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The Horse Gene Map Ernest Bailey and Matthew M. Binns INTRODUCTION TABLE 1 The first contribution to the horse gene map was identification of linkage between 6-phosphogluconate dehydrogenase and the K blood group system (Sandberg 1974). During the next 20 years, additional linkage groups were identified and the map was periodically reviewed (Sandberg and Andersson 1993). Gene mapping discoveries were incidental to studies of new genetic systems for parentage analyses in horses. The concept of creating a comprehensive gene map for the horse appeared to be too expensive, requiring more resources than were available for research on horses. This changed in the early 1990s with advances in biotechnology and as the successes associated with the human genome project became publicized. During the early 1990s, inexpensive techniques became widely available for identifying, sequencing, and mapping large numbers of genes. This area of research became known as "genomics." Cattle, pigs, chickens, and sheep were among the first species for which scientists developed comprehensive gene maps. These maps were developed through efforts by individual laboratories as well as through collaborative efforts between laboratories. Following the successes of those programs and inspired by potential applications of gene maps for horses, scientists conducted a workshop in October 1995 to collaborate on constructing a gene map for the horse. This workshop marked the beginning of genomics research on horses. REASONS FOR MAPPING THIS SPECIES The domestic horse belongs to the genus Equus, which is composed of 9 extant species including horses, donkeys, hemiones, and zebras (Table 1). The horse evolved in North America approximately 3.5 to 5 million yr ago and migrated to the Asian/European continent across the Bering Strait before becoming extinct in the Americas between 20,000 and 10,000 yr ago (Simpson 1951). The horses and donkeys populating North America today are descendants of those reintroduced from Europe, Africa, and Asia over the last 500 years. Ernest Bailey, Ph.D., is with the M. H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky. Matthew M Binns, B.Sc, Ph.D., is with the Centre for Preventive Medicine, Animal Health Trust, Suffolk, United Kingdom. Volume 39, Numbers 2 and 3 1998 Extant species of Equus? Genus/species Equus przewalskii Equus caballus Equus asinus Equus hemionus onager Equus hemionus kulan Equus kiang Equus grevyi Equus burchelli Equus zebra hartmannae Common name Chromosome no. (2N) Mongolian wild horse Domestic horse Domestic ass/donkey 66 64 62 Persian wild ass 56 Kulan Kiang, Asian wild ass Grevy's zebra Burchelli's zebra, common zebra Hartmann's mountain zebra 54/55 51/52 46 44 32 ^Adapted from Lear 1997. The evolution of Equus is an example of evolution associated with rapid karyotypic change. Member species are clearly delineated by differences in chromosome numbers, ranging from 32 in the Hartmann's zebra (Equus zebra hartmannae) to 66 for Przewalski's horses (Equus przewalskii) (Table 1). Equid species can be induced to hybridize under artificial conditions; however, most of the hybrids are sterile, probably due to chromosome instability during meiosis. The most common hybrid is the domestic mule, produced by crossing a male donkey (jack) with a female horse (mare). Mules have 63 chromosomes, compared with 64 for horses and 62 for donkeys. The horse was domesticated approximately 6000 yr ago and was a primary source of power and transportation until the advent of the combustion engine. Diverse breeds of horses have been developed worldwide for diverse applications. Horses range in size from the heavy European draft horses such as the Percheron, Clydesdale, and Belgian horse to the diminutive pony breeds such as the ancient Exmoor pony, the well-known Shetland pony, and the modern pony breed known as the American Miniature Horse. Genetic diversity is great among horses despite a long history of selection. Although founder stallions have had a profound influence on some breeds, horse breeders have used crossbreeding extensively until modern times. Furthermore, genetic diversity has been maintained because horse popula171 tions have been large, and great numbers of mares have been used in founding most breeds. The extent of variation among horses is reflected by the power of the early blood typing programs to resolve questions of parentage using biochemical and blood group markers. These horse blood typing tests were able to exclude 95 to 98% of incorrect paternities in most breeds when using between 