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We constructed a 400K WG tiling oligoarray for the horse and applied it for the discovery of copy number variations (CNVs) in 38 normal horses of 16 diverse breeds, and the Przewalski horse. Probes on the array represented 18,763 autosomal and X-linked genes, and intergenic, sub-telomeric and chrY sequences. We identified 258 CNV regions (CNVRs) across all autosomes, chrX and chrUn, but not in chrY. CNVs comprised 1.3% of the horse genome with chr12 being most enriched. American Miniature horses had the highest and American Quarter Horses the lowest number of CNVs in relation to Thoroughbred reference. The Przewalski horse was similar to native ponies and draft breeds. The majority of CNVRs involved genes, while 20% were located in intergenic regions. Similar to previous studies in horses and other mammals, molecular functions of CNV-associated genes were predominantly in sensory perception, immunity and reproduction. The findings were integrated with previous studies to generate a composite genome-wide dataset of 1476 CNVRs. Of these, 301 CNVRs were shared between studies, while 1174 were novel and require further validation. Integrated data revealed that to date, 41 out of over 400 breeds of the domestic horse have been analyzed for CNVs, of which 11 new breeds were added in this study. Finally, the composite CNV dataset was applied in a pilot study for the discovery of CNVs in 6 horses with XY disorders of sexual development. A homozygous deletion involving AKR1C gene cluster in chr29 in two affected horses was considered possibly causative because of the known role of AKR1C genes in testicular androgen synthesis and sexual development. While the findings improve and integrate the knowledge of CNVs in horses, they also show that for effective discovery of variants of biomedical importance, more breeds and individuals need to be analyzed using comparable methodological approaches.

We report a high-quality draft sequence of the genome of the horse (Equus caballus). The genome is relatively repetitive but has little segmental duplication. Chromosomes appear to have undergone few historical rearrangements: 53% of equine chromosomes show conserved synteny to a single human chromosome. Equine chromosome 11 is shown to have an evolutionary new centromere devoid of centromeric satellite DNA, suggesting that centromeric function may arise before satellite repeat accumulation. Linkage disequilibrium, showing the influences of early domestication of large herds of female horses, is intermediate in length between dog and human, and there is long-range haplotype sharing among breeds.

Chemokine receptors (CCRs) play an essential role in the initiation of an innate immune host response. Several of these receptors have been shown to modulate the outcome of viral infections. The recent availability of complete genome sequences from a number of species provides a unique opportunity to analyze the evolution of the CCR genes. A phylogenetic analysis revealed that the CCR2 gene evolved in concert with the paralogous CCR5 gene, but not with another paralogous gene, CCR3, in the opossum, platypus, rabbit, guinea pig, cat, and rodent lineages. In addition, evidence of concerted evolution of the CCR2 and CCR5 genes was observed in chicken and lizard genomes. A unique CCR5/2 gene that originated by unequal crossing over between the CCR2 and CCR5 genes was detected in the domestic horse. The CCR2, CCR5, and CCR5/2 genes were mapped to ECA16q21 using fluorescent in situ hybridization (FISH). Single-nucleotide polymorphisms identified in the equine CCR5 gene and characterized within 5 horse breeds provide haplotype markers for future case/control studies investigating the genetic bases of horse susceptibility to infectious diseases.

Telomeres, specialized structures present at the ends of linear eukaryotic chromosomes, function to maintain chromosome stability and integrity. Telomeres shorten with each cell division eventually leading to replicative senescence, a process thought to be associated with age-related decline in immune function. We hypothesized that shortened PBMC telomere length is a factor contributing to immunosenescence of the aged horse. Telomere length was assessed in 19 horses ranging in age from 1 to 25 years. Mitogen-induced 3H-thymidine incorporation, total serum IgG, and pro-inflammatory cytokine expression was also determined for each horse. Relative telomere length (RTL) was highly correlated with overall age. RTL was positively correlated with 3H-thymidine incorporation and total IgG. Expression of pro-inflammatory cytokines was negatively correlated with RTL. These measures were also correlated with age, as expected. However, RTL was not correlated with immunosenescence and inflammaging in the oldest horse.

