18 5 2 18 25 12 10 28 X 3 11 1 30 11 25 6 24 10 1 1 12 X 16 X 1 28 1 26 2

Kakoi et al. (1998) (F) Kakoi et al. (1998) (F) Kakoi et al. (1998) (F) Kakoi et al. (1998) (F) Kakoi et al. (1998) (F) Kakoi et al. (1998) (F) Kakoi et al. (1998) (F) Kakoi et al. (1998) (F), Hirota et al. (1997) Hirota et al. (1997) (F) Shiue et al. (1999) (S) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S), International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S)

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Name

Location

Reference

UM010 UM011 UM012 UM015 UM019 UM022 UM031 UM033 VHL020 VIASH07 VIASH17 VIASH21 VIASH34 VIASH39 VIASH64 #31 #32 #33 #34 #35 #36 #37 #38 #39 #40 #41

14 20 24 6 23 23 26 8 30 7 2 31 1 29 20 15q22 13q12 22q24 26q23 3q33 8q11 26q23 7p12 28q23 22q24 11p11

Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Bowling et al. (1997) (S), Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) International Equine Gene Mapping Workshop (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Shiue et al. (1999) (S) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F) Hirota et al. (1997) (F)

*Indicates those markers whose location has been changed according to recommendations at The International Equine Gene Mapping Workshop, 3–5 June, Uppsala, 1999.

insert clones ranging from phage and cosmid to bacterial artificial chromosomes (BACs) (Breen et al., 1997b; Godard et al., 1997, 1998, 1999a). Recently, 41 sequence-tagged sites (STSs) from a cosmid library (Hirota et al., 1997) and and 46 new Type I loci from a BAC library (Godard et al., 1999b) were isolated and FISH mapped to equine chromosomes. By now, according to published data, over 331 DNA markers (Table 9.4), 37 genes and 11 EST sites (Table 9.5) have been localized to equine chromosomes using the in situ techniques. Until now, all in situ hybridization experiments in horses have been at the metaphase chromosome level and usually one probe is mapped at a time. As far as is known, there is only one report about the use of double-colour FISH for ordering markers on ECA3 and on the homologous chromosomes in donkey (Raudsepp et al., 1999). Fine mapping approaches like fibre FISH or interphase FISH have not yet found application in equine genome analysis. The FISH technique has been also used to map rRNA genes (Millon et al., 1993; Deryusheva et al., 1997) the location of which had been shown earlier

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19p13.3 2p22–p21 6q12–q14 6p21.3 6q25.1 21q22.3 6p25.1–p24.3 Xq28 16q13 16q21 19q13.1 16p13.3 11p15 4q12 2q21 3q21–q23; 3p21.3–p21.2

20q24 20q14–q22 31q15–q17 26q17 20q13 Xq29 3p15 10pter 13qter 12q14* 3q21 15q21 16q23

Oestrogen receptor v-ets avian erythroblastosis virus E2 oncogene homologue Coagulation factor XIII, A1 polypeptide ? Glutamic–oxaloacetic transaminase 2

Glycose phosphate isomerase α-Globin gene complex Insulin-like growth factor II Tyrosine kinase Transmembrane receptor for mast/stem cell growth factor Lactase Lactotransferrin

COL9A1 ELA

ESR1 ETS2 F13A F18 GOT2

GPI HBA IGF2 KIT

Harbitz et al. (1990) (R) Oakenfull et al. (1993) (F) Raudsepp et al. (1997) (F) Lear et al. (1998c) (F), Raudsepp et al. (1999) (F) Godard et al. (1998) (F) Lear et al. (1999) (F)

206

LCT LTF3

Godard et al. (1998) (F) Godard et al. (1998) (F) Lear et al. (1999) (F) Lear et al. (1999) (F) Millon et al. (1993) (F) Godard et al. (1998) (F)

4q11–q13 20q11.2–q12

3q14.3 22q15–16 28q18–qter 28q18–qter 7pter 15q25

Albumin Agouti (mouse)-signalling protein β-Lactoglobulin 1 β-Lactoglobulin 2 Complement component 3 Carbamoylphosphate synthetase 2, asparate transcarbamylase, and dihydroorotase Collagen, type IX, alpha 1 Major histocompatibility complex

ALB ASIP BLG1 BLG2 C3 CAD

Godard et al. (1998) (F) Ansari et al. (1988) (R), Mäkinen et al. (1989) (R) Lear et al. (1998a) (F) Lear et al. (1998b) (F) Godard et al. (1998) (F) Tozaki et al. (1998) (F) Lear et al. (1998c) (F)

Reference and method

Human homology

Location

Name

A list of all the genes mapped by in situ hybridization in the horse. Human homology is retrieved from the GenBank database.

Genes

Table 9.5.

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6p21.2 8q11

19q13.1

15q27 3q21 2p 7p16–p15 24q15–q16 20q24 9p12 1pter 27cen 28cen 31cen 10p15 10pter 26qter* 16q23 28q13*

Ornithine decarboxylase 1 Platelet-derived growth factor receptor, α polypeptide 6-Phosphogluconate dehydrogenase Progesterone receptor Protease inhibitor 1 (anti-elastase, α-1-antitrypsin)

pim-1 oncogene DNA protein kinase catalytic subunit, candidate gene for SCID

rRNA (rDNA)

Ryanodine receptor 1 (calcium release channel gene) Superoxide dismutase 1 Transferrin Thymopoietin

ODC1 PDGFRA PGD PGR PI (AAT)

PIM1 PRKDC, DNA-PK RNR

RYR1 (CRC) SOD1 TF4 TMPO

Chowdhary et al. (1992) (R), Godard et al. (1998) (F) Godard et al. (1999b) (F) Lear et al. (1999) (F) Godard et al. (1999b) (F)

Deryusheva et al. (1997) (F), Millon et al. (1993) (F)

Godard et al. (1998) (F) Raudsepp et al. (1999) (F) Gu et al. (1992) (R) Lear et al. (1998a) (F) Godard et al. (1998) (F), Lear et al. (1999) (F) Godard et al. (1998) (F) Bailey et al. (1997) (F)

Raudsepp et al. (1999) (F) Godard et al. (1998) (F) Godard et al. (1998) (F) Lear et al. (1998b) (F) Godard et al. (1998) (F)

*Indicates those genes whose location has been changed according to recommendations at The International Equine Gene Mapping Workshop, 3–5 June, Uppsala, 1999.

21q22.1 3q21 12q22

16q24.3 6q12 15q22–qter 21q22.3 14q11.2 14q13.1 2p25 4q11–q12 1p36.3–p36.13 11q22.1–q22.3 14q32.1

3p12 10q12–q13 1 26q17 1q26–q27

Melanocortin 1 receptor Malic enzyme 1, soluble Mannose phosphate isomerase Myxovirus (influenza) resistance 1 Nucleoside phosphorylase

MC1R ME1 MPI MX1 NP

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by NOR-banding (see Cytogenetics section). However, while silver staining reveals only the location of actively transcribed rRNA genes, FISH shows the precise chromosomal location of all rDNA loci. This might be the reason why in one of the latest rDNA FISH studies a hitherto unknown NOR was shown on ECA27 (Deryusheva et al., 1997). Further, as mentioned in the Cytogenetics section of this chapter, using a human telomeric repeat probe, telomeric regions were detected on horse chromosomes (de La Seña et al., 1995). The telomeres were found at the expected locations on the chromosomes (at the ends and in some cases close to the centromere) and no intercalary site was detected. A terminal location of telomeric repeats was reported recently for eight extant equid species (Lear et al., 1999d). However, some chromosomes of the common zebra (Equus burcelli) and Hartmann’s mountain zebra (Equus zebra) also showed interstitial telomeric sites. The latter are supposed to be relics of ancient telomere–telomere fusions. Further, some of the heterochromatic short arms and large centromeric segments in donkey showed cross-hybridization with the telomeric probe. A new development in FISH mapping is the use of probes from an evolutionarily distantly related species, a method termed heterologous FISH. Except for the use of a human Mega-YAC clone on the cat (Wienberg and Stanyon, 1997) and three HSA2 YAC clones on common shrew chromosomes (Dixkens et al., 1998), there are no published data showing application of individual large-sized genomic clones for FISH across distantly related species. Pooled rather than individual human PAC clones have been used on pig chromosomes to map the LCAT gene on SSC6 (Frengen et al., 1997). In the light of this, it is noteworthy that recently individual porcine genomic clones were used for the localization of MC1R, KIT and PDGFRA genes in horse and donkey (Raudsepp et al., 1999). The possibility of using FISH for large genomic clones across species certainly provides a new perspective to develop the equine cytogenetic comparative map. In situ hybridization in horses, as in other species, has enabled linkage and synteny groups to be located in specific horse chromosomes. Precise physical localization of markers has, on the other hand, been instrumental in integrating genetic linkage and physical maps (Breen et al., 1997b; Godard et al., 1997). In situ hybridization based physical order and relative distances between loci on ECA3 has allowed comparisons to be made between linkage and FISH mapping data (Raudsepp et al., 1999). The equine cytogenetic map generated through in situ hybridization has, as in other species, provided a visual basis for detecting segmental/chromosomal homology across species by demonstrating physical order and distances of syntenic/linked genes. The technique has thus been useful in enhancing our knowledge about the comparative organization of the horse genome in relation to other mammalian species, both distantly and closely related. Recently, five genes and two equine microsatellites were FISH mapped in a closely related species, the donkey (Raudsepp et al., 1997, 1999), providing the first comparative map information between the genomes of the two equid species.

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Flow sorting of chromosomes This technique separates individual chromosomes of a given species using a fluorescence-activated cell sorting system (FACS). Because the exhibited fluorescence of a chromosome is proportional to the DNA content and AT/GCrich regions, it is possible to separate individual chromosomes (Lebo, 1982; Lebo and Bastian, 1982). The procedure results in the generation of a flow karyotype, distinguishing each chromosome (Lebo, 1982). Once separated and collected, DNA from individual chromosomes can be either directly amplified using, for example, degenerated oligonucleotide primers (DOP; Telenius et al., 1992), or used for library construction (Collins et al., 1991). In both cases, whole chromosome-specific DNA is available as a composite probe for FISH applications in clinical cytogenetics, comparative painting, cloning and gene mapping. In horses, this technique has hardly been used. The only report available concerns isolation of the X chromosome by bivariate chromosome sorting for generation of an X-specific painting probe. The paint was used for the detection of X chromosome-related numerical aberrations in mares with fertility problems (Breen et al., 1997a). Though there are distinct flow karyotypes in a variety of species such as the mouse (Rabbits et al., 1995), the pig (Langford et al., 1993; Yerle et al., 1993), the dog (Langford et al., 1996), sheep (Burkin et al., 1997) and the cat (Wienberg et al., 1997), as yet there are none in the horse.