12 and 15 genetic systems (Bowling and Clark 1985). By comparison, comparable exclusion power was not attained for human or canine paternity cases until the introduction of DNA-based tests. Horses remain economically important and have been used increasingly for sports and recreation during the last 80 years. More than 70% of the approximately 6.9 million horses in the United States are involved in showing or recreational uses. The estimate of 7.1 million Americans who participate in the horse industry includes those who are employed in connection with horses, those who own or use horses as recreation, and those who provide related services. Even more people participate as spectators at horse racing meets and horse shows. The horse has a similar economic impact in Europe, Australia, and parts of Asia and Africa (Barents Group 1996). CURRENT MAP STATUS The development of the horse gene map has involved an international gene mapping workshop, database development, international agreement on a standard karyotype, and comparative mapping. The techniques used and the accomplishments resulting from use of those techniques (such as linkage mapping and physical mapping studies—fluorescence in situ hybridization [FISH1] and synteny mapping) are described below. International Equine Gene Mapping Workshop As noted in the Introduction, the International Equine Gene Mapping Workshop had a large impact on gene mapping research for the horse. Construction of a comprehensive gene map requires more resources than are available to any of the laboratories conducting genomics research on horses. To address this problem, the first workshop meeting was held by the Dorothy Russell Havemeyer Foundation in October 1995. At the conclusion of the meeting, the 70 participating scientists from 23 countries agreed to collaborate over a 5-yr period to share resources and construct a gene map for the horse. Some of the results of these collaborations are described immediately below and in Approaches Used to Develop the Map. 'Abbreviations used in this paper: FISH, fluorescence in situ hybridization; SCID, severe equine combined immunodeficiency disease. 172 Database The gene map of the horse is developing so rapidly that any published list of markers will soon be out of date. The status of the gene map for the horse is best determined by consulting the databases available through the World Wide Web. As information is published, the data are entered onto the databases. Addresses of the 2 databases in current development through the auspices of the International Equine Gene Mapping Workshop are as follows: Roslin, Scotland—http://www.ri.bbsrc.ac.uk/cgibin/arkdb/browsers/browser.sh?species=horse Jouy-en-Josas, France—http://locus.jouy.inra.fr/cgibin/horsemap/Horsemap/main.pl Standard Karyotype Chromosomes are identified in most species based on chromosome banding patterns produced after chemical treatments. During 1996, the Dorothy Russell Havemeyer Foundation hosted a workshop meeting to establish a new international standard. That standard was published in 1997 (ISCNH 1997) and has been important for accurate physical mapping of genes to chromosomes. Comparative Mapping The genome organization among mammals is similar, and the human gene map is best characterized. Knowing the regions of the horse genome that correspond to the different regions of the human genome makes it possible to use data from the human genome studies to predict the genomic organization of the horse without actually sequencing the horse genome. This area of research is called comparative gene mapping. This approach offers significant opportunities to draw on the research from other species and to more quickly apply the map to identify economically important genes. The first comprehensive comparative gene mapping study for the horse is described in the ZOO-FISH section below. APPROACHES USED TO DEVELOP THE MAP The genome organization of the horse is similar to that of other mammals. The approaches used in genome studies have included development of genetic tests for biochemical markers, blood groups, lymphocyte alloantigens, and DNA markers. The markers have also been used for investigations involving linkage mapping, cytogenetic studies, and synteny mapping. Studies are under way in several laboratories using chromosome flow sorting, chromosome microdissection, and bacterial artificial chromosome libraries. These techniques are being used together to provide valuable, indepen- ILAR Journal dent confirmation of map positions and to extend the borders and resolution of the horse gene map. Linkage Maps A linkage map