Animal Reproduction Science 107 (2008) 208218

Potential applications of equine genomics in

dissecting diseases and fertility
Bhanu P. Chowdhary , Nandina Paria, Terje Raudsepp
Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences,
Texas A&M University, College Station, TX 77843-4458, USA
Available online 29 April 2008

Abstract
Following the recent development of high-resolution gene maps and generation of several basic tools and
resources to use them in analyzing traits that are economically important to horse owners, genome analysis
in horses is witnessing a shift towards developing an ability to analyze complex traits. The likelihood of this
happening in the very near future is great, mainly because of the recent availability of the whole genome
sequence in the horse. The latter has triggered the development of novel tools like SNP-chip and expression
arrays that will permit rapid genome-wide analysis. While these tools will be used for a range of multifactorial disease traits, attempts are underway to develop focused tools that can target reproduction, fertility
and sex determination. For this, a catalog of sex and reproduction related (SRR) genes is being developed in
horses. A recently developed dense map of the horse Y chromosome will provide genes that are expressed
exclusively in males and, therefore, have an impact on stallion fertility. Overall, these advances in equine
genome analysis hold promise for improved diagnosis and treatment of various conditions in horses.
2008 Elsevier B.V. All rights reserved.
Keywords: Horse; Gene maps; Diseases; Reproduction; Fertility

1. Analysis of the equine genome: brief overview of the current status

Knowledge regarding the organization of equine genome has exponentially increased during
the past decade. From less than 50 markers mapped by 1997, today the horse gene map contains
5000 mapped markers, the majority of which are physically ordered, and all are assigned to
This paper is part of the Special issue entitled Proceedings of the 5th International Symposium on Stallion Reproduction, Guest Edited by Terttu Katila.
Corresponding author. Tel.: +1 979 458 0519; fax: +1 979 845 9972.
E-mail address: bchowdhary@cvm.tamu.edu (B.P. Chowdhary).

0378-4320/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2008.04.010

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209

specific chromosomes. This remarkable improvement in mapping the genome is attributed to the
development of several novel tools and resources and implementation of a range of advanced
mapping techniques in recent years. The nuclear genome of the horse is compacted in 32 pairs
of chromosomes (2n = 64) of which 31 pairs are autosomes and the remaining two are sex chromosomes (XX in the females and XY in the males). Of the autosomes, 13 pairs are meta- or
submeta-centric and the remaining are acrocentric. High-resolution gene maps for all chromosomes (autosomes as well as sex chromosomes) have been developed. It is noteworthy that horse is
the only domesticated animal species where a detailed map of the Y chromosome is also available.
Like with genome mapping projects in other species, application of a combination of meiotic,
synteny, cytogenetic and radiation hybrid mapping techniques resulted in the development of
a comprehensive map of the equine genome. Though the use of the horse mouse somatic cell
hybrid (SCH) panels was discontinued in year 2000, it had already contributed in a significant way
by assigning 500 markers to various syntenic groups that were mapped to almost all autosomes
and the X chromosome (reviewed by Chowdhary and Raudsepp, 2008). Two reference families
the International Horse Reference Family Panel (IHRFP) comprised of half sib families (Guerin
et al., 1999) and the Animal Health Trust (AHT) family panel comprised of three-generation
full-sibs (Swinburne et al., 2000) are primary contributors to the horse meiotic maps. The latest
iterations of maps generated using these resources show that the AHT map contains 742 markers
(734 microsatellites and eight gene based markers) that are arranged into 32 linkage groups
mapped to individual autosomes and the X chromosome. The markers are spaced on average at
3.7 centiMorgans (cM) intervals and the total span of the map is 2772 cM. Compared to this,
the IHRFP (Penedo et al., 2005) contains 776 markers, of which 626 were linearly ordered, and
the remaining 140 markers were placed in bins to specific regions of the chromosomes. The total
length of the map is 3740 cM and the markers are distributed on average at 6.3 cM intervals.
Though the two maps share the majority of the markers, 200 markers are unique to each map.
The generation of a 5000rad horse hamster RH panel in 2001 and its use to develop a physical
and comparative map of ECA11 (Chowdhary et al., 2002) marked the beginning of a new era that
first provided a medium-density RH and comparative map of the horse genome (258 type I and
472 type II markers; Chowdhary et al., 2003) and eventually culminated in the development of
high-resolution maps for all autosomes and the X chromosome that jointly contain 4103 markers
(Raudsepp et al., unpublished results). The overall resolution of the map is one marker every
775 kilobases. The map integrates available genetic linkage (920 markers shared with the AHT
and IHRFP linkage maps) and RH mapping data into a physically ordered map for all equine
chromosomes except the Y chromosome. The assignment and orientation of various linkage
groups is strongly supported by 1144 fluorescence in situ hybridisation (FISH) mapped markers.
The comparative part of the map aligns 1904 equine loci with sequence information available for
eight diverse vertebrate species (human, chimp, cow, dog, mouse, rat, opossum, and chicken).
This high-resolution map is a valuable tool for the isolation of genes and/or markers associated
with economically important traits like disease resistance/susceptibility, growth, reproduction,
and performance (e.g., Ward et al., 2003; Klukowska-Rotzler et al., 2006).
Presently, 1150 loci have been mapped by fluorescence in situ hybridization to equine chromosomes, providing an average of one cytogenetically mapped marker every 2.5 Mb of the
genome. This extensive coverage has been extremely useful in accurate physical alignment and
orientation of various types of maps (synteny, genetic linkage, RH, etc.) to specific chromosomes
or chromosomal regions in the horse. A number of multicolor FISH on metaphase/pro-metaphase
chromosomes, interphase chromatin and mechanically stretched DNA fibers (Chowdhary et al.,
2003; Raudsepp et al., 2004) have also been conducted in the horse. These experiments have been