Chromosome microdissection Chromosome-specific painting probes can also be successfully generated using a procedure referred to as ‘microdissection’ (e.g. see Fig. 9.2). Although the technique was initiated 18 years ago by isolating DNA from Drosophila polytene chromosomes (Scalenghe et al., 1981), the method was modified and improved during recent years by introduction of PCR (Lüdecke et al., 1989; Senger et al., 1990), use of universal primers (Telenius et al., 1992) and topoisomerase treatment (Guan et al., 1993). Now it has developed into one of the most direct means for isolating DNA from any chromosomal region of an organism (Cannizzaro, 1996). Nowadays, whole chromosomes, arms, regions or even a single band ranging from 5 to 100 Mb can be microdissected readily, and the DNA amplified for various purposes. In humans, chromosomal microdissection has found a broad range of applications. Microdissected whole chromosomal or partial probes can be used as paints for fine cytogenetic analysis (reviewed by Ried et al., 1998), generating microlibraries for regions involved in cancer (e.g. see Zhang et al., 1995), for isolating microsatellites, sequence-tagged sites and genes (Gingrich et al., 1996; Meltzer et al., 1997; Yu et al., 1997). As compared with this, chromosome microdissection has, as yet, found very limited use in horse genome analysis. Recently, chromosome-specific

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B.P. Chowdhary and T. Raudsepp Chromosome

Glass needle attached to micromanipulator Microdissected segment Before microdissection

After microdissection

Collection buffer

PCR amplification

Cytogenetic analysis chomosome painting

Direct cDNA capture

Chromosome-specific DNA

Zoo-FISH

Cloning chromosome specific library ESTs, genes

Microsatellites

Unique sequences

Mapping

Linkage mapping

SCH/RH mapping

Screening genomic libraries

FISH

Fig. 9.2. Schematic representation of the chromosome microdissection approach showing possible applications in genome analysis.

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paints for all equine meta- and submetacentric autosomes and the sex chromosomes were generated by microdissection (Raudsepp and Chowdhary, 1999). These composite probes were then used for cross-species chromosome painting (discussed in the next section). Further, microdissection and microcloning techniques were used to construct chromosome-specific libraries for two equine chromosomes, ECA6 and ECA12 (Chowdhary et al., 1998b). The microlibraries were screened with different oligonucleotide probes. As a result, three and 12 microsatellite-containing clones were isolated, respectively, from the two libraries. This is a useful material for chromosome-specific linkage analysis and for screening genomic libraries to find clones suitable for FISH. Besides this, there is another preliminary report about microcloning ECA1 into a phage vector (Bowling et al., 1998). Thus, even though application of microdissection for horse genome analysis is in its very early stages, the initiative is expected to be a useful tool for an orderly development of the equine gene map. This is particularly true for those chromosomes to which no or very few markers are assigned.

Chromosome painting Chromosome painting is a technique where flow-sorted or microdissected whole chromosomes or their parts are used as composite probes in FISH experiments. Because the probe is a cocktail of DNA representing numerous sites of the originating chromosome, the observed signal is an aggregation of several hybridization sites uniformly covering (‘painting’) the chromosome or region. The method was developed primarily to study individual human chromosomes in clinical and cancer cytogenetics (Lichter et al., 1988; Pinkel et al., 1988; for a review, Ried et al., 1998). The use of chromosome painting in farm animal clinical cytogenetics has been very limited, and has been even more scarce in the horse. As mentioned earlier, there is only one report about the use of chromosome-specific paints for detection of X chromosome numerical aberrations in the horse (Breen et al., 1997a). The only other example where equine chromosome-specific probes were used for painting is when microdissected paints from individual meta- and submetacentric and the sex chromosomes were tested to confirm their origin (Raudsepp and Chowdhary, 1999). Attempts are underway currently to use the X-specific microdissected paint to study aberrations in equine oocytes (M. Switonski, personal communication).

Comparative chromosome painting When chromosome painting is carried out across species, it is termed comparative chromosome painting or Zoo-FISH. Now this approach has become an integral part of the construction of comparative maps between different mammalian species (reviewed by Wienberg and Stanyon, 1995, 1997;

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Chowdhary, 1998b). Generally, when cross-species genome homology is examined, conservation of synteny/linkage between genes is taken as a measure of homology for segments lying between them. However, this extrapolation of information may or may not be correct. Through developments during the last decade, the mapping data can, to a large extent, be supported through comparative chromosome painting. Although the latter has some limitations with regards to accuracy, it none the less helps in delineating fairly precisely homologous chromosomal regions between species (for a review, see Chowdhary, 1998b). Depending on the evolutionary distance between the species involved, Zoo-FISH can be classified into two major categories: (i) between evolutionarily closely related species belonging to the same mammalian order or family; and (ii) between evolutionarily distantly related species In the following discussion, we shall focus on the comparative chromosome painting work carried out in the horse and other related equid species. The utility of this approach in enhancing our knowledge on the comparative status of the equine genome will be highlighted.

Zoo-FISH within equids This type of comparative painting is possible only if chromosome-specific paints are available for one of the species within an order or family. For example, human chromosome-specific paints have long been used successfully in several studies to address questions pertaining to comparative genome organization and karyotype evolution in over ten different primate species (Wienberg and Stanyon, 1997; Chowdhary et al., 1998a). With the availability of flow-sorted or microdissected chromosome-specific paints for more species, comparative painting studies have now been carried out also within suids, cervids, rodents, marsupials and bovids (for a review, see Raudsepp, 1999). The findings of all these investigations have helped mainly in understanding karyotype evolution within these orders. Further, in some cases, they have also helped in direct transfer of genetic information from the developed/partially developed maps of one species to those which are less developed. As mentioned in the section on microdissection and flow sorting, chromosome-specific paints for almost half of the horse karyotype are available currently (Raudsepp and Chowdhary, 1999, for all meta- and submetacentric autosomes and the sex chromosomes; Breen et al., 1997a, for the X chromosome). The former set of paints was used recently on donkey chromosomes to Fig. 9.4 (opposite). Detailed homology between microdissected horse (ECA) chromosomes and the corresponding donkey (EAS) chromosomes. The ideograms of G-banded equine chromosomes were taken from ISCHN (1997). Ideograms for donkey chromosomes were prepared from a set of G-banded karyotypes, and are shown only for chromosomes/arms detected to be homologous to the horse genome. The arrows between the horse and donkey chromosomes define regions of homology. Vertical line(s) beside the equine chromosomes specifies whether the homology is restricted to the designated equine arm or to the whole chromosome. A vertical line beside EAS1q (F) and EAS8q (L) shows the actual region of homology with ECA4 and ECA8, respectively.

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J

F

M

q EAS28 p

p EAS5

q

q

ECA1

EAS2

ECA4

EAS1

ECA3

ECA2

G

B

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p EAS3 EAS3 K

ECA9

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EAS20

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ECA6 EAS22

EAS25 ECA12

EAS17

D

ECA11

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I

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E

EAS5

ECA8 ECAY

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ECA10 EAS24

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study how the two karyotypes are organized in relation to each other. It may be worthwhile mentioning at this stage the background of this investigation. The horse and donkey are considered to have diverged from a common ancestor about 3–10 million years ago (Lindsay et al., 1980; Xu et al., 1996). On an evolutionary time scale, the period is short and it is expected that the karyotype/genome organization of such related species should be fairly similar. For example, among bovids (Allard et al., 1992) and the majority of the primates (Dutrillaux, 1979; Comparative Genome Organization, 1996; Wienberg and Stanyon, 1997), the comparative karyotype structure is fairly preserved, although species within each of the groups diverged from their common ancestor about 16–17 and 5–15 million years ago, respectively (Allard et al., 1992; Arnason et al., 1996). Further, the fact that all equids can interbreed freely and give viable, though usually infertile, offspring (Allen and Short, 1997) strongly suggests that the karyotypes of equids should not differ much from each other. However, cytogenetic studies have shown significant morphological differences in the chromosomes of different equid species (Ryder et al., 1978; Power, 1984). The application of chromosome-specific paints for the 15 equine chromosomes detected a total of 21 homologous chromosomes/segments in the donkey karyotype (Raudsepp and Chowdhary, 1999; see Fig. 9.3 (Frontispiece) and Table 9.7). One of the interesting facts which emerged from the horse–donkey comparative painting is that 29 equine chromosomal arms corresponded to 29 asine arms. Although two bi-armed chromosomes, ECA4 and ECA7, show homology with a single arm each in donkey (EAS1q and EAS20, respectively; Fig. 9.3, see Frontispiece), a balance in the numbers remained because the two arms of ECA5 and ECA6 are homologous to a total of three arms each in the donkey karyotype. Very often, the use of Y chromosome-specific paints across species, even between closely related ones, produces no results. Among the primates, in some cases, the human Y-specific paint shows a clear hybridization signal (Wienberg et al., 1992; Koehler et al., 1995; Bigoni et al., 1997; Müller et al., 1997), while in others there is no hybridization (Consigliere et al., 1996; Richard et al., 1996; Sherlock et al., 1996; Morescalchi et al., 1997). The equine Y chromosome paint showed distinct hybridization on EASY. However, additional signals on the centromeres and/or telomeres of some asine chromosomes and the heterochromatic region of EASXq were also observed (Raudsepp and Chowdhary, 1999). Comparison of Zoo-FISH results with banding homology between the chromosomes of the two equids (see Fig. 9.4) revealed that only four horse chromosomes (ECA1, -9, -12 and -13) show a moderate to high degree of banding pattern homology with the respectively painted chromosomes in donkey. In general, about 60% of the segments homologous between the two species do not show clear correspondence in their banding patterns. Comparative painting thus for the first time discloses molecular homology between a large part of the horse and donkey karyotypes. This is significant because other approaches, mainly cytogenetic, were hitherto unable precisely to define

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homology between their chromosomes, even though the two species are evolutionarily closely related. Zoo-FISH between human and horse genomes Closely related species share a high degree of sequence homology which makes the use of chromosome-specific paints straightforward within the group. Extension of the concept of chromosome painting from closely to distantly related species was realized with modifications to the FISH protocol (for details, see Scherthan et al., 1994). After this success, human chromosomespecific libraries have been used extensively on the chromosomes of a wide range of mammals (15 species belonging to seven orders), among which several are domesticated/farm animals (for a review, see Chowdhary, 1998b; Chowdhary et al., 1998a; Raudsepp, 1999). In the first attempt to delineate gross chromosomal homology between horse and human, whole chromosome paints (WCPs) for individual human chromosomes (22 autosomes and sex chromosomes) were used separately on horse metaphase chromosomes in a Zoo-FISH experiment. All human probes, except the Y chromosome, hybridized to one or more chromosomes in the horse (Fig. 9.5; see Frontispiece). Altogether 44 conserved segments were detected (Raudsepp et al., 1996). The majority of human chromosomes are homologous to whole chromosomes, chromosome arms or large segments in the horse karyotype (see Table 9.6). This reflects a fairly high degree of synteny conservation between the two genomes. Immediately thereafter, two more studies reported use of HSA3, -4, -14 and -16 paints on horse chromosomes (Rettenberger et al., 1996; Lear and Bailey, 1997b). The findings of all three studies are in full accordance with each other. Recently, depending on the position of the NOR on the chromosomes and some gene localizations, minor changes in the equine Zoo-FISH homologues were agreed upon at the Second International Equine Gene Mapping Workshop, 1999 (our unpublished results). Now ECA26 corresponds to HSA21 while ECA28 corresponds to HSA12 and 22. The results are the opposite of those of Raudsepp et al. (1996), and are presented in the revised format in both Fig. 9.5 (Frontispiece) and Table 9.6. Some equine chromosomes/regions, i.e. ECA6p, 12, 13p, 27 and 31, however, did not hybridize with any human WCP. This was attributed to a possible variation in the representation of coding sequences between the human chromosome-specific libraries, a phenomenon which can occur while libraries are grown. However, later, in a different study, homology between ECA12 and HSA11 was revealed (Raudsepp et al., 1997). With the development of the horse physical gene map (synteny and in situ hybridization analysis), it is expected that human homologies for other ‘blank’ regions on horse chromosomes will be known. For example, with regards to 6p, there are already indications that it corresponds to part of HSA2q. Comparison of human–horse Zoo-FISH results with the genes so far mapped in both species shows that, in most cases, the two data sets agree very closely with each other (see Fig. 9.5; Frontispiece). However, as is evident in