allows us to identify DNA markers that are closely linked to genes for inherited diseases, performance traits, or other inherited characteristics for the horse. A first male linkage map was published based on investigations of 140 markers in 8 half sibling families with a total of 263 offspring (Lindgren and others 1998). Based on segregation of markers from the stallions, 100 markers were found in 25 linkage groups, 22 of which could be mapped to 18 autosomes. At the time of this writing, 2 additional linkage maps are being developed concurrently and were presented in poster format at the 26th International Conference on Animal Genetics (Auckland, New Zealand, August 1998). Swinburne and others (1998) tested 180 markers on a family composed of 5 grandparents, 5 parents, and 41 progeny. This family structure is unusual for a horse and was constructed with the use of 2 pairs of identical twin mares covered by a single stallion and embryo recovery at 30 days to increase the number of available offspring. DNA-based genetic marker testing was used. This study identified 27 linkage groups of which 20 could be assigned to 19 chromosomes, including the X chromosome. A third linkage map is being constructed at the time of this writing on the basis of an international collaboration. The International Equine Gene Mapping Workshop includes 20 laboratories that have tested 162 markers on 448 offspring distributed among 12 families (Guerin and others 1998). Of these markers, 124 were found in 29 linkage groups and appear to be distributed among 25 of the 31 autosomes of the horse. The main tool of the workshop is the International Equine Gene Mapping Reference Families, which will be a resource for continued development of the linkage map by diverse laboratories. The maps constructed by Lindgren and others (1998) and Guerin and others (1998) take advantage of the large number of offspring produced by individual stallions. Most genetic markers will be informative when multiple families are used. However, it is not possible to map genes to the sex chromosomes. The advantage of the family used by Swinburne and others (1998) is the reduced cost of testing fewer individuals and the ability to map markers on the X chromosome and place them on the Y. All 3 maps make extensive use of microsatellite DNA markers, and there is considerable overlap in the markers used, facilitating comparison of maps and eventual development of consensus on the linear relationship of genetic markers. FISHing Horse Genes Before 1995, only 5 genes had been mapped to horse chromosome, including HBA, MHC, GPI, CRC, and PGD (Table 2). Volume 39, Numbers 2 and 3 1998 TABLE 2 DNA markers mapped to horse chromsomes Chromosome Markers Reference 1q2.1 1q12->q13 1q13 1q16->q17 1q14 Iq14 1p16->p15 1p1.2 1q1.l 2p 2p1.3->p1.4 2p14->p15 2p17->p18 3p15 3q21->q22.1 3p13->p14 3q12, 3q22 4p12->p13 4q21 4q27->cen15 5 6q21 7pter 7p15 7p1.1->p1.2 7p2.1 8 9p12 9q16->q18 9? 9q15->ql6 9p13->p14 9p15 9p1.2 1 Opter 10pter ECA2 ASB12 ASB13 ASB8 SGCV02 SGCV25 HP27.1 ECA6 ECA13 PGD ECA3 ASB17 ASB11 GOT2 KIT SGCV18 SGCV33 ASB23 ASB3 ASB22 SGCV23 None ASB14 C3 PR ECA5 ECA12 None DNApk ASB4 ASB5 SGCV17 SGCV20 SGCV32 ECA14 GPI CRC Sakagami and others 1995 Breen and others 1997 Breen and others 1997 Breen and others 1997 Godard and others 1997 Godard and others 1997 Lear and others 1998b Hirotaand others 1997 Hirotaand others 1997 Gu and others 1992 Tozaki and others 1995 Breen and others 1997 Breen and others 1997 Lear and others 1997 Lear and others 1997 Godard and others 1997 Godard and others 1997 Lear and others 1998b Breen and others 1997 Breen and others 1997 Godard and others 1997 10p13 10q21->q23 11q12 11pl4 11p12 11 pi .1 12q13 12p13 1 3qter 13q13->q15 13q12 13q11->q12 13p1.2 14 15q21 15q21.3->q23 15q21.3->q23 15q24 15q23 16q14->q16 ASB6 ASB9 SGCV13 SGCV22 SGCV24 ECA10 IGF2 SGCV10 HBA ASB16 SGCV03 ASB37 #2->33 None ASB15 ASB19 ASB2 SGCV06 SGCV21 ASB7 Breen and others 1997 Millon and others 1993 Lear and others 1997 Hirota and others 1997 Hirota and others 1997 Bailey and others 1997 Breen and others 1997 Breen and others 1997 Godard and others 1997 Godard and others 1997 Godard and others 1997 Hirota and others 1997 Harbitz and others 1990 Chowdhary and others 1992 Breen and others 1997 Breen and others 1997 Godard and others 1997 Godard and others 1997 Godard and others 1997 Hirotaand others 1997 Raudsepp and others 1997 Godard and others 1997 Oakenfull and others 1993 Breen and others 1997 Godard and others 1997 Lear and others 1998b Hirota and others 1997 Breen and others 1997 Breen and others 1997 Breen and others 1997 Godard and others 1997 Godard and others 1997 Breen and others 1997 173 TABLE 2 {Continued) Chromosome Markers Reference 16q24->q25 17 18q21 18q2.3->q2.4 19q21 19 19q22->q23 19q2.6 19q2.7->q2.8 2Oq14->q22 HP7.2 None SGCV07 ECA7 SGCV01 SGCV08 ASB25 ECA4 ECA9 MHC Learetal. 