The equine ascarid parasite Parascaris equorum is well known as a ubiquitous parasite infecting foals. A sibling species, Parascaris univalens, was first described over 130 years ago, but very little attention has been given to its existence and possible implications for anthelmintic resistance, clinical disease, or host age spectrum. P. univalens only possesses one germ line chromosome pair as opposed to two for P. equorum, but the two species are otherwise considered morphologically identical. For the present study, live worms obtained from the University of Kentucky parasitology horse herd were dissected and identified using karyotyping techniques. With no exception, all specimens (n=30) were identified to be P. univalens. Further, the karyotyping technique was adapted to ascarid eggs derived from fecal samples and carried out on samples collected from 25 Thoroughbred foals from three farms in Central Kentucky. P. equorum was not identified among these, whereas P. univalens was found in 17 samples, with the remaining being inconclusive. The mitochondrial genome was sequenced, assembled, and annotated from one male worm identified as P. univalens, and comparison with available sequence reads labeled as P. equorum revealed only 0.16 % nucleotide differences. However, it is unlikely that the sequences available in public databases have been unequivocally identified to species level by karyotyping. Taken together, these data suggest that P. univalens is likely the main species now observed in equines and that perhaps the designation Parascaris spp. should be used unless cytological characterization has confirmed the species.

Horses, asses, and zebras belong to a single genus, Equus, which emerged 4.0-4.5 Mya. Although the equine fossil record represents a textbook example of evolution, the succession of events that gave rise to the diversity of species existing today remains unclear. Here we present six genomes from each living species of asses and zebras. This completes the set of genomes available for all extant species in the genus, which was hitherto represented only by the horse and the domestic donkey. In addition, we used a museum specimen to characterize the genome of the quagga zebra, which was driven to extinction in the early 1900s. We scan the genomes for lineage-specific adaptations and identify 48 genes that have evolved under positive selection and are involved in olfaction, immune response, development, locomotion, and behavior. Our extensive genome dataset reveals a highly dynamic demographic history with synchronous expansions and collapses on different continents during the last 400 ky after major climatic events. We show that the earliest speciation occurred with gene flow in Northern America, and that the ancestor of present-day asses and zebras dispersed into the Old World 2.1-3.4 Mya. Strikingly, we also find evidence for gene flow involving three contemporary equine species despite chromosomal numbers varying from 16 pairs to 31 pairs. These findings challenge the claim that the accumulation of chromosomal rearrangements drive complete reproductive isolation, and promote equids as a fundamental model for understanding the interplay between chromosomal structure, gene flow, and, ultimately, speciation.

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.

Chromosomal aberrations in the horse are known to cause congenital abnormalities, embryonic loss, and infertility. While diagnosed mainly by karyotyping and FISH in the horse, the use of SNP array comparative genome hybridization (SNP-CGH) is becoming increasingly common in human diagnostics. Normalized probe intensities and allelic ratios are used to detect changes in copy number genome-wide. Two horses with suspected chromosomal abnormalities and six horses with FISH-confirmed aberrant karyotypes were chosen for genotyping on the Equine SNP50 array. Karyotyping of the first horse indicated mosaicism for an additional small, acrocentric chromosome, although the identity of the chromosome was unclear. The second case displayed a similar phenotype to human disease caused by a gene deletion and so was chosen for SNP-CGH due to the ability to detect changes at higher resolutions than those achieved with conventional karyotyping. The results of SNP-CGH analysis for the six horses with known chromosomal aberrations agreed completely with previous karyotype and FISH analysis. The first undiagnosed case showed a pattern of altered allelic ratios without a noticeable shift in overall intensity for chromosome 27, consistent with a mosaic trisomy. The second case displayed a more drastic change in both values for chromosome 30, consistent with a complete trisomy. These results indicate that SNP-CGH is a viable method for detection of chromosomal aneuploidies in the horse.

Peripheral blood samples from a 13-year-old Thoroughbred mare were submitted for chromosome analysis. The mare had a poor reproduction record only producing four live foals during her 10 years as a broodmare. She had remained barren or experienced early embryonic loss in the other 6 years. Chromosomal analysis revealed the mare carried a rare nonreciprocal translocation involving chromosomes 2 and 13 [64,XX,t(2;13)]. Fluorescence in situ hybridization with probes specific for horse chromosomes 2 and 13 were used to confirm the nonreciprocal translocation. Both conventional and molecular cytogenetic techniques are important for identifying and characterizing chromosomal abnormalities in horses with poor reproductive performance, particularly in mares experiencing repeated early embryonic loss.

Toll-like receptors 3, 7, and 8 (TLR3, TLR7, and TLR8) were studied in the genomes of the domestic horse and several other mammals. The messenger RNA sequences and exon/intron structures of these TLR genes were determined. An equine bacterial artificial chromosome clone containing the TLR3 gene was assigned by fluorescent in situ hybridization to the horse chromosomal location ECA27q16–q17 and this map location was confirmed using an equine radiation hybrid panel. Direct sequencing revealed 13 single-nucleotide polymorphisms in the coding regions of the equine TLR 3, 7, and 8 genes. Of these polymorphisms, 12 were not previously reported. The allelic frequency was estimated for each single-nucleotide polymorphism from genotyping data obtained for 154 animals from five horse breeds. Some of these frequencies varied significantly among different horse breeds. Domain architecture predictions for the three equine TLR protein sequences revealed several conserved regions within the variable leucine-rich repeats between the corresponding horse and cattle TLR proteins. A phylogenetic analysis did not indicate that any significant exchanges had occurred between paralogous TLR7 and TLR8 genes in 20 vertebrate species analyzed.