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B.P. Chowdhary and T. Raudsepp Table 9.6. Conserved chromosomal segments detected on equine metaphase chromosomes after Zoo-FISH with individual human chromosome-specific libraries (CSLs). Human CSLs

Homologous chromosome segments in horse

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

2p; 5; 30 1q; 15; 18 16; 19 2q; 3q 14; 21 10q; 20 4 9 23; 25 1p; 29 7p, q; 12p, q 1q; 6q; 8p; 28* 17 1qter; 24 1q 3p; 13q 11 8q 10p; 7qcent 22 26* 1pter; 8pter; 28ter* X

*Indicates changes recommended at The International Equine Gene Mapping Workshop, 3–5 June, Uppsala, 1999.

other species, it is expected that with the expansion of the equine gene map, some discordance with the Zoo-FISH data will surface. This is attributed primarily to the resolution limitations of Zoo-FISH where small segments of cross-hybridization might easily go undetected. Refinement of the Zoo-FISH observations mentioned above can be done either by reverse painting, wherein horse individual chromosome paints can be used as probes on human metaphase chromosomes, or by the use of subchromosomal probes. The latter approach was used by Chaudhary et al. (1998) where arm-specific paints (ASPs) from HSA2, -5, -6, -16 and -19 were generated and used on horse chromosomes. The results showed that individual arms of HSA2 and HSA5 are not conserved as separate segments (Fig. 9.6a and b). The human synteny appears to be broken most probably at HSA2q13 and HSA5q13 (for details, see Chaudhary et al., 1998). Recent gene assignments in horses (Godard et al., 1998) are in agreement with these observations, although partial discordance is also observed. Further, contrary to the above findings, Zoo-FISH with ASPs from HSA6, -16 and -19 was in accordance with

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the hypothesis that individual arms of the human chromosomes are conserved as separate blocks (see Fig. 9.6c, d and e) as is indicated in a detailed comparative analysis spanning several distantly related species (Chowdhary et al., 1998a; Chowdhary, 1998b). The findings thus provided a first hand insight into the comparative organization of horse and human genomes but also helped indirectly to relate the horse genome to other mammalian genomes. The results have been of significance in the horse because, in the absence of a proper gene map, the comparative information has served as a framework to extrapolate information from the developed gene maps. Further, the findings have also acted as a reference point to compare all new gene assignments in the horse. Lastly, the Zoo-FISH observations have also contributed in assigning linkage or syntenic groups to specific horse chromosomes. In human–horse Zoo-FISH studies (Raudsepp et al., 1996; Lear and Bailey, 1997b), it was predicted that equine linkage group II (LG II) is most likely to be located on ECA3. Recent gene mapping data (Lear et al., 1998c; Godard et al., 1998; Raudsepp et al., 1999) strongly support this prediction. Similarly, it also proposed that an equine syntenic group (NP, MPI, IDH2; Williams et al., 1993) maps to ECA1q (Raudsepp et al., 1996). Recent FISH mapping of the NP locus to ECA1q26–q27 (Godard et al., 1998) confirms this. At present, only one linkage group containing type I markers (APOA1 and APOA4) is not anchored to a specific horse chromosome (Kakoi and Gawahara, 1997). Because both these genes are mapped to HSA11, it is expected that the linkage group is located on either ECA7 or ECA12. Zoo-FISH between human and other Equidae genomes Besides human–horse Zoo-FISH studies, there have been some recent reports of chromosomal homologies between humans and other equid species. Lear and Bailey (1997a) painted probes from HSA4, -8, -9, -16 and -21 to horse, donkey and Hartmann’s zebra chromosomes. The results enable the comparison of homologous regions in the three equid species and help to clarify some of the complex chromosomal rearrangements which are most likely to have occurred during karyotype evolution. Further, the defined homology between human–horse (Raudsepp et al., 1996) and horse–donkey karyotypes (Raudsepp and Chowdhary, 1999) makes it possible for the first time to compare indirectly a significant part of the asine genome with that of humans. Some of these homologies are supported by direct human–donkey Zoo-FISH results using HSA4-, 16p-, 16q- and 19q-specific paints, while some are supported through comparative gene mapping data (Raudsepp et al., 1997, 1999; T. Raudsepp and B.P. Chowdhary, unpublished). A general overview of the available correspondence between donkey, horse and human chromosomes is summarized in Table 9.7. Organization of the equine genome versus other mammalian genomes As compared with the human karyotype, horse chromosomes do not show any extraordinary variation for the conserved chromosomal arms/segments, unlike

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other mammalian species (reviewed by Chowdhary et al., 1998a). However, it needs to be mentioned that for some chromosomes, for example HSA9, the horse does appear to be an exception. While all (or most) studied species show a single chromosome or segment corresponding to this human chromosome, the horse shows two complete acrocentric chromosomes as homologues (see Fig. 9.3; Frontispiece). Similarly for HSA4, the horse is the only Table 9.7. Likely homologies between donkey (EAS) and human (HSA) chromosomes as deduced from human–horse (ECA) and horse–donkey Zoo-FISH data.

Clearly evident

Indirectly deduced

Donkey

Horse

Human

EAS1q* EAS3p EAS3q EAS12 EAS13 EAS14 EAS17 EAS26 EAS28 EASX EAS2p EAS2q EAS5q EAS5p + 8q* EAS16 + 25 EAS19 + 22 EAS20 EAS24

ECA4 ECA2q ECA3q ECA9 ECA11 ECA13q ECA12 ECA10p ECA3p ECAX ECA1p ECA1q ECA2p ECA8 ECA5 ECA6 ECA7 ECA10q

HSA7 HSA4 (direct painting) HSA4 (direct painting) HSA8 HSA17 HSA16p (direct painting) HSA11 (part) HSA19q (direct painting) HSA16q (direct painting) HSAX HSA10 + 22 HSA2 + 12 + 14 + 15 HSA1 (part) HSA12 + 22 + 18 (parts) HSA1 (part) HSA12 + part of another HSA? HSA11 + 9 (parts) HSA6 (part)

Those which are clearly evident could be inferred either from whole chromosome/arm-specific homology between human–horse and horse–donkey chromosomes or through the use of human arm-specific paints and comparative mapping data; indirectly deduced represent those where precise homology was not evident because human–horse painting data did not indicate the exact region(s) of the human chromosomes which were homologous to the horse chromosomes. *represents the non-heterochromatic part of the chromosomes. Fig. 9.6 (opposite). Schematic drawings showing homologous chromosomal regions detected in the horse (ECA) karyotype, using arm-specific paints (ASPs) from five human (HSA) chromosomes, i.e. HSA2 (a), HSA5 (b), HSA6 (c), HSA16 (d) and HSA19 (e). Each arm of the five human chromosomes is denoted with a specific pattern, and the same pattern is used for those regions of horse chromosomes detected to be homologous to individual paints. For each chromosome, the species abbreviation is given above while the chromosome number is given below. Beside the chromosomes, a list of genes hitherto mapped is presented (only comparative data in humans). Vertical lines beside the human chromosomes show the region most likely to be homologous to the specified (follow arrows) equine chromosomes. The band (q13) shown on the left of HSA2 (a) and HSA5 (d) indicates the most probable site where two evolutionarily conserved blocks on these chromosomes meet. The question mark (?) besides ECA7 (e) indicates that this segment was not painted by either of the ASPs used. The results are, therefore, proposals which need further verification.

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species (except the common shrew and bovids) which has two homologous segments. This result is of even greater significance when the same human chromosome paints only a single chromosome in the donkey and Hartmann’s zebra (Raudsepp and Chowdhary, 1999; Lear and Bailey, 1997a), suggesting that the proposed fission of the equine homologues to HSA4 occurred only in the horse and not in other equids. It would be interesting, therefore, to check more Equidae karyotypes to understand further how the homologue of HSA4 evolved in this mammalian group. With regards to neighbouring or contiguous segment combinations (genomic regions which are present as separate chromosomes in humans but are syntenic in a wide range of evolutionarily distantly related species), the horse, as compared with a wide range of evolutionarily diverged species, appears to be a partial exception where two of the four neighbouring syntenies (HSA3/21 and HSA16/19) are disrupted (Chowdhary et al., 1998a). Even in a fairly rearranged genome such as that of mouse (as compared with other mammalian species), traces of most of the syntenic combinations are present. However, it needs to be pointed out that the HSA3/21 synteny is also disrupted in the rabbit (Korstanje et al., 1999), and HSA16/19 in the donkey (Raudsepp and Chowdhary, 1999; T. Raudsepp and B.P. Chowdhary, unpublished observations).

Future Prospects There are several ways in which to look at a genome. In the present chapter, we first tried to look at the equine genome through the eyes of a cytogeneticist and then examined the progress made in the field of physical gene mapping. How research progresses in the two fields during the coming years depends on what the goals are and how much has been achieved. New technological developments and shifting demands of mankind for the welfare of horses may also, from time to time, alter these goals. During the ‘golden era’ of cytogenetics, the volume of data and the diversity of techniques applied in the horse were rather limited. This can be ascribed to a combination of circumstances, the main one being the secret environment under which the equine industry functions – a trend similar to other industries where economic stakes are high. Though several authors in different reports (e.g. Haynes and Reisner, 1982; Bowling et al., 1987) have reiterated ways in which to increase accessibility to equine material for cytogenetic analysis, it has been difficult to percolate the idea through the domain of private horse owners associations that cytogenetic analysis can act as an important tool in initial screening, at least for some of the basic but more common chromosome-related problems in the horse. We are still of the opinion that under a coded system of examination (which is practised for several investigations in horse as well as other species including humans), a simple and routine cytogenetic investigation, especially of animals used for breeding purposes, will certainly be beneficial to horse breeders. The sooner