1998b 20q2.2 21q13 21q13 22q19 23q19 24q15 25 26q17->q18 26q17->q18 27 28 ECA8 SGCV14 SGCV16 SGCV19 SGCV04 HP13. None MX ETS None None None None ER 29 30 31q Xq2.8 #4->6 Godard and others 1997 Hirota and others 1997 Godard and others 1997 Godard and others 1997 Lear and others 1998b Hirota and others 1997 Hirota and others 1997 Ansari and others 1988; Makinen and others 1989 Hirota and others 1997 Godard and others 1997 Godard and others 1997 Godard and others 1997 Godard and others 1997 Lear and others 1998b Lear and others 1998a Lear and others 1998a Lear and others 1997 Hirota and others 1997 With the advent of FISH and cloning of horse genes, a total of 73 genes have been mapped to chromosomes as indicated in Table 2. Many of these mapped markers were microsatellite DNA sequences cloned into cosmid or lambda vectors. These markers are useful because they can be used to integrate the physical and linkage maps (Breen and others 1997; Godard and others 1997). Mapping genes encoding proteins is useful to compare the organization of the horse map with the maps for other species (O'Brien and others 1993). ZOO-FISH The first comprehensive genome map for the horse was a "comparative map" reported in 1996 (Raudsepp and others 1996). These scientists used the technique of ZOO-FISH (also known as chromosome painting) to identify regions of homology between the human and horse genome. The results are summarized in Table 3. Based on their study, the number of chromosome rearrangements that occurred since divergence of the common ancestor of people and horses was relatively limited, especially when compared with the large number of rearrangements found between the mouse and human genomes. For example, chromosome 9 of the horse appears to correspond entirely to the genome for hu174 TABLE 3 Conserved chromosomal segments detected on equine metaphase chromosomes after zoological fluorescence in situ hybridization (ZOOFISH) with individual human chromosome-specific libraries (CSLs). None of the human chromosome paints hybridized to ECA6p, ECA12, ECA13p, ECA27, ECA31,orECA-Ya Human CLS for chromosome no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Conserved horse chromosome segments revealed by ZOO-FISH 2p, 5, 30 l q , 15, 18 16, 19 2q, 3q 14, 21 10q, 20 4 9 23,25 1p, 29 7p, q 1q, 6q, 8p, 26 17 1qter, 24 iq 3p, 13q 11 8q 10p, 7qcent 22 28 1 pter, 8pter, 26ter X a Adapted from Raudsepp and others 1996. man chromosome 8. However, the resolution of this technique does not detect inversions within chromosome regions or translocations of small chromosome segments. Although most of the ZOO-FISH assignments are being confirmed by subsequent physical mapping studies in other laboratories, a few discrepancies have been found and will be the subject of future reports. Regardless of future clarification and refinements to this map, this work was a major stimulus for research and a major landmark for gene mapping. Synteny Mapping Somatic cell hybrid panels can be a powerful tool for gene mapping research. This tool is based on hybridization of horse lymphocytes with a mouse cell line, subsequent random and partial loss of horse chromosomes during culture to produce sets of unique cell lines, each with a different subset of horse chromosomes. The distribution of 2 genes compared across a large number (20 to 80) of these lines will indicate whether the genes occur on the same or different ILAR Journal chromosomes. If 2 genes are present on the same chromosome, they will tend to have the same distribution among the cell lines; if they are on different chromosomes, then their distribution will appear unrelated. Two reports have described the development of synteny mapping panels for the horse, identifying several new synteny groups including biochemical markers (Williams and others 1993) and microsatellite markers (Bailey and others 1995). Recently, a third synteny panel has been developed and used to develop a comprehensive synteny map for the horse composed of microsatellite markers, randomly amplified polymorphic DNA markers, and DNA-based markers for genes encoding proteins (Shiue and others 1998). This report identified synteny groups associated with 21 of the horse chromosomes and evidence that 12 other synteny groups were associated with the remaining 12 chromosomes. Synteny mapping provided a rapid and