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pivotal in orienting and ordering closely located or tightly linked markers and central in developing a BAC contig map of the pseudoautosomal region (PAR, Raudsepp and Chowdhary, in press)
and the male-specific region of the Y (MSY; Raudsepp et al., 2004). The approach has also been
used to anchor and orient 18 unassigned supercontigs from the whole genome sequence, thus
assisting assembly of the horse genome (Wade et al., 2008).
The landmark comparison between horse and human chromosomes by Zoo-FISH (Raudsepp
et al., 1996; Chowdhary et al., 1998) laid the foundation for whole genome comparison between
equine and other mammalian genomes. The map was later verified and partially redefined by
horsehuman reciprocal painting. The comparisons were further refined through reverse chromosome painting (Yang et al., 2004) and development of improved comparative maps for individual
chromosomes as well as the whole genome (reviewed by Chowdhary and Raudsepp, 2008). More
recently, the second generation RH map used a total of 2000 gene-specific loci to finely align the
horse genome with the sequenced genomes of a range of mammals (Raudsepp et al., unpublished
results).
Though BAC contig maps over small chromosomal regions like the proximal one-quarter
of horse chromosome 21 (ECA21; Brinkmeyer-Langford et al. in press) and the major histocompatibility locus (ELA; Gustafson et al., 2003; Tallmadge et al., 2005) have been developed
in the recent past, a more comprehensive effort is presently underway to develop a physical
map of the entire horse genome based on a combination of fluorescent fingerprinting and end
sequencing of 180,000 BAC clones from the 11X coverage CHORI-241 equine BAC library
(http://www.tiho-hannover.de/einricht/zucht/hgp/index.htm).
The Broad Institute at MIT and Harvard collaborated with the International Equine
Genome Sequencing Consortium to sequence the female horse genome (The equine whole
genome sequence). Using the whole genome shotgun (WGS) approach, a 6.8X coverage
of the genome was obtained. The version 2 draft assembly contains on average N50 contig
size 112 kb and N50 supercontig size of 46 Mb for the estimated 2.68 Gb equine genome
(http://www.broad.mit.edu/ftp/pub/assemblies/mammals/horse/Equus2/). Roughly 92% of the
sequence has been ordered and oriented on the chromosomes using available genetic linkage
maps, the second generation RH map and FISH mapping data. The genome sequence is currently
being annotated by Ensembl.
2. Whole genome SNP-chip and expression oligoarrays: new tools for equine genome
The availability of the whole genome sequence has opened the door to the development of novel
powerful tools that could facilitate rapid analysis of the entire genome. One of these tools includes
the genome-wide SNP-chip that facilitates rapid mapping of diseases to specific chromosomal
region(s) and allows isolation of candidate genes. The Broad Institute at MIT and Harvard in
collaboration with the Equine Genome Sequencing Consortium has isolated single nucleotide
polymorphisms (SNPs) within the genome assembly of the sequenced female horse Twilight
and by comparing 100,000 WGS reads from each of the seven horse breeds including: Akhal
Teke, Icelandic, Arabian, Andalusian, Quarterhorse, Thoroughbred, and Standardbred. Using
funds from Morris Animal Foundation and USDA, a 60 K SNP-chip has been developed after
carefully analyzing over 1.5 million SNPs obtained from re-sequencing of specific regions
from various breeds. The chip will be available through Dr. James Mickelson at the University of
Minnesota. Next, the sequence of the equine genome has also been used by researchers at Texas
A&M University to develop a 21,000 element 70-mer oligoarray that represents the majority
of the expressed equine sequences. This array will be available through Dr. Bhanu Chowdhary