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such examinations are conducted (of foals/hinnies earmarked as future breeding individuals), the easier it will be to circumvent big financial losses which the breeders might incur by rearing, for example, an XO animal for some years, beyond the onset of breeding age (5–7 years). The horse breeders need to realize that in such cases, a small expenditure of around US$100–150 for early cytogenetic analysis of potential breeding individuals could prove to be an investment rather than an expenditure in curtailing the likelihood of chromosome-related problems. Further, such investigations will be an important resource for enhancing our knowledge of the status of chromosome abnormalities in different breeds of horses, and devising ways to overcome the problem. Equine cytogeneticists still thrive on the hope that horse breeding associations might one day recommend mandatory cytogenetic evaluation, at least of the breeding animals. The benefits of similar investigations in pigs and cattle carried out routinely in some countries can provide a good example. Because of the situation with equine cytogenetics during the past 6–8 years (fewer and fewer cases are being analysed every year), it may not be a pessimistic overstatement that this important tool is on the verge of gradually fading into oblivion because the number of people conversant with this art is reducing. Although hope of its revival lies in the hands of horse farmers, it will be interesting to see whether the equine cytogeneticists can develop better, faster, appealing and more accurate methods of analysis so that the owners can be persuaded to adopt them. In spite of encouraging steps taken by some groups in this direction (e.g. Pailhoux et al., 1995; Meyers-Wallen et al., 1997; Breen et al., 1997a), more input is needed to match DNA diagnostic tests for certain inherited diseases/traits, which will attract the attention of the breeders. The last few years represent a period of significant breakthroughs in equine genome analysis. After having waited a fairly long time for the attention of animal geneticists, gene mapping in the horse has now received a robust start. The major credit for this goes to the joint efforts of an international consortium of researchers, who have been able to lift the map from a non-existent status to one with around 600 markers (projected figures). This in no way means that the work is even ‘partly’ done. On the physical gene mapping front, progress has indeed been rapid, and closely matches similar efforts carried out to construct the linkage map. In spite of these efforts, the equine physical gene map is not even a basic map mainly because: (i) the distribution of markers along the chromosomes is still very uneven and (ii) the majority of the in situ hybridization-mapped markers are type II. Based on experience in the construction of gene maps in other livestock species, especially cattle and pigs, a balanced approach to the development of the equine gene map, with a reasonable number of loci of both types, is something which will have to be contemplated in the horse. Although the available human–horse Zoo-FISH data have served efficiently as a framework for comparison with other species (directly with humans and indirectly with others; Raudsepp et al., 1996; Chowdhary et al., 1998a), efforts need to be directed towards increasing the number of gene localizations in order to increase the utility of the map for

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comparative purposes. If the available gene mapping data are any measure of this ‘balance’, horse geneticists are more or less on track, or at least better off compared with their counterparts working with cattle and pigs. During the past 2–3 years, synteny mapping has made a major contribution to the equine physical gene map, and it is expected that at least in the next few years, SCH panels will continue to occupy the centre stage of physical gene mapping. A recent report by Bowling et al. (1999) showing mapping of over 500 markers (~200 of which represent specific genes) is certainly a vital boost to our knowledge of the assignment of these markers to specific horse chromosomes. The work is unquestionably the most concerted effort to enhance the comparative worth of the equine gene map. The knowledge should act as a base to organize in situ hybridization experiments such that the distribution of the markers along the length of the chromosomes can be examined. Further, synteny mapping of orthologous genes using CATS or TOASTs primer pairs on SCH panels has opened up a new avenue for cross-species genome comparisons. The approach has already been used in some of the very recent studies in the horse (Caetano et al., 1999a,b; Terry et al., 1999). Whether this advancement will continue to attract the attention of equine geneticists will depend, to a large extent, on how versatile the technique will be. Irrespective of their contribution, the primers have been rather difficult to work with, although it must be added that the horse is a species where the success rate of mapping these primer sets is higher than in other livestock species (A.T. Bowling, personal communication). Systematic FISH mapping of markers from the linkage map is also an important endeavour that needs to be initiated to align physical and genetic linkage maps and to generate a consensus map of the horse genome. The work will, for the first time, provide information on the genetic length of the equine genome, presuming that markers used for this purpose reasonably cover the genome, especially the ends of the chromosomes. Considering the rate at which new markers are being added to the equine genetic linkage map, it is anticipated that concurrent FISH mapping will enable alignment of the two maps early in the year 2000. Comparative chromosome painting has played a vital role in deducing homologies between the horse and human karyotypes (Raudsepp et al., 1996). However, through these initial experiments, about 8–10% of the equine genome did not show correspondence with its human counterpart. Recent Zoo-FISH experiments (Raudsepp et al., 1997), together with new gene mapping data (Lear et al., 1998a; our unpublished results), have already shown that three of the unpainted segments (i.e. ECA6p, ECA12 and 31) are indeed homologous to parts of the human genome. It is expected, therefore, that more efforts will be made in the near future to patch this ‘gap’ between the two genomes. Recent use of large-sized cross-species probes for heterologous FISH mapping appears to be a viable option to map specific genes in the horse. In the absence of equine gene probes, it is expected that this alternative might

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be useful in mapping evolutionarily conserved sequences. The availability of PACs, BACs and YACs in a number of species will make this task possible. Whether this approach will gain popularity will depend mainly on the development and characterization of equine cDNA libraries (a necessary but time-consuming project), which if produced, will be the preferred way to map coding sequences in the horse. Microdissection of equine chromosomes is a relatively new addition to the tools for studying the horse genome. Although the technique has proven to be extremely useful in humans (Guan et al., 1993; for a review, see Cannizzaro, 1996), it is too early to say whether equine geneticists are prepared to invest intensively and embark on this approach. To date, there are two preliminary reports about construction of horse microlibraries from ECA1, -6 and -12 (Bowling et al., 1998; Chowdhary et al., 1998b). However, at present, it appears that the use of microdissected chromosome or arm-specific material will be restricted mainly to comparative analysis within the equids/ perissodactyls or even across other orders. It is expected that in the near future, chromosome-specific paints for the acrocentric equine chromosomes will also be available, which will add to the ongoing efforts in studying karyotype evolution within the equids. The present resolution of the physical gene map in the horse is very low. Under these circumstances it may seem that there are no reasons why fine mapping techniques such as interphase mapping, fibre-FISH, DNA combing, etc., will be applied in the immediate future. However, with studies already concentrating on traits of specific interest, the need for these approaches will soon be felt. Among other physical gene mapping techniques, characterization and analysis of the recently constructed radiation hybrid panels in the horse will remain a priority. Considering that such developments have contributed significantly in making the cattle and pig gene maps more informative and integrated than before, horse geneticists will certainly make substantial efforts in this direction in the near future.

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Womack, J.E., Johnson, J.S., Owens, E.K., Rexroad, C.E. III, Schläpfer, J. and Yang, Y. (1997) A whole genome radiation hybrid panel for bovine gene mapping. Mammalian Genome 8, 854–856. Xu, X., Gullberg, A. and Arnason, U. (1996) The complete mitochondrial DNA (mtDNA) of the donkey and mtDNA comparisons among four closely related mammalian species-pairs. Journal of Molecular Evolution 43, 438–446. Yadav, B.R. and Balakrishnan, C.R. (1982) Trisomy of the X chromosome in a Murrah buffalo. Veterinary Record 111, 184–185. Yang, Y.P. and Womack, J.E. (1998) Parallel radiation hybrid mapping: a powerful tool for high-resolution genomic comparison. Genome Research 8, 731–736. Yang, Y.P., Rexroad, C.E. III, Schläpfer, J. and Womack, J.E. (1998) An integrated radiation hybrid map of bovine chromosome 19 and ordered comparative mapping with human chromosome 17. Genomics 48, 93–99. Yerle, M., Schmitz, A., Milan, D., Chaput, B., Monteagudo, L., Vaiman, M., Frelat, G. and Gellin, J. (1993) Accurate characterization of porcine bivariate flow karyotype by PCR and fluorescence in situ hybridization. Genomics 16, 97–103. Yerle, M., Pinton, P., Robic, A., Alfonso, A., Palvadeau, Y., Delcros, C., Hawken, R., Alexander, L., Beattie, C., Schook, L., Milan, D. and Gellin, J. (1998) Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs. Cytogenetics and Cell Genetics 82, 182–188. Yu, J., Tong, S., Shen, Y. and Kao, F.T. (1997) Gene identification and DNA sequence analysis in the GC-poor 20 megabase region of human chromosome 21. Proceedings of the National Academy of Sciences USA 94, 6862–6867. Yunis, J.J., Soreng, A.L. and Bowe, A.E. (1987) Fragile sites are targets of diverse mutagens and carcinogens. Oncogene 1, 59–69. Zalensky, A.O., Tomilin, N.V., Zalenskaya, I.A., Teplitz, R.L. and Bradbury, E.M. (1997) Telomere telomere interactions and candidate telomere binding protein(s) in mammalian sperm cells. Experimental Cell Research 232, 29–41. Zhang, J., Cui, P., Glatfelter, A.A., Cummings, L.M., Meltzer, P.S. and Trent, J.M. (1995) Microdissection based cloning of a translocation breakpoint in a human malignant melanoma. Cancer Research 55, 4640–4645. Zuhlke, C., Thies, U., Braulke, I., Reis, A. and Schirren, C. (1994) Down syndrome and male fertility: PCR-derived fingerprinting, serological and andrological investigations. Clinical Genetics 46, 324–326.

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Glossary

Glossary

Additive gene effects. The average effect on phenotype when one allele is replaced by another. Allele. One of a pair, or series of alternative forms of a gene that can occur at a given locus on homologous chromosomes. Anaerobic threshold. This is the upper limit of the exercise power which elicited the anaerobic metabolism of the muscles. In horses, this critical power can be estimated during an exercise test at increasing speed by analysing the blood lactate concentration. Lactate is the final product of the anaerobic metabolism which can be used as a marker of the anaerobic metabolism contribution. One of the purposes of a training programme is to improve the anaerobic threshold by stimulating metabolism adaptations to a specific exercise. Ancestor. Any individual from which an animal is descended. Autosome. Any chromosome that is not a sex chromosome. Back-cross. The cross produced by mating a first-cross animal back to one of its parent lines or breeds. BLUP. Best linear unbiased prediction. A standard statistical method for estimating breeding values in (large) populations. BLUP accounts for genetic relationships and adjusts for systematic fixed effects. Breeding value. The mean genetic value of an individual as a parent. It can be estimated as the average superiority of an individual’s progeny relative to all other progeny under conditions of random mating. Centromere. Spindle–fibre attachment region of a chromosome. Chromosome. A thread-like structure of a DNA molecule complexed with RNA and proteins in the nucleus of a cell, which carries genes.

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Codominant alleles. Alleles, each of which produces an independent effect in heterozygotes. Collection of the gait. Speed, stride frequency and stride length of the gait are reduced. The amplitude of the vertical displacement increases. The vertical movements of the body seem to be elastic with the contribution of the tendon elasticity. Combining ability. The mean performance of a line when involved in a crossbreeding system. General combining ability is the average performance when a breed or line is crossed with two or more other breeds or lines. Specific combining ability is the degree to which the performance of a specific cross deviates from the average general combining ability of two lines. Composite. A line developed from a crossbred foundation. Conformation. General structure, form or outline of a horse’s body. Congenital. A condition present at birth but not necessarily hereditary. Covariance. The measure of degree to which two measurements vary together. A covariance is positive when two measurements tend to increase together. A covariance is negative when one measurement increases and the other measurement tends to decrease. Cribbing (crib biting). Specific behaviour in horses, which involves arching the neck while grasping a horizontal surface with the incisors and aspirating air into the pharynx and upper oesophagus with a characteristic sound. Crossbreeding. Matings between animals of different breeds or lines. Crossing over (crossover). The process during meiosis when chromosomal segments from different members of a homologous pair of chromosomes break, and part of one will join a part of the other so that two gametes form possessing new combinations of genes. The frequency of crossover between two loci is proportional to the physical distance between the loci. Deletion. A mutation involving a loss of either one or a number of base pairs or a chromosomal fragment. Descendant. An individual descended from specific other individuals. Diallele cross. When both males and females from each breed (or line) in a set of breeds (or lines) are mated to males and females of each breed (or line) in the set including their own breed (or line). Diastema. A gap between the front incisors and rear grinders in horses. Dominant. Applied to one member of an allelic pair of genes which has the ability to express itself wholly or largely at the exclusion of the expression of the other member.