Background: The mammalian OAS/RNASEL pathway plays an important role in antiviral host defense. A premature stop-codon within the murine Oas1b gene results in the increased susceptibility of mice to a number of flaviviruses, including West Nile virus (WNV). Mutations in either the OAS1 or RNASEL genes may also modulate the outcome of WNV-induced disease or other viral infections in horses. Polymorphisms in the human OAS gene cluster have been previously utilized for case-control analysis of virus-induced disease in humans. No polymorphisms have yet been identified in either the equine OAS1 or RNASEL genes for use in similar case-control studies., Results: Genomic sequence for equine OAS1 was obtained from a contig assembly generated from a shotgun subclone library of CHORI-241 BAC 100I10. Specific amplification of regions of the OAS1 gene from 13 horses of various breeds identified 33 single nucleotide polymorphisms (SNP) and two microsatellites. RNASEL cDNA sequences were determined for 8 mammals and utilized in a phylogenetic analysis. The chromosomal location of the RNASEL gene was assigned by FISH to ECA5p17-p16 using two selected CHORI-241 BAC clones. The horse genomic RNASEL sequence was assembled. Specific amplification of regions of the RNASEL gene from 13 horses identified 31 SNPs., Conclusion: In this report, two dinucleotide microsatellites and 64 single nucleotide polymorphisms within the equine OAS1 and RNASEL genes were identified. These polymorphisms are the first to be reported for these genes and will facilitate future case-control studies of horse susceptibility to infectious diseases.

The structures of the canine, rabbit, bovine and equine EIF2AK2 genes were determined. Each of these genes has a 5' non-coding exon as well as 15 coding exons. All of the canine, bovine and equine EIF2AK2 introns have consensus donor and acceptor splice sites. In the equine EIF2AK2 gene, a unique single nucleotide polymorphism that encoded a Tyr329Cys substitution was detected. Regulatory elements predicted in the promoter region were conserved in ungulates, primates, rodents, Afrotheria (elephant) and Insectifora (shrew). Western clawed frog and fugu EIF2AK2 gene sequences were detected in the USCS Genome Browser and compared to those of other vertebrate EIF2AK2 genes. A comparison of EIF2AK2 protein domains in vertebrates indicates that the kinase catalytic domains were evolutionarily more conserved than the nucleic acid-binding motifs. Nucleotide substitution rates were uniform among the vertebrate sequences with the exception of the zebrafish and goldfish EIF2AK2 genes, which showed substitution rates about 20% higher than those of other vertebrates. FISH was used to physically assign the horse and cattle genes to chromosome locations, ECA15q24-q25 and BTA11q12-15, respectively. Comparative mapping data confirmed conservation of synteny between ungulates, humans and rodents.

A first-generation radiation hybrid (RH) map of the equine (Equus caballus) genome was assembled using 92 horse x hamster hybrid cell lines and 730 equine markers. The map is the first comprehensive framework map of the horse that (1) incorporates type I as well as type II markers, (2) integrates synteny, cytogenetic, and meiotic maps into a consensus map, and (3) provides the most detailed genome-wide information to date on the organization and comparative status of the equine genome. The 730 loci (258 type I and 472 type II) included in the final map are clustered in 101 RH groups distributed over all equine autosomes and the X chromosome. The overall marker retention frequency in the panel is approximately 21%, and the possibility of adding any new marker to the map is approximately 90%. On average, the mapped markers are distributed every 19 cR (4 Mb) of the equine genome--a significant improvement in resolution over previous maps. With 69 new FISH assignments, a total of 253 cytogenetically mapped loci physically anchor the RH map to various chromosomal segments. Synteny assignments of 39 gene loci complemented the RH mapping of 27 genes. The results added 12 new loci to the horse gene map. Lastly, comparison of the assembly of 447 equine genes (256 linearly ordered RH-mapped and additional 191 FISH-mapped) with the location of draft sequences of their human and mouse orthologs provides the most extensive horse-human and horse-mouse comparative map to date. We expect that the foundation established through this map will significantly facilitate rapid targeted expansion of the horse gene map and consequently, mapping and positional cloning of genes governing traits significant to the equine industry.

The U2 linkage group of horses includes the genes albumin (ALB), vitamin D binding protein (GC), mitochondrial glutamate oxaloacetate transaminase 2 (GOT2), and haptoglobin (HP) which are found on two human chromosomes, namely, 4 (HSA 4) and 16 (HSA 16). Likewise these genes are also found on two different chromosomes in mice, rats, and cattle. Chromosome painting demonstrated that only horse chromosome 3 (ECA 3) hybridized with whole chromosome paints for both HSA 4 and HSA 16. This indicated that the equine U2 linkage group occurs on ECA 3, spanning the centromere. This technique will be useful to study the chromosome rearrangements associated with speciation of the genus Equus.