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Table 1a
Equine genetic disorders with available genetic tests
Trait/condition

Locus

Chromosome

Glycogen storage disease IV, GBED

Hereditary equine regional dermal asthenia, HERDA
Herlitz junctional epidermolysis bullosa, H-JEB
Hyperkalemic periodic paralysis, HYPP
Overo lethal white foal syndrome, OLWFS
Polysaccharide storage myopathy, PSSM
Severe combined immunodeficiency, SCID

GBE1
PPIB
LAMC2
SCN4A
EDNRB
GYS1
PRKDC

ECA26q
ECA1
ECA5p
ECA11p
ECA17q
ECA10
ECA9p

from April 2008. The array will allow analysis of gene expression patterns between affected
and unaffected individuals, and will lay the foundation to study the entire equine transcriptome
in pursuit of genes and genetic mechanisms governing normal and perturbed physiological and
developmental conditions. Overall, the two new tools will add a new dimension to equine genetics
research by permitting study of particularly complex traits for which current tools and resources
were insufficient.
3. Genomics and genetic diseases in horses some recent advances
3.1. Genetic disorders
Gene maps and comparative genomic approaches have largely contributed to the identification and analysis of genes governing monogenic traits including disorders of the immune system
(SCID, Shin et al., 1997), metabolic disorders in skeletal muscle (Rudolph et al., 1992; Ward et
al., 2003; Dranchak et al., 2007; McCue et al., 2008), hereditary skin abnormalities (HERDA,
Tryon et al., 2007), and disorders in brain (cerebellar abiotrophy, Brault et al., unpublished). In
recent years, the availability of gene maps and genome scanning panels has led to the mapping
of various diseases to specific chromosomal regions or bands through tightly linked markers
(Tables 1a and 1b). An example of such a condition is the susceptibility locus for a hereditary
eye disorder anterior segment dysgenesis that was mapped to ECA6 using linked microsatelTable 1b
Equine genetic disorders with known linked markers
Trait/condition

Locus

Chromosome

Anterior segment dysgenesis, ASD

Cerebellar abiotrophy
Degenerative suspensory ligament desmitis, DSLD
Endurance performance
Epitheliogenesis imperfecta
Fertility (sperm-egg fusion)
Insect bite hypersensitivity, IBH
Laminitis
Osteochondrosis and navicular disease
Recurrent airway obstruction
Recurrent exertional rhabdomyolysis, RER
Sex reversal

ms
DMAP1, PRNPIP
ms
ACE
LAMA3
CRISP1
HMS01
KIT downregulation
8 candidate genes, SNPs
ms
ms, gene exclusion
Y deletion including SRY

ECA6
ECA2p
ECA14qter
ECA11
ECA8
ECA20q
ECA15
ECA3
Five chromosomes
ECA13
ECA4/ECA12?
ECAY