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Dressage. The act or technique of guiding a horse through a set of paces or manoeuvres by imperceptible movements on the part of the rider. Dressage traits. A set of morphological, behavioural and locomotor characteristics required for executing standardized gaits and exercises that are defined by academic riding schools and competition rules in dressage. Duplication. A mutation involving a gain of either one or a number of base pairs or a chromosomal segment. EBV. Estimated breeding value. An estimate of the mean genetic value of an individual for a quantitative trait. Economic trait loci. Loci that have effects on traits of economic importance. Economic value. A measure of the contribution which an individual trait makes to the overall economic value of an animal. Elasticity of the gait. Amplitude and suppleness of the vertical movements of the body at trot, passage and piaffe. Environmental correlation. Situation when two traits tend to change in the same or different direction as a result of environmental effects. Environmental variance. Variation in phenotype which results from a variation in environmental effects. Epistasis. Intergenic interaction, when alleles at one locus affect the expression of alleles at another locus. F1. Animals resulting from crossing parents from different lines or breeds. F2. Animals resulting from matings among F1 parents. Full sibs. Individuals having the same male and female parents. Gaits. The horse’s way of moving, characterized by particular footfall patterns and particular speeds. The movements of a horse’s legs in movement, such as the walk, trot, pace, toelt, gallop, etc. Two types of gait can be distinguished by the symmetry or asymmetry of the limb movement sequence with respect to time and the median plane of the horse: symmetric gaits (walk, pace, trot) and asymmetric gaits (canter, gallop). Within each gait, there exist continuous variations. Among the normal variations of the trot, the speed of the gait increases from collected to extended trot. Passage and piaffe are two dressage exercises derived from the collected trot. Gait speed. To increase the gait speed, the amplitude of the steps becomes larger and the duration of the limb cycle is reduced in order to repeat the limb movements more frequently. The stride frequency (SF) and stride length (SL) are the two main components of the gait speed. The mean speed (V) can be estimated by the product of the mean stride parameters: V = SF × SL. To increase the gait velocity progressively, the horse linearly increases the length of its strides.

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Gallop. Canter and gallop refer to the same gait at increasing speed: the canter is a three-beat gait at a slow speed and the gallop is a four-beat gait at a higher speed. At the canter, the diagonal stance phase is synchronized, while at the gallop the footfalls of the diagonal are disassociated. The first hindlimb hits the ground before the diagonal forelimb. The gallop is the fastest equine gait of racing horses such as Thoroughbreds and Quarter Horses. These two gaits are composed of asymmetric movements of the hind and forelimbs. Because of the asymmetry, each limb is named differently: the leading limb is the last one of the limb pair to leave the ground. The contralateral limb is called the non-leading or trail limb. Consequently, there are two possible symmetric footfall sequences: right lead canter and left lead canter and, similarly, right lead gallop and left lead gallop. In free conditions, the horse prefers to canter or gallop on the right lead to go into a right curve, and vice versa, and will change the lead gallop when necessary to keep in balance. Gamete. A sperm or egg cell containing the haploid (1n) number of chromosomes. Gametic imprinting. An allele-specific reversible epigenetic modification dependent upon the parental origin of the allele, which can cause differences in development. Gene. A functional hereditary unit that occupies a fixed location on a chromosome, has a specific influence on phenotype and is capable of mutation to various allelic forms. Generation interval. The average age of the parents when the progeny that will replace them are born. Genetic correlation. A measure indicating the degree to which two traits tend to change in the same or a different direction as a result of genetic effects. Genetic drift. Changes of allele frequencies in populations due to random events. Genetic drift is highly pronounced in small populations. Genetic variance. Variation in phenotype which results from variation in genetic composition among individuals. Genome. A complete set of chromosomes (hence genes). Genotype. The genetic constitution of an organism. Genotype–environment interaction. Situation when the difference in performance between two genotypes depends upon the environment in which performance is measured. This may be a change in the magnitude of the difference or a change in rank of the genotypes. Half sibs. Individuals that share one common parent. Heritability. Degree to which a given trait is controlled by genotype. The proportion of total phenotypic variation that is attributable to additive genetic variation.

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Heterosis. The degree to which the performance of a crossbred animal is better or worse than the average performance of the parents. Heterozygote, adj. heterozygous. An organism with unlike members of any given pair or series of alleles, which consequently produces unlike gametes. Homologous chromosomes. Chromosomes which occur in pairs and are similar in size, shape and genetic content. One homologous chromosome comes from the male parent and another from the female parent. Homozygote, adj. homozygous. An organism whose chromosomes carry identical members of a given pair of genes. The gametes are therefore all alike with respect to this locus. Inbreeding. Matings among related individuals which result in progeny that have more homozygous gene pairs than the average of the population. Inbreeding coefficient. A measurement of the increase in homozygosity, each unit is equal to a 1% increase in homozygosity relative to the average homozygosity in the base population. Inbreeding depression. The decreased performance normally associated with accumulation of inbreeding. Many recessive genes result in undesired traits or decreased performance when they are expressed. Inbred animals have more recessive genes in the homozygous condition that are expressed and result in reduced performance or undesired traits. Inversion. A mutation in which the order of genes in a chromosomal segment has been reversed. Karyotype. The appearance of the metaphase chromosomes of an individual or species which shows the comparative size, shape and morphology of the different chromosomes. Kinematics. Study of the changes in the position of the body segments in space during a specified time. The motions are described quantitatively by linear and angular variables which relate time, displacement, velocity and acceleration. No reference is made in kinematics to the cause of motion. Kinetics or dynamics. Study of the cause of the motion, which can be explained by the force applied to the body, its mass distribution and its dimensions. Kinetics are concerned with forces, energy and work, which are also in relation to kinematic variables such as acceleration and velocity. Limb alignment. Posture or mode of standing especially with respect to angles of joints in the horse’s legs, leg (bone) alignment. The alignment affects how the weight of the body is distributed over all four legs. The legs have to be evaluated from the front (forelegs) and back (hindlegs) as well as from both sides of the animal. ‘Leg stances’ is another term for limb alignment.

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Linebreeding. Mating of selected individuals from successive generations to produce animals with a high relationship to one or more selected ancestors. It is a form of inbreeding. Linkage. Association of genes that are physically located on the same chromosome. A group of linked genes is called a linkage group. Locus, pl. loci. A fixed position on a chromosome occupied by a given gene or one of its alleles. Major gene. A gene that has an easily recognizable and measurable effect on a trait. Map (cytogenetic). A diagram showing the internal structure of chromosomes (dark and light bands) and the relative location of genes on a chromosome. Map (genetic). A diagram showing linkage distances between genes and markers on chromosomes. These distances are measured in centiMorgans (cM). Map (physical). A diagram showing physical distances between genes and markers on chromosomes. These distances are measured in numbers of nucleotides. Marker. Specific and identifiable sequences of the DNA molecule. The markers may or may not be functional genes. MAS. Marker-assisted selection. Selection for a trait of interest (usually quantitative), based on the genotype for linked testable genetic markers. Mating systems. The rules which describe how selected breeds and/or individuals will be paired at mating. Meiosis. The process including two successive nuclear and cell divisions by which the chromosome number of a germ cell becomes reduced to half the diploid (2n) or somatic number and results in the formation of haploid cells (1n), which finally become eggs or sperm cells. Microsatellites. Short arrays of simple sequence DNA repeats, typically 1–4 bp, which are interspersed throughout the genome. Minisatellites. Long tandemly repeated usually untranslated sequences spread throughout genome. Minisatellites are effective tools for DNA fingerprinting. Mitosis. Nuclear and cell division resulting in the formation of two daughter cells with the diploid (2n) number of chromosomes identical to the parental cell. Mutation. A random change in DNA structure. The term is used most often in reference to point mutations (changes within a gene), but can refer to chromosomal changes.

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Overlap time. Duration of the double support of diagonal or lateral limbs at the canter and gallop. Pace. A two-beat gait characterized by alternate weight-bearing on two legs of the same side, which move simultaneously. This lateral symmetric gait is used in harness racing mainly in North America and Australia. A small temporal asynchrony of the lateral as the hooves impact and lift off could occur at high speed. The maximum speed can be higher than at the flying trot (see Trot). Pedigree. A diagram showing relationships between the members of an extended family and an inheritance pattern for a specific trait. Penetrance. The proportion of individuals with a particular genotype that expresses the corresponding trait. Performance capacity. A horse’s ability to perform in the discipline it is trained for. It is a combination of genetic, physical and emotional characteristics and can be measured through the competition results and statistical data of the horse. Performance index. The phenotypic index which is computed annually for each discipline to quantify the level of performance of the horse. For example, in France it is calculated using a logarithmic transformation of the average annual earnings and it is corrected for age, sex and the year of competition. The scale is linear and the indexes are distributed normally. The mean of the horse population is set to 100 and the standard deviation is 20. An index of 140 indicates a high level of performance and 60 a poor level of performance. Phenotype. Characteristics of an individual that are observable, such as size, shape, colour or performance. Phenotypic correlation. A situation where two traits tend to change in the same or different direction as a net result of genetic and environmental effects. Phenotypic variation. Variation in phenotype which is caused by genetic and environmental effects. Pleiotropy. The developmental phenomenon where an allele affects two or more characters. QTL. Quantitative trait loci. Gene(s) located within a certain region of a chromosome, which have significant effects on a quantitative trait. Qualitative trait. A trait that generally can be classified into a limited number of categories, and the animal can be said to ‘possess’ the quality or not. Examples include hair colour, some inherited disease, etc. Quantitative trait. A trait that is represented by an almost continuous distribution of measurements. Examples include average daily gain, racing capacity, etc.

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Random mating. A mating system in which animals are assigned as breeding pairs at random without regard to genetic relationship or performance. Recessive. Applied to one member of an allelic pair which lacks the ability to manifest itself when the other, dominant member is present. Reciprocal cross. When males of breed A are mated to females of breed B and males of breed B are mated to females of breed A. Recombination. A genetic process that generates new allele combinations on the same chromosome (haplotypes) which were absent in preceding generations. Recombination is based upon meiotic and/or mitotic crossing over. Recurrent selection. A method of selection for combining ability or heterosis. Selection within one line is based on performance of crossbred progeny from matings with a ‘tester’ line. Regularity of the gaits. This is related to the conformation and stances of the legs. A regular gait is straight when evaluated from front and back, and no defects exist due to incorrect limb alignment, and the movements are balanced. In a regular gait, the stride to stride variability of the limbs should be low. Repeatability. The proportion of total phenotypic variation that is attributable to variation caused by genetic and permanent environmental effects. It is a measure of the degree to which early measures of a trait can predict later records of the same trait. Rideability. Ability of the horse to give accurate responses to the signals of the rider. Selection. Any natural or artificial process favouring the survival and propagation of certain individuals in a population. Selection criteria. The character(s) upon which selection decisions are based. Selection differential. The difference in mean performance of the selected group of animals relative to the mean performance of all animals available for selection. Selection index. The combining of measurements from several sources into an estimate of genetic value. More than one measurement on a trait, and/or measurements of the trait on relatives, and/or the measurements on more than one trait are combined into a single estimate of overall genetic value. Selection intensity. The proportion of animals selected to be parents relative to the total number available for selection. The smaller the proportion selected, the higher the selection intensity.