inexpensive method to map markers to chromosomes and confirm or predict other physical or linkage map assignments. SCIENTIFIC CONTRIBUTIONS OF THE MAP Use of the newly invented horse gene map has been limited to date. Bailey and coworkers (1997) used markers developed for the gene map in a genome screening study to investigate linkage with the gene causing severe equine combined immunodeficiency disease (SCID1) in horses. That study identified microsatellite marker HTG-8 as being closely linked to the SCID gene. Synteny mapping demonstrated that a candidate gene identified by Wiler and coworkers (1995), DNA-PK, was syntenic to HTG-8; a phage clone containing part of the DNA-PK gene was mapped to equine chromosome 9 using FISH (Bailey and others 1997). Subsequently, Shin and coworkers (1997) identified a 5-base deletion in the DNA-PK gene that was responsible for SCID in horses. ANTICIPATED FUTURE CONTRIBUTIONS OF THE MAP Genetics is important to horse breeders who continue to select characteristics of the horse related to gait, behavior, size, color, and performance. Horses have changed over the last 50 years. Modern horses run faster than those of previous generations, although some controversy exists in this area. Gaffney and Cunningham (1988) noted that record-breaking performances at classic thoroughbred races have become increasingly less common. At the same time, they found that horses, on average, continued to exhibit genetic gain for racing performance. However, although breeders have effectively selected for performance, many horses do not perform because of health problems (Jeffcott and others 1982). For example, chondrodysplasias (developmental bone diseases) are thought to have a significant hereditary component and to affect 10 to 25% of horses across a wide range of breeds Volume 39, Numbers 2 and 3 1998 (Jeffcott 1996). Limited and usually indirect selection is applied to hereditary aspects of developmental bone diseases, allergic diseases, infectious diseases, and behavior in horses. As noted above, health problems are important to horse performance, but horse breeders do not select for health at the expense of selecting for performance. Therefore, genetic studies on diseases of the horse will likely be the primary focus of genomics research in horses. USES OF THE MAP AND ACCESSIBILITY The order Perissodactyla includes species of rhinoceros and tapirs as well as the members of the genus Equus. The evolution of the Equidae has been 1 of the standard models used for education in biology. The horse gene map will provide tools for investigations of phylogeny within Perissodactyla, providing valuable information about the process of evolution. Furthermore, the addition of the horse gene map to the developing comparative map information for diverse species is important because this species is the best characterized member of Perissodactyla to date. CONCLUSION The horse industry is very important to the economies of many countries of the world through the use of horses for recreational riding, showing, and racing. Genetics is important to horsemanship, and a gene map for the horse will be useful not only for investigating performance characteristics but also, more importantly, for understanding hereditary aspects of health in horses. Although limited funds delayed the start of gene mapping research for the horse, current work on the gene map of the horse at the time of this writing is based on collaborative activities of more than 25 laboratories associated with the International Equine Gene Mapping Workshop and through independent research at those laboratories. Linkage, physical, and comparative maps are under construction using diverse techniques and resources, including reference families, synteny mapping, FISH, and ZOO-FISH. More specific information about the map is available through 2 World Wide Web databases. ACKNOWLEDGMENTS The International Workshop was supported by the Dorothy Russell Havemeyer Foundation, Inc. Work at the Animal Health Trust was supported by the Horse Racing Levy Board and the Childwick Trust. Work at the Gluck Equine Research Center was supported by Fares Farms, Inc., The Grayson-Jockey Club Research Foundation, Inc., the Morris Animal Foundation, Inc., and Invitrogen Corporation. This report (98-14-37) was prepared in connection with a project of the University of Kentucky Agricultural Experiment Station. 175 REFERENCES Ansari HA, Hediger R, Fries R, Stranzinger G. 1988. Chromsomal localization of the MHC of the horse by in situ hybridization. Immunogenetics 28:362-364. 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