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lite markers (Andersson et al., 2008). More recently, a genome scan panel was used on 500
Hannoverian horses that led to the mapping of the susceptibility loci for osteochondrosis and
navicular disease to five different equine chromosomes (Distl et al., 2008). Similarly, a candidate locus for recurrent airway obstruction was assigned to ECA13 (Jost et al., 2007). Further,
in a comparison between laminitis-affected horses and healthy controls, the likely involvement
of the KIT locus was evaluated (Brooks and Bailey, 2008). The gene was found to be 2-fold
downregulated in the affected horses. Presently, genes and mutations/variations within the genes
have been discovered for some diseases. Consequently, diagnostic tests are available for them
(see Tables 1a and 1b).
The past few years have witnessed a surge in the discovery of single nucleotide polymorphisms.
Consequently, a number of association studies have been carried out to establish relationship
between SNPs and diseases. For example, SNPs have been identified in LAMC2an insertion
mutation that causes Herlitz junctional epidermolysis in two French draft horse breeds (Milenkovic
et al., 2003); cyclophilin B (PPIB)known to carry a missense mutation in HERDA individuals
(Tryon et al., 2007); and SLC26A2a candidate gene for chondrodysplasia (Hansen et al., 2007).
Among the immune system genes related to susceptibility/resistance to various infectious diseases,
SNPs have been reported in IL4R (Solberg et al., 2004), TNFalpha (Brown et al., 2006), and OAS1
(Rios et al., 2007). Recently, Hamann et al. (2007) reported a SNP in CRISP3 that was considered
to have a significant effect on stallion fertility. Among the performance related genes, SNPs have
been identified and analyzed in potassium and amino acid transporters SLC12A4 and SLC7A10
(Hanzawa et al., 2002) and lactate transport genes MCT1 and CD147 (Reeben et al., 2006).
Many of the equine genetic disorders, however, are multigenic and have an environmental
component. New tools and resources like the whole genome sequence, SNP-chip, expression
arrays, etc., will permit a level of genome-wide analysis previously considered difficult if not
improbable. Study of a range of complex equine conditions that will benefit from these tools and
resources leading to improved ways for their prevention and treatment include chronic obstructive
pulmonary disease, sweet-itch, osteochondrosis dessicans, laminitis, exercise-induced pulmonary
hemorrhage, susceptibility and resistance to infectious diseases, inflammation, stress, performance
or athletic ability, endurance, exercise intolerance, conformation, behavior, fertility, etc.
4. Prospects to study the genetics of reproduction and fertility
4.1. Overview
Stallion and mare fertility are traits of paramount importance to horse breeders and to the entire
equine industry due to the huge economic impact they have on stud fees and foal crop. Several
organized studies have been conducted during past decades to understand the role of various
environmental, behavioral and physiological factors affecting horse fertility (Madill, 2002; Leeb
et al., 2005). However, very little is known about genetic factors and their role in regulating fertility.
It is estimated that from fertilization until sexual maturation, over 1000 genes spatiotemporally
control the development of mammalian testes and ovaries (Cederroth et al., 2007; Wilhelm et al.,
2007). Mutations in these genes can result in abnormal sexual development causing a range of
reproductive abnormalities that adversely affect fertility. Normal functioning of adult gonads is
regulated by additional 1000 genes. Overall, it is estimated that about 10% of all the genes are
involved in human and mouse male fertility (Carrell, 2007), however, much less is known about
genes associated with female reproduction (Barnett et al., 2006).