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Selection objective. The character(s) which are intended to be modified by selection. Sex chromosomes. The X or Y chromosomes. Sex-influenced. Traits in which the expression depends on the sex of the individual. Sex-limited. A trait that can be expressed only in one sex, such as milk production. Sex-linked. Genes that are located on a sex chromosomes (X or Y). Stride. The full cycle of limb motion. Since the pattern is repeated, the beginning of the stride can be at any point in the pattern and the end of that stride at the same place in the beginning of the next pattern. Each complete limb cycle includes a stance phase when the limb is in contact with the ground and swing phase when the limb is not in contact with the ground. The duration of the stride is equal to the sum of the stance and swing phase durations. Stride frequency. Corresponds to the number of strides performed per unit of time. The stride frequency is equal to the inverse of stride duration and it is usually expressed in stride s−1 or in Hertz. Stride length. Corresponds to the distance between two successive hoof placements of the same limb. To increase its velocity, the horse can switch gait from walk to trot and from trot to canter and then extend the canter to gallop. Each gait can be also extended by changing the spatial and temporal characteristics of its strides. Takh. Nearly extinct eastern subspecies of the wild horse, Equus ferus przewalskii. Reintroduction of the takh to its native Mongolian habitat is the current objective. Tarpan. Extinct wild ancestor of the modern domestic horses, Equus ferus ferus. Tarpan-like horses were reconstructed in Germany by the Heck brothers. Toelt. A gait with a four-beat rhythm, in which the foot sequence is: left hind, left fore, right hind, right fore, etc. Toelt is a running gait without suspension, and at least one foot always touches the ground. Toelt is called alternatively ‘running walk’, ‘rack’ or ‘singlefoot’. The fast four-beat gaits are also called ‘broken’ gaits; they are intermediate between trot and pace. Translocation (reciprocal). Exchange of chromosome fragments between non-homologous chromosomes. Translocation (Robertsonian). Fusion of two non-homologous chromosomes.

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Treadmill. Under laboratory conditions, the treadmill provides an excellent means of controlling the regularity of the gaits because the velocity and slope of the treadmill belt are entirely fixed by the operator. In order to analyse the gait of a horse without stress, some pre-experimental exercise sessions are required to accustom it to this unusual exercise condition. On a treadmill, the locomotion is exactly the same as on the ground; the stride length is slightly increased in flat and inclined exercise at trot and gallop. Consequently, exercise on a flat treadmill generates a lower cardiac and blood lactate response than exercise on the track at the same velocities. Trot. A two-beat gait characterized by alternate weight-bearing on diagonal pairs of legs. This is a springing gait with a suspension phase (no ground contact) between two diagonal supports. Among the normal variations of the trot of saddle horses, the speed of the gait increases from collected to extended trot. Passage and piaffe are two dressage exercises derived from collected trot. In harness trotters, the trot is very extended and is called the flying trot. It is a four-beat gait because there is an asynchrony of the impact and/or lift off the diagonal. In most cases, the hindlimb touches the ground first. As for the racing trot, there is an asynchrony of the hoof impact and hoof lift off. Type. Special conformational characteristics representative for a certain horse breed. Usually, visible breed characteristics which make one breed distinctive from another. A horse has a certain type according to its breed, sex and purpose (riding, harness, work, etc.). Variance. A measure of the dispersion of the mean value in a population Walk. The slowest gait with a four-beat rhythm and evenly distributed weight on the four feet. The foot sequence is: left hind, left fore, right hind, right fore, etc. In walk, either two or three feet touch the ground simultaneously. This is probably one of the most complex gaits because of the overlap and lag phase variability. Zygote. The cell produced by the fusion of mature gametes (egg and sperm).

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Index

Note: all entries refer to the domestic horse, Equus caballus, unless otherwise stated. abnormal behaviour, 293–301 Index

cribbing, 294–298 self-mutilation, 298, 300 sexual, 300–301 stall walking, 294, 298 trailer problems, 301 weaving, 294, 298 wood chewing, 295–296 achdari, 5, 16 aggregate genotype, 476–479, 486 aggression, 288–290 agricultural and draught use, 28, 36, 40, 41, 48, 389, 396, 402 breeding objectives, 479 allantois, 317, 318, 359 allele frequency markers, 92–97, 98, 164–167, 245–246, 480 comparisons, 42–47, 480 terminology guidelines, 503–504 American Livestock Breeds Conservancy (ALBC), 398, 406–407 amplified fragment length polymorphisms (AFLPs), 246 anaerobic threshold, 415, 448–449, 477 antibodies, 85–86, 89, 91, 354–355 production, 139–140 see also immunoglobulin antigens, 129, 130–131, 134–135, 134–136, 142, 322

artificial insemination (AI), 36, 314, 323, 326–329, 405, 434 semen storage and freezing, 327, 328–329, 333 Artiodactyla, 132 asses, 5, 7, 9–11, 117, 172 ancestors, 7 cytogenetics summary, 174–175 taxonomy, 13, 16–18 see also donkey; wild asses assisted reproductive technologies (ART), 36–37 Astrohippus, 4 asynchronous twin conception, 311–312 banding techniques, 173–178 C-banded, 177, 370 G-banded, 173, 176–178, 370 NOR-banded, 177–178 Q-banded, 173, 177–178 R-banded, 173, 177–178, 185 T-banded, 177 behaviour, 281–306 activity patterns, 284 feeding, 284 foal, 287–288 maternal, 285–287 sexual, 284–285 517

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518 biochemical loci, 130–131 6-phosphogluconate dehydrogenase, 92, 96 A1B-glycoprotein, 92–93 acid phosphatase, 92–93 albumin, 92–93 aspartate aminotransferase, 92–93, 165 carbonic anhydrase, 92–94 catalase, 92, 94 ceruloplasmin, 92, 94 complement component 3, 92–93 creatine kinase, 165 fucosidase alpha, 92, 95 glucosephosphate isomerase, 92, 95 haemoglobin alpha, 92, 95 haptoglobin, 92, 95 lactate dehydrogenase, 96 lactoglobulin beta II, 92, 97 malic enzyme 1, 92, 96 mannosephosphate isomerase, 92, 96 NADH diaphorase, 92, 94 peptidase A, 92, 96 phosphoglucomutase, 92, 96 plasminogen, 92, 96 protease inhibitor, 92, 96–97 red cell protein, 92, 97 serum carboxylesterase, 92, 94 serum protein 3, 43, 92, 97 transferrin, 92, 97, 114 vitamin D-binding protein, 92, 95 biochemical polymorphism, 91–97, 246 biodiversity, see conservation blastocysts, 331–332, 344–346, 350–4 blood circulation physiology, 416 blood group systems, 43, 46, 86–108, 87–89, 98–100, 112, 160–161 polymorphism, 192 study history, 86–87 blood marker utilization, 246 blood protein polymorphism, 160–161, 166, 192 blood transfusion, 91 blood typing, 114, 398–399 BLUP, 414, 474, 477, 492–494 animal model use, 483–485 EBV selection of trotters, 488–489 genetic improvement applications, 485–486 used to estimate EBV, 481–483 bone marrow transplants, 138

Index breed registration, 36–38, 115, 157, 327, 400–401, 474 show judging, 440–441 show measurements, 464 breed societies, 167, 221, 287 breeding objectives, 474–480 quantitative traits, 480–481 suitability, 167–168 technology, 326–334 for breed conservation, 405 use of conformation traits, 463–464 breeds Akhal Teke, 393, 401 American Belgian, 396 American Cream Draft, 393, 401, 407 American Paint, 158–159 American Saddlebred, 290–292, 479 American Standardbred, 99 Andalusian, 450–451 Anglo-Arabians, 448 Appaloosa, 37, 282, 290–291, 393 type changes, 402–403 Arabian, 33, 34, 37, 38, 44–46, 111, 159, 161, 285, 286–287, 290–291, 293, 299, 393, 442, 450–451, 479 Bavarian coldblood, 442 Bavarian warmblood, 115, 444 Belgian, 288, 393 Caspian pony, 33, 47, 393, 407–408 Chilote Horse, 100 Chinese miniature, 400 Choctaw, 408 Cleveland Bay, 397 Clydesdale, 393, 400 Connemara, 37 Dutch trotters, 421 Dutch warmblood riding, 441–443, 443 Exmoor pony, 393 Finnhorse, 418 Finnhorse Standardbred trotter, 442–443, 444, 455–456, 456 earnings heritability, 421 Florida Cracker, 393, 397, 399 Freibergers, 142 French Anglo-Arabian, 451 French Saddlebred, 288, 293 French trotter, 449 earnings heritability, 421 Friesians, 115

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Index gene loci comparisons, 42–47 genetic relatedness, 42–47 German trotters, 485 Hackney, 397 Haflinger, 443 Hannover, 295, 442–443, 444, 455 Holsteiner, 442, 444 Icelandic, 37, 143, 393, 401, 442–443, 455, 479, 485, 494 genetic improvement, 491 Irish Draught, 397 Japanese native horse, 111, 297, 300 landrace development, 392–393, 399 Lipizzaner, 38, 39, 44–46, 111, 400 Lusitano, 38–39 Miniature, 39–40, 44–46, 290–291 Morgan, 37–38, 40, 44–46, 162, 290–291, 295, 395 type changes, 402 New Forest ponies, 288 Nokota, 399 Nordic trotters, 494 Noriker, 403 North Swedish trotter, 111, 165, 296, 419 earnings heritability, 421 Norwegian Fjord, 40–41, 44–46, 115 Norwegian trotter, 419 earnings heritability, 421–422 Oldenburg, 403 Paint, 37, 159, 287, 290–291, 393 Pantaneiro, 100, 399–400 Paso Fino, 37, 41, 44–46, 479 Percheron, 41, 44–46, 393 Posavina, 100, 407 Purebred Spanish, 38–39, 44–46 Quarter Horse, 33, 110, 115, 159, 161, 162, 163, 164, 283, 287, 290–293, 299, 393, 397, 451, 485, 494 genetic improvement, 490–491 learning ability, 448 Timeform improvements, 414, 415 type changes, 402–403, 403 Saddlebred, 287–288, 295 Selle Français, 448 Shagya, 37 Shetland pony, 34, 40, 159, 323, 443 Shire, 323, 400 Sorraia, 407

519 Standardbred, 33, 115, 139, 162, 164, 181, 285, 288, 290–292, 295–296, 299, 418–419, 454 earnings heritability, 421–422 Standardbred trotter, 163, 165, 446, 459 Suffolk, 400 Swedish Standardbred, 164, 165, 456, 489 Swedish Standardbred trotter, 494 Swedish trotter, 33, 110–111 Swedish warmblood, 160, 442–443, 444, 456 Swiss warmblood, 143 Tennessee Walking Horse, 37, 115, 290–292, 479 Thoroughbred, 37, 41–42, 44–46, 111, 115, 162, 164, 283, 287, 288, 299, 393, 441, 443, 486 behaviour, 290–292 blood circulation physiology, 416 breeding objectives, 476–477 breeding techniques, 327, 329 chimeric, 187–188 conformation and performance, 455 crossbred foals, 397 crossbreeding for sport, 41, 478 durability, 457 earnings heritability, 413 genetic improvement, 414, 490 learning ability, 448 locomotor performance evaluation, 459–460 muscle characteristics, 451 muscle metabolism, 415–416 performance and race length, 417–418 performance traits heritability, 412–414 reproductive problems, 308, 311 stereotypies, 293–296 Timeform rating, 414–416, 490 Trakehner, 42, 44–46, 295, 442, 444 Welsh pony, 288, 395 Yakut, 34 see also coat colour and breeds; feral horses breeds registration, 36–38 Bulmer effect, 492