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.

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.

Cytogenetic analyses of horses have benefited the horse industry by identifying chromosomal aberrations causing congenital abnormalities, embryonic loss and infertility. Technical advances in cytogenetics enabled the identification of chromosome specific aberrations. More recently, advances in genomic tools have been used to more precisely define chromosome abnormalities. In this report we review the history of equine clinical cytogenetics, identify historical landmarks for equine clinical cytogenetics, discuss how the current use of genomic tools has benefited this area, and how future genomics tools may enhance clinical cytogenetic studies in the 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.

The availability of a contig of bacterial artificial chromosome (BAC) clones spanning the equine major histocompatibility complex (MHC) made possible a detailed analysis of horse MHC class I genes. Prior to this study, only a single horse MHC class I gene had been sequenced at the genomic level. Although many (∼60) MHC class I cDNA sequences had been determined and published, from this information, it was not possible to determine how many class I loci are expressed in horses or to assign individual sequences to allelic series. In this study, 15 MHC class I genes were identified in BAC subclones and fully sequenced. Because the BAC library donor horse had been bred for homozygosity at the MHC, these 15 genomic clones represent distinct MHC class I genes and pseudogenes and not alleles at a smaller number of loci. For five of the genes, cDNA sequences from these loci had previously been identified. Two additional expressed class I genes were discovered, bringing the known total of different equine MHC class I genes (loci) expressed as mRNA to seven. Expression of all seven loci was detected by reverse transcriptase–polymerase chain reaction in adult, fetal, and placental tissues. The remaining eight genes were designated as pseudogenes. This work resulted in moderate expansion of the horse MHC BAC contig length, and the remaining gap was shortened. The information contained in these equine MHC class I sequences will permit comparison of MHC class I genes expressed across different horse MHC haplotypes and between horses and other mammalian species.

A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of the coding sequence similar to those of the beta(2)-m gene of other species. The beta(2)-m gene was localized to horse chromosome ECA1q23-q25 by fluorescent in situ hybridization. This was confirmed by synteny mapping on a (horse x mouse) somatic cell hybrid panel. The sequence and intron/exon boundaries determined were used to design PCR primers to amplify and sequence the coding region of the beta(2)-m gene in other equids, including five breeds of domestic horse, one Przewalski's horse, five domestic donkeys and five zebras. A high degree of conservation was found among equids, illustrated by >98% (349/354) identity at the nucleotide level and 95% (113/118) at the amino acid level, because of non-synonymous nucleotide substitutions. The promoter detected in the region upstream of the coding sequence was subcloned and used in chloramphenicol acetyl transferase (CAT) assays to demonstrate the presence of a functional promoter. This study provides tools for the analysis of regulation of not only the horse beta(2)-m gene, but also for any genes dependent upon beta(2)-m for expression.

The aim of this study was to increase the number of type I markers on the horse cytogenetic map and to improve comparison with maps of other species, thus facilitating positional candidate cloning studies. BAC clones from two different sources were FISH mapped: homologous horse BAC clones selected from our newly extended BAC library using consensus primer sequences and heterologous goat BAC clones. We report the localization of 136 genes on the horse cytogenetic map, almost doubling the number of cytogenetically mapped genes with 48 localizations from horse BAC clones and 88 from goat BAC clones. For the first time, genes were mapped to ECA13p, ECA29, and probably ECA30. A total of 284 genes are now FISH mapped on the horse chromosomes. Comparison with the human map defines 113 conserved segments that include new homologous segments not identified by Zoo-FISH on ECA7 and ECA13p.

BLAST searches of 61 equine microsatellite sequences revealed two related families of retroposons. The first family included seven markers, all of which showed significant homology to the Equine Repetitive Element-1 (ERE-1) Short Interspersed Nucleotide Element (SINE) sequence. Length of homology ranged from 76 to 171 bases with identities to the ERE-1 consensus sequence ranging from 71% to 83%. The second family referred to as Equine Repetitive Element-2 (ERE-2) has a consensus sequence that showed homology to ERE-1 over approximately 60 bases. These 60 bases comprised subunit I. Sequence comparisons for the two retroposons led to the identification of a subunit II, subunit III, as well as the tRNAser subunit. The subunit structure of ERE-1 was tRNAser-I-II. By contrast, the subunit structure of ERE-2 was I-III-III. The nine markers related to ERE-2 showed homology lengths ranging from 84 to 163 bases with identities ranging from 75% to 99%. In addition to being present in microsatellites, ERE-2 appeared in three separate equine genes. It occurred in an intron of DNA-PK, in an untranslated region as well as in the promoter of PGHS, and in the coding region of PAM. The amino acids corresponding to the ERE-2 sequence in PAM were not present in the human or mouse PAM homologs. These amino acids associated with the ERE-2 sequence were present on the cytosolic side of the transmembrane domain of the PAM enzyme. Microsatellite markers in the ERE-1 and ERE-2 families were found throughout the genus equus and also for rhinoceros, indicating that the appearance of both retroposons predates the divergence of equids from the other perissodactyls. The markers did not amplify in human or bovine DNA. This indicated that ERE-1 and ERE-2 are, at least, perissodactyl specific.