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4.2. Current status

Very few reproduction and fertility related genes have been analyzed in horses (Ing et al., 2004;
Leeb et al., 2005; Hamann et al., 2007) and even fewer have been analyzed for their function and
interactions. The remarkable progress made during the last decade to develop a high-resolution
map of the horse genome (Chowdhary et al., 2003, Raudsepp et al. unpublished results), recently
led to the sequencing of the genome (Wade et al., 2008). This resource provides a unique opportunity to initiate organized genome-wide search for sex and reproduction related (SRR) genes
in horses. A project is currently underway at Texas A&M to obtain a comprehensive catalogue
of these genes so that SNP and expression based formats could be developed to simultaneously
interrogate these genes to understand normal genetic regulation of fertility and sex determination,
as well as, to analyze the role of these genes in abnormal or disease conditions of the reproductive
system. The eventual goal of generating such formats will be to identify candidate genes and their
variations/mutations that are responsible for different reproduction related problems in mares and
stallions. While the whole genome sequence will provide access to a substantial proportion of
female and male fertility genes in the horse, it will not furnish any information on transcripts
encoding various genes residing on the Y chromosome (because the genome sequenced is from a
female individual), several of which are critical for male reproductive development and fertility.
Hence, obtaining information on the gene content of the Y chromosome will be essential to make
the collection of genes to study male fertility and sex determination comprehensive.
4.3. Y chromosome and male fertility
Recent studies in humans and mice have clearly shown that a significant proportion of the
genes controlling testicular development, spermatogenesis, sperm motility and other important
male reproductive functions are located on the Y chromosome (Toure et al., 2004; Ellis and Affara,
2006). The critical role of the Y chromosome in male fertility is further underscored by the fact
that about 1015% of idiopathic male infertility in humans is caused by mutations on the Y
chromosome and that the available genetic diagnostic tests to evaluate human male infertility are
exclusively based on Y chromosome markers (Peterlin et al., 2004). Hence studies are underway
to develop a complete map of the Y chromosome that could provide complete information on
the structure and function of male-specific genes and could also serve as a knowledgebase for
identifying genetic indicators that could be used to monitor and improve fertility in stallions.
4.4. A map of the horse Y chromosome
A comprehensive map of the euchromatic region of the horse Y chromosome was recently
developed. The map covers the pseudoautosomal region as well as the male-specific region of
the Y (Raudsepp et al., 2004; Raudsepp and Chowdhary, in press). The map of the equine PAR
has 129 physically ordered markers (110 STSs and 19 genes) contained in 71 BAC clones that
are arranged into two contigs spanning the region. Furthermore, the pseudoautosomal boundary
(PAB) has been precisely demarcated in the horse (Raudsepp and Chowdhary, in press). Following
human/chimp and mouse, horse is the only species where PAR is described in such details and
the PAB is defined. Interestingly, a 200 kb region was discovered in the middle of the PAR that
is also present in the male-specific region of the Y. Duplication like this is a novel observation in
mammals. Next, the first generation map of the horse Y chromosome (Raudsepp et al., 2004) has
been further expanded (our unpublished data). The current map incorporates 400 BAC clones

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Fig. 1. Schematic overview of the BAC contig map of the horse Y chromosome.

that are arranged into five contigs containing 300 sequence tagged sites (STSs) and 32 genespecific markers (Fig. 1). The existing four gaps do not exceed a total of 1 Mb as revealed by
interphase FISH analysis. Overall, the map lays an important foundation for complete sequencing
of the euchromatic region of the Y chromosome and to undertake discovery of genes important
for stallion fertility.
4.5. Application of the map to study sex reversal in horses
Sex reversal is a condition where genetic sex does not agree with the phenotypic sex, i.e. females
have a male chromosome constitution (64XY) and males have a female chromosome constitution
(XX). The condition is a concern for animal breeders/owners because the discrepancy between
phenotypic and genetic sex causes behavioral abnormalities and infertility (Pailhoux et al., 1995;
Makinen et al., 1999; Bugno et al., 2003). The development of a high-resolution map for the horse
Y chromosome (Raudsepp et al., 2004) has provided new tools (clones and markers) to study the
molecular causes of XY sex reversal syndrome in more detail. It appears that XY mares can be
both SRY-negative and SRY-positive (our preliminary observations). Molecular analysis of SRYnegative mares indicates that there is a deletion on their Y chromosome which involves not only the
SRY locus but at least six additional sequence tagged sites around it (Fig. 2, our unpublished data).
Ongoing complete sequencing of the BAC clones spanning this region is expected to demarcate
the actual boundaries of the deletion and reveal that in addition to SRY which other genes are
deleted.
Further, we recently identified two SRY-negative XY mares with unusually small Y chromosomes. Molecular analysis revealed that in one animal about 23 Mb from the proximal part of
male-specific Y is deleted, while in another the entire euchromatic region has been lost (Fig. 2).
Overall, regardless of the size of Y chromosome deletion, all SRY-negative mares lack the male sex
determination gene which explains their female phenotype. However, what causes SRY-positive
XY sex reversal condition, remains unclear. STS content analysis indicates that the Y chromosomes of these mares are intact and contain the same markers as Y chromosomes in normal males
(our unpublished observations). It is, therefore, likely that SRY-positive sex reversal is caused
by mutations in other genes involved in the complex cascade of gene interactions during sex
determination (Wilhelm et al., 2007).
4.6. Discovery of male fertility genes
Realizing the huge economic importance of stallion fertility in equine industry, targeted studies
of Y-specific genes in horses (ECA) were initiated which was aimed at isolating Y chromosome
genes in horses with a long-term goal to understand their role/function in regulating male fertility.
Direct cDNA capture (Lovett et al., 1991; Del Mastro and Lovett, 1997) from normal, adult horse