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520 cattle blood groups, 86–87 centiMorgans (cM), 244 chromosome Y sex-determining region (SRY), 368–369, 370–371 chromosomes, 127–128 aberrations, 180–191 aberrations – autosomal, 181–184, 184 aberrations – fragile sites, 191 aberrations – sex linked, 182, 185–191 chimaerism, 89–90, 183, 187–188, 370, 181 mosaicism, 183, 187–188, 370 numerical aberrations, 182, 186–187 sex reversal, 183, 188–191 structural aberrations, 182, 185–186 C-banded, 177, 370 centromeres and satellites, 112–113 comparative painting, see Zoo-FISH fluorescence-activated cell sorting (FACS), 209 G-banded, 173, 176–178, 370 length measurement, 178 meiotic, 178–180 female, 179 interspecific hybrids, 180 male, 179 synaptonemal complex analysis, 179–180 microdissection, 209–211, 223 NOR-banded, 177–178 painting, 211 Q-banded, 173, 177–178 R-banded, 173, 177–178, 185 reverse painting, 216 Robertsonian translocation, 32–33 T-banded, 177 X, 158–159, 369–371 inactivation, 372 Y, 47, 158, 367–368, 370 cleavage, 356 cloning, 145–146, 357, 364–365, 370 calcyclin, 352 phospholipase A2 (PLA2), 352 SRY gene, 369 coat colour and breeds American Saddlebred, 54 Andalusian, 39 Appaloosa, 67 Arabian, 54, 63 Cleveland Bay, 54

Index Cremollo, 60 Curly, 54 Exmoor, 54 Friesian, 54 Haflinger, 54 Icelandic, 62 Knabstrup, 67 Lipizzaner, 54 Lusitano, 54 Miniature, 54, 67 Morgan, 54 Noriker, 67 Norwegian Fjord, 61 Paint, 54, 61 Palomino, 54, 59–60 Paso Fino, 54 Perlino, 60 Pinto, 54 Quarter Horse, 54, 61, 62 Shetland, 62 Standardbred, 63 Suffolk, 54 Tennessee Walking Horse, 54, 61 Thoroughbred, 54, 63 coat colour genes, 37, 53–68, 53–70, 245 Agouti, 54–56, 55, 57, 59, 366 albino phenotype, 60 Chestnut, 55, 58–59, 114 dilution genes, 54–56, 57, 59–62 Champagne, 61 Cream, 59–60 Dun, 40, 60–61 Silver (dapple), 62 dominant white, 63, 159 Extension, 54–56, 57, 58, 366 genotypes and phenotypes, 56 Grey, 54, 54–56, 55, 57 identification techniques, 68 mutations, 365–366 albino, 366 melanocyte morphology, 366 white, 365 Overo lethal white syndrome (OLWS), 74, 158–159, 161, 365, 390 Roan, 55, 58–59, 158–159 spotting Leopard (tiger, appaloosa), 67 Overo, 64, 66–67 Roan, 64–65 Tobiano, 64, 65–66

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Index White, 63–64 spotting loss, 395 Tobiano, 58–59 variant loss, 395 codon differences, 165 colour blindness, 282 Committee on Genetic Nomenclature of Sheep and Goats (COGNOSAG), 499–501, 504–505 comparative anchor-tagged sequences (CATS) primers, 193–194 comparative gene map, 248–250, 273–274 complementary DNA (cDNA), 133, 352 clones, 136–137, 197 libraries, 117–118, 132, 136, 244 conformation, 157, 439–472 limb, 441, 446, 454–455, 456, 457, 460, 461–462 measured traits, 444–447 heritability, 445 physiological traits, 447–443 quantitative evaluation, 446, 456 relationship with performance, 453–456 scored traits, 441–444 heritability, 442–443 soundness and durability, 457 trait breeding programmes, 463–464 conservation, 112, 388–410 breed design and genetic pool, 393–394 changes in equine use, 388–389, 396–397 coat colour variant loss, 395 feral horses, 394–395, 399 future developments, 408–409 gamete preservation, 405–406 genetic erosion, 388, 389, 391, 395–397 genetic insurance policy, 388–390 geographic aspects, 399–400 governmental action, 406 herd book importance, 398 historical aspects, 390 non-governmental organizations, 406–407 political aspects, 400 population choice, 397–399 population history, 398 population theoretical aspects, 404 priorities, 397 rare breeds, 99–100 scientific value, 390–391

521 selection philosophy, 401–403 wild horse preservation, 390–391 crossbreeding, 7–8, 14, 35–36, 40, 180, 213, 322, 371–372, 397, 398 hinnies, 371–372 mules, 371–372 cytogenetics, 171–241 banding techniques, 173–178, 214 future prospects, 220–223 reduction in use, 221 study history, 172 developmental genetics, 344–385 diastema, 11, 30 digestive system, 29 Dinohippus, 4, 6, 9 diseases, 126–127, 157–170 environmental, 161–163 see also morphological traits and inherited disorders DNA anonymous markers, 198–205 fingerprinting, 110–111, 398–399 globin genes, 117 microsatellites, see microsatellites minisatellites, see minisatellites mitochondrial, see mitochondrial DNA (mtDNA) polymorphisms, 246 satellite repeats, 109–114 satellite sequences, 110–113, 135–137 single nucleotide polymorphisms, see single nucleotide polymorphisms (SNP) tetranucleotide repeats, 112 DNA fingerprinting, 409 Dolichohippus, 6 domestic animals nomenclature, 9 domestic horse distribution, 35–48 future use and conservation, 48 genetic comparison with Przewalski’s horse, 32–35 introgression, 47–48 karyotype evolution, 32–33 origins, 26, 28–29 domestication and human mobility, 31–32, 391–392 genetic aspects, 25–51

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522 domestication continued history, 30–32 reasons and pre-conditions, 29–30 donkey, 112–113, 135–136, 137, 180, 208, 288, 322–323, 371, 372 cytogenetics summary, 174–175 karotype, 212–214, 217, 219, 220 see also asses EMBL Database, 116–117 embryo development embryonic period, 346–348 fetal period, 348 vesicular, 344–346 embryo transfer, 36, 324, 329–333, 371, 405 in vitro fertilization (IVF), 333 intracytoplasmic sperm injection (ICSI), 333 monozygotic twin production, 313, 332 ovary response to exogenous eCG, 329–331 oviducal transport time, 331–332 environmental behaviour influences, 296, 297–299 enzyme electrophoresis, 192 Equidae, 1, 2–6, 109–111, 112–113, 133, 133 early development, 2–4 generic limits, 5–6 genetics, 4–5 Equinae, 3 equine chorionic gonadotrophin (eCG), 311, 319–322, 324–325, 329–331, 348, 355, 357, 358 Equine Gene Mapping Workshop (EGMW), 250 equine lymphocyte antigen (ELA) non-MHC, 138–139 paternally inherited, 139 see also major histocompatibility complex Equini, 3–4 Equioidea, 2 Equus, 4 cytogenetics summary, 174–175 early history, 6–9 taxonomy, 9–22 Equus (burchelli) quagga, see quagga Equus africanus africanus (Nubian wild ass), 17–18

Index Equus africanus somaliensus (Somali wild ass), 17, 18 Equus asinus, see asses Equus ferus ferus, see tarpan Equus ferus przewalskii, see Przewalski’s horse Equus ferus sylvestris, see tarpan Equus Hemionus, 6, 8, 9–11 see also E. hemionus Equus hemionus, see onager Equus hemionus hemionus, 15, 172, 174 Equus hemionus kulan, 15 Equus hemippus (achdari), 5, 16 Equus Hippotigris, see zebra Equus khur (khur), 13–14, 15–16 Equus kiang, see kiang Equus quagga, see zebra, plains Equus quagga burchelli, see quagga Equus quagga quagga, see quagga Equus zebra, see zebra erythrocyte antigens, 86–89 estimated breeding values (EBV), see BLUP expressed sequence tags (ESTs), 116–117 FAO, 398 FAOSTAT database, 35 feral horses, 288, 391, 394–395, 406 Australian brumby, 394 Cerbat, 394 Colonial Spanish, 399, 406, 408 herd blood groups, 99 Kiger, 394 Misaki pony, 394 Pryor Mountain, 394, 406 Sable Island, 394 Sulphur, 394 gait, 37, 41, 427–428, 446–447, 452, 454, 456, 460, 494 asymmetry, 459 breeding programme, 463–464, 479 collected, 462–463 gametes, 244–245 genetic resource conservation, 405–406 imprinting, 372 gene diversity across breeds, 42–47 gene mapping – physical, 68, 71, 80, 191–241, 244

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Index comparative, 248–250, 273–274 future prospects, 220–223 history, 191–192 in situ hybridization (FISH), 192, 193, 197–208, 221–222 marker distribution, 221 radiation hybrid maps (RH), 196–197 SCH panels, 222 somatic hybrid cell panel analysis (SCH), 198–205 synteny maps, 192–196 gene nomenclature mapped loci, 504–505 generation interval, 463, 487–488 genes Axin, 360 dominant autosomal inheritance, 163 dominant coat colour lethality, 158 housekeeper, 351–352 Hox, 361–362, 363 marker alleles, 164–167 Pax, 362, 364 protein structures, 124 proto-oncogenes, 351 recessive autosomal inheritance, 161, 163 T-box, 360–362, 364 genetic distance, 44–45, 165–167 genetic diversity, 99 genetic drift, 395–396, 493 genetic effects additive, 486–488, 492, 493 marker-assisted selection (MAS), 494 non-additive, 163, 486 quantitative traits loci (QTL), 163, 164, 474, 494 selection within and outside generations, 492–493 selection within small populations, 493–494 genetic erosion, 395–397, 396–397 genetic evaluation, 480–486 genetic improvement, 473–497 genetic nomenclature, 499–505 horse allele guidelines, 503–504 horse locus guidelines, 501–502 ruminants, 500–501 sheep guidelines, 500 genetic resources, see conservation genetic response to selection, 486–494

523 genetic risk factors, 163–167 genome mapping, 109–110, 112–113, 115, 390 analysis, 160–161 comparison of equids, 208 genotype, 475–476, 477, 478 terminology guidelines, 504 geographic region studies, 37, 99, 392, 394, 399–400 hemiones, 6, 7, 9–11, 13–14, 16 taxonomy, 13–14 Hippidion, 4 Hippomorpha, 2 hormones follicle-stimulating hormone (FSH), 309, 319, 321 luteinizing hormone (LH), 309–312, 319, 321, 355 oestrogen, 324–326, 358 serum eCG concentration, 357 hyperimmune serum, 144 HYPP, 167 identity testing, 98 immune response, 126, 140 immunogenetics, 123–155 immunoglobulin, 124–155, 139–140, 143–145 heavy chain loci, 144 light chain loci, 145 inbreeding depression, 492–493 Institute for Ancient Equestrian Studies, 26 International Society for Animal Genetics (ISAG), 33–34, 87 karotype, 26, 171–178, 212, 249, 370 khur, 13–14 kiang, 5, 13–14 kinematics, 460, 461–462 kulan, 15 lameness, 457 learning ability, 283–284, 447–448 linkage maps, 111, 217, 243–274, 256–272 group II, 249 group III, 249

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524 linkage maps continued group IV, 249 group V, 249 pedigreed families, 246–247 locomotion, 452–456 locus terminology guidelines, 501–502 lymphocytes, 129–131 mixed lymphocytes culture, 134 major histocompatibility complex (MHC), 125–143 gene map, 127 MHC class I, 127–133 MHC class II, 127–129, 133–137 MHC class III, 127–128, 137–138 typing, 138 meat production, 396 meiosis, 178–180, 247, 367–368, 372 Mendelian Inheritance in Animals database, 72–73 microsatellites, 35, 42–43, 43, 46, 109, 110, 111–112, 114, 161, 193, 198–205, 246, 248, 250, 255–267, 273 military use of animals horses, 31, 38, 42 mules, 371 milk proteins, 97 minisatellites, 110–111 fingerprinting, 115 mitochondrial DNA (mtDNA), 5, 12, 13, 18, 34–39, 47, 116, 117 mitogen-activated protein (MAP), 365 molecular genetics, 109–121 research resources, 118 morphogenesis gastrulation, 360–361 genetic control, 360–366 Hox genes, 361–362 muscle development, 364–365 notochord formation, 361 organogenesis, 362–363 testicular development, 367–368 morphological traits and inherited disorders, 71–84, 161–163 adrenal agenesis, 370 adynamia episodica hereditaria, 75, 114, 159 agammaglobulinaemia, 75, 114, 146, 159 androgen insensitivity syndrome, 78

Index aniridia with cataract, 76 ataxia (wobbles), 79 atlanto-occipital fusion, 79 autoimmune disease, 124 cerebellar disorders, 79 chronic bronchitis, 141–142 collagen defect, 79 connective tissue disease, 79 cryptoorchid, 190, 301, 370 curly coat, 79 deformed extremity bones, 159 degenerative myeloencephalopathy, 79 dihydrotesterone receptor deficiency, 78 dominant white lethality, 114, 158–159 epidermolysis bullosa junctionalis, 79 epitheliogenesis imperfecta, 76 exertional rhabdomyolosis syndrome (Rha), 163, 167 exostosis, multiple, 76 Gamstorp disease, 75, 114, 159 Gilbert syndrome, 76 glucose-6-phosphate dehydrogenase deficiency, 76 gonadal agenesis, 370 gonadal dysgenesis, 75, 158, 187, 188, 189 gonadal hypoplasia, 75, 158, 187, 188, 189 haemophilia A, 76, 159 hemeralopia (night blindness), 79 hermaphroditism, 370 hyperbilirubinaemia, 76 hyperkalaemic periodic paralysis (HYPP), 75, 159, 365 Impressive syndrome, 75, 114, 159 infertility, 405 insect bite dermal hypersensitivity (sweet itch), 126, 140–141, 143 intestinal aganglionosis, 366 laminitis, 141–142 laryngeal hemiplegia (roaring defect), 79, 142–143, 160, 163 lethal dominant roan, 77 lethal dominant white, 77 megacolon, 74 multiple exostosis, 159 myoclonus, 77 narcolepsy, 79 neonatal isoerythrolysis, 90–91 orthopaedic, 443, 457

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Index osteochondrosis, 79, 163 Overo lethal white syndrome, 74, 158–159 palmar osteochondral fragments (POF), 165–167 patellar dislocation, 79, 159 periodic paralysis II, 75, 114, 159 polycythemia, 167 polysaccharide storage myopathy (PSSM), 163 pseudo-hermaphrodite, 185, 186, 187, 190 pseudohaemophilia, 78 red foot disease, 79 rhabdomyolysis, 77 Roan, 159 sarcoid tumour susceptibility, 126, 140–143 severe combined immunodeficiency disease (SCID), 75, 114, 138, 146, 159 sex reversal, 370 sex reversal XY female, 75, 158, 188–191 sex-linked, 158, 163 single locus, 71–72, 72–78 sterility, 7–8, 186, 370 Stringhalt, 79 Swyer syndrome, 75, 158, 189 T-lymphocyte cytotoxicity, 147 testicular feminization, 78 tumour necrosis, 129 ulnar and tibial malformation, 78 von Willebrand disease, 78 muscles characteristics, 459–461 development, 364–365 fibre, 459, 461 metabolism, 415, 451 myosin heavy chains, 451, 460, 462 nervous system, 447–448 neurotransmitters, 282 non-MHC lymphocyte alloantigens, 138 nuclear gene detection, 33–34 onager, 5, 13–14, 135, 137 Persian, 133 taxonomy, 15–16

525 Onohippidion, 4 oocytes, 179, 180, 309, 333, 348–349, 356, 367, 372 paraspecies, 9 parentage testing, 33–34, 42, 47, 92, 99, 111 blood typing, 98, 114 DNA-based, 114–116 PCR analysis, 135, 192–193 Perissodactyla, 1–2, 132–133, 132–133 phenotype, 391–392, 398–399 terminology guidelines, 503–504 photometric evaluation, 446 phylogenetic tree, 5 phylogeny, 1–22 physiological traits behavioural tests, 447–448 breeding programmes, 463–464 energetics, 448–450 gait, see gait muscle characteristics, 450–451 relation to performance, 457–463 plasma proteins, 91–97 Pliohippus, 4 polymerase chain reaction (PCR), 4, 35, 111, 115–116, 161, 248, 255, 369 polymorphisms, blood and milk nomenclature, 504–505 populations, 37 breeding objectives, 475, 476–480 breeding selection from small populations, 493–494 genetic response selection, 486–491 genetic variability, 98–100 pre-implantation development cytoskeletal organization, 351 embryonic gene expression, 349, 350–353 gametic imprinting, 354–357 genome activation, 349–350 growth factors, 352 maternal regulation, 348–349 proto-oncogenes, 351 trophoblast gene expression, 353–355 pregnancy, 130, 308–341 hybrid gestation period, 371–372 maternal recognition, 358 placenta development, 358–360 protein databases, 115, 117

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526 protein histochemical stains, 85–86 protein markers, 42–46 protein polymorphisms, 85–86, 160–161 Przewalski’s horse, 12, 26–27, 29–30, 44–46, 116, 136, 172, 180, 288, 297–298, 388, 391 comparison with domestic horse, 32–35 cytogenics summary, 174–175 karotype, 32 major histocompatibility complex (MHC), 133 mitochondrial DNA, 12 quagga, 4, 6, 8–9, 20–21, 172 cytogenetics summary, 174–175 taxonomy, 19–22 racing horses, 412–426, 458–460, 486, 488–491 breeding goals, 476–477 performance limits, 414–415 see also Thoroughbred random amplified polymorphic DNA (RAPDs), 114, 193, 246 Rare Breeds Survival Trust (RBST), 398, 406–407 recombination, 244–245, 247 red blood cell antigens, 129, 160 red blood cell proteins, 43 reproduction, 130, 307–341 for breed conservation, 405 breed conservation assistance, 405 embryogenesis, 313–314, 315, 316–318 see also embryo development embryonic capsule, 317–318, 346, 353, 358 endometrial cups, 311–312, 319–322, 324–325, 346, 348, 355, 358–359, 360, 371 endometrosis, 316, 323–324 foal bilateral gonadectomy, 326 inherited ELA antigens, 139 intraspecies, 322–324, 357 intrauterine growth retardation (IUGR), 316 membrane differentiation, 318–323 oestrogens of pregnancy, 324–326 ovarian function, 312–313

Index oviducal function, 308, 313–314 pituitary LH secretion, 310–312 placentation, 323–324 prolonged spring oestrus, 310–311 seasonality and cyclicity, 308–312 uterine structure, 314–316 restriction fragment length polymorphism (RFLP), 13, 114, 116, 131–132, 135, 144, 161, 246 rhinocerus, 1, 2, 113, 133 Safeguard for Agricultural Varieties in Europe (SAVE), 406–407 SCID, 167 serological studies, 130–131, 133, 134–135 serum proteins, 43 sex determination, 366–371 gonad differentiation, 366–368 SRY gene, 368–369 single nucleotide polymorphisms (SNP), 110, 113–114, 116 single-strand conformational polymorphisms (SSCPs), 246 somatic cell hybrid panel (SCH), 192, 193, 194, 244, 247–248, 255, 274 Southern blots, 110, 115, 131–132, 135, 192, 255 spermatozoa gender selection, 333–334 intracytoplasmic injection (ICSI), 333 longevity, 327–328 sport horses, 39, 41, 42, 48, 396, 427–434, 430–434 breeding objectives, 477–479 competitions, 428–429 crossbreeding, 486 dressage, 462–463, 478 endurance riding, 461 health as breeding goal, 478–479 international genetic evaluation, 485 locomotor performance evaluation, 461–463 longevity as breeding goal, 478–479 multiple-trait genetic evaluations, 430, 434 non-recorded horse bias, 429 performance and conformation, 454 performance measurement, 427–429, 457–463

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Index performance traits genetic correlations, 432–434 performance traits heritability, 429–432 show-jumping, 461–462, 478 soundness, 457 three-day eventing, 461, 478 trait breeding programmes, 463–464 SSCP study, 136 standardization, 393–394 starch gel electrophoresis, 85–86 stereotypic breed differences, 294–297 sterility, 7–8, 14, 186, 370 synteny, 222, 247–250, 255–272 systematics, 1–24 T-cell receptors (TCR), 145–146 takh, see Przewalski’s horse tapir, 1, 2, 133 tarpan, 12, 26, 27–30, 29, 32, 172, 388, 391 governmental action in conservation, 406 temperament, 288–293 sire effects, 292–293 testicular development, 367–368 toelt, 479, 494 treadmills, 448, 460 trotters, 418–426 age-related performance, 422 BLUP evaluation, 477 earnings heritability, 419–422 non-recorded horse bias, 426 genetic response to selection, 488–489 locomotor performance evaluation, 458–459 overall performance measurement, 423–426 performance and conformation, 454 random regression, 425–426

527 soundness, 457 speed heritability, 418–419 trainer effect, 424–425 vascular anastomosis, 89 Western blotting, 129 wild asses African, 11, 17 Asian, 13–14 Indian (khur), 5, 7, 8, 13–14, 15–16, 174 Mongolian, 15, 172, 174 Nubian, 17–18 Persian, 3, 7, 8, 174 Somali, 5, 17, 18, 174 Syrian (achdari), 5, 16 Tibetan (kiang), 13–14, 174 Transcaspian, 172, 174 Turkmenian (kulan), 15 wild horses, see feral horses zebra, 5, 6, 9–11, 30, 117, 133, 135, 172, 180 ancestors, 6 Grant’s, 174 Grevy’s, 7, 10, 18, 22, 112–113, 133, 174 Hartmann’s mountain, 10, 18, 19, 174–175, 208, 220 plains, 18, 19–20 taxonomy, 18–22 Zoo-FISH, 211–220, 221–222, 250–255, 273 equine vs. other mammalian genome, 217–220 human vs. horse genome, 214–217 human vs. other Equidae genomes, 217 within equids, 212–214

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