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215

Fig. 2. Varying degree of Y chromosome deletions found in SRY-negative mares. Top: Chromosomes from (A) an XY
mare with apparently normal Y chromosome where SRY gene is deleted, (B) XY mare where only the heterochromatic
region of the Y chromosome is retained (arrow); (C) XY mare where only a portion of the euchromatic region of the
Y chromosome is retained (arrow). Bottom: Schematic drawing of the horse Y chromosome showing heterochromatic
(striped) and euchromatic (solid grey) regions. The latter includes the male-specific region of the Y (MSY) and the
pseudoautosomal region (PAR); the two are separated by the pseudoautosomal boundary (PAB). A summary of the BAC
contig map over the euchromatic region is presented showing number of known genes in each contig.

testis using flow sorted Y chromosome and 183 Y-specific BAC clones as selectors identified
29 male-specific genes and transcripts of which 13 correspond to known mammalian Y-linked
genes, e.g., DBY, SMCY and SRY, six were retrotransposed from autosomal and mitochondrial
locations, and ten are novel and equine specific (unpublished data). All genes/transcripts were
physically ordered on the ECAY BAC contig map (Raudsepp et al., 2004) by STS content analysis
and FISH. Eleven of the transcripts, including the six novel genes, are ampliconic. Expression
analysis of all genes/transcripts on nine selected body tissues, viz., brain, kidney, heart, skeletal
muscle, liver, lungs, spleen, seminal vesicle and testis from normal adult horses showed that
19 of the transcripts had either ubiquitous (expressed in all tissues) or intermediate expression
(expressed in testis and some other tissues), and ten transcripts are exclusively expressed in testis.
The latter have multiple copies on the Y chromosome and are potential candidates for future
studies aimed at understanding the regulation of stallion fertility.
5. Conclusion
Recent developments in mapping and sequencing the equine genome offers possibilities to
study Mendelian and complex traits in ways that were not available earlier. Novel tools like the
60 K SNP-chip and expression microarray will lay the groundwork for several new studies aimed
at understanding genetic mechanisms regulating the normal biology of the horse and identifying genetic factors and their relative contribution in the causation and progression of diseases.
Concurrently, generating resources for study of complex disease traits (e.g. family material) and

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accurate definition of the diseases will be an important pre-requisite to initiate such studies.
While reproduction and fertility are complex traits that are influenced by environment to a varying extent, the tools and resources presently being developed hold promise for the identification
and analysis of genetic factors that are intrinsically involved in regulating them. Knowledge of
these factors will be critical in advancing our understanding regarding various reproduction and
fertility related problems in horses, and will be essential for devising improved diagnostic and
therapeutic approaches in future.
Conict of interest statement
None.
Acknowledgements
This work was supported by USDA-NRI grants 2006-04801, Link Endowment, Morris Animal
Foundation, Havemeyer Foundation and Grayson Jockey Club.
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