Lavender Foal Syndrome (LFS) is a lethal inherited disease of horses with a suspected autosomal recessive mode of inheritance. LFS has been primarily diagnosed in a subgroup of the Arabian breed, the Egyptian Arabian horse. The condition is characterized by multiple neurological abnormalities and a dilute coat color. Candidate genes based on comparative phenotypes in mice and humans include the ras-associated protein RAB27a (RAB27A) and myosin Va (MYO5A). Here we report mapping of the locus responsible for LFS using a small set of 36 horses segregating for LFS. These horses were genotyped using a newly available single nucleotide polymorphism (SNP) chip containing 56,402 discriminatory elements. The whole genome scan identified an associated region containing these two functional candidate genes. Exon sequencing of the MYO5A gene from an affected foal revealed a single base deletion in exon 30 that changes the reading frame and introduces a premature stop codon. A PCR–based Restriction Fragment Length Polymorphism (PCR–RFLP) assay was designed and used to investigate the frequency of the mutant gene. All affected horses tested were homozygous for this mutation. Heterozygous carriers were detected in high frequency in families segregating for this trait, and the frequency of carriers in unrelated Egyptian Arabians was 10.3%. The mapping and discovery of the LFS mutation represents the first successful use of whole-genome SNP scanning in the horse for any trait. The RFLP assay can be used to assist breeders in avoiding carrier-to-carrier matings and thus in preventing the birth of affected foals.

Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3 ؉ T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3؉ T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a resistant CD3 ؉ T lymphocyte phenotype using both Illumina Equine SNP50 BeadChip and Sequenom's MassARRAY system identified a common, genetically dominant haplotype associated with the susceptible phenotype in a region of equine chromosome 11 (ECA11), positions 49572804 to 49643932. The presence of a common haplotype indicates that the trait occurred in a common ancestor of all four breeds, suggesting that it may be segregated among other modern horse breeds. Biological pathway analysis revealed several cellular genes within this region of ECA11 encoding proteins associated with virus attachment and entry, cytoskeletal organization, and NF-B pathways that may be associated with the trait responsible for the in vitro susceptibility/resistance of CD3 ؉ T lymphocytes to EAV infection. The data presented in this study demonstrated a strong association of genetic markers with the trait, representing de facto proof that the trait is under genetic control. To our knowledge, this is the first GWAS of an equine infectious disease and the first GWAS of equine viral arteritis.Equine arteritis virus (EAV) is a small enveloped virus with a positive-sense, single-stranded RNA genome of 12.7 kb and belongs to the family Arteriviridae (genus Arterivirus, order Nidovirales) (16,64). EAV is the causal agent of equine viral arteritis (EVA), a disease of equids (12, 13). The vast majority of EAV infections are inapparent or subclinical (5, 6, 68). However, some acutely infected horses may develop any combination of the following clinical signs: pyrexia, depression, anorexia, dependent edema (scrotum, ventral trunk, and limbs), conjunctivitis, lacrimation and swelling around the eyes (periorbital or supraorbital edema), respiratory distress, urticaria, and leukopenia (5, 6). During natural outbreaks of the disease, the virus can cause abortion in pregnant mares, with abortion rates varying from 10 to 71%, depending on the virus strain (5, 6). Following EAV infection, a variable proportion of stallions (30 to 70%) can become persistently infected and continuously shed the virus in their semen (55,68). The mechanism of persistence of EAV in the male reproductive tract is not clear. However, studies have established that persistence of EAV in stallions is testosterone dependent (42,52). Moreover, the prevalences of EAV infection differ markedly among different breeds of horses (68), strengthening the assumption of genetic influence on susceptibili...

Genetic markers are important resources for individual identification and parentage assessment. Although short tandem repeats (STRs) have been the traditional DNA marker, technological advances have led to single nucleotide polymorphisms (SNPs) becoming an attractive alternative. SNPs can be highly multiplexed and automatically scored, which allows for easier standardization and sharing among laboratories. Equine parentage is currently assessed using STRs. We obtained a publicly available SNP dataset of 729 horses representing 32 diverse breeds. A proposed set of 101 SNPs was analyzed for DNA typing suitability. The overall minor allele frequency of the panel was 0.376 (range 0.304-0.419), with per breed probability of identities ranging from 5.6 × 10 to 1.86 × 10 . When one parent was available, exclusion probabilities ranged from 0.9998 to 0.999996, although when both parents were available, all breeds had exclusion probabilities greater than 0.9999999. A set of 388 horses from 35 breeds was genotyped to evaluate marker performance on known families. The set included 107 parent-offspring pairs and 101 full trios. No horses shared identical genotypes across all markers, indicating that the selected set was sufficient for individual identification. All pairwise comparisons were classified using ISAG rules, with one or two excluding markers considered an accepted parent-offspring pair, two or three excluding markers considered doubtful and four or more excluding markers rejecting parentage. The panel had an overall accuracy of 99.9% for identifying true parent-offspring pairs. Our developed marker set is both present on current generation SNP chips and can be highly multiplexed in standalone panels and thus is a promising resource for SNP-based DNA typing.

Champagne coat color in horses is controlled by a single, autosomal-dominant gene (CH). The phenotype produced by this gene is valued by many horse breeders, but can be difficult to distinguish from the effect produced by the Cream coat color dilution gene (CR). Three sires and their families segregating for CH were tested by genome scanning with microsatellite markers. The CH gene was mapped within a 6 cM region on horse chromosome 14 (LOD = 11.74 for θ = 0.00). Four candidate genes were identified within the region, namely SPARC [Secreted protein, acidic, cysteine-rich (osteonectin)], SLC36A1 (Solute Carrier 36 family A1), SLC36A2 (Solute Carrier 36 family A2), and SLC36A3 (Solute Carrier 36 family A3). SLC36A3 was not expressed in skin tissue and therefore not considered further. The other three genes were sequenced in homozygotes for CH and homozygotes for the absence of the dilution allele (ch). SLC36A1 had a nucleotide substitution in exon 2 for horses with the champagne phenotype, which resulted in a transition from a threonine amino acid to an arginine amino acid (T63R). The association of the single nucleotide polymorphism (SNP) with the champagne dilution phenotype was complete, as determined by the presence of the nucleotide variant among all 85 horses with the champagne dilution phenotype and its absence among all 97 horses without the champagne phenotype. This is the first description of a phenotype associated with the SLC36A1 gene.

White spotting phenotypes in horses are highly valued in some breeds. They are quite variable and may range from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for white spotting phenotypes in horses. For the present study, we investigated an American Paint Horse family segregating a phenotype involving white spotting and blue eyes. Six of eight horses with the white-spotting phenotype were deaf. We obtained whole-genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous~63-kb deletion spanning exons 6-9 of the MITF gene (chr16:21 503 211-21 566 617). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. PCR-based genotyping revealed that all eight available affected horses from the family carried the deletion. The finding of an MITF variant fits well with the syndromic phenotype involving both depigmentation and an increased risk for deafness and corresponds to human Waardenburg syndrome type 2A. Our findings will enable more precise genetic testing for depigmentation phenotypes in horses.

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SummaryExtreme lordosis, also called swayback, lowback or softback, can occur as a congenital trait or as a degenerative trait associated with ageing. In this study, the hereditary aspect of congenital swayback was investigated using whole genome association studies of 20 affected and 20 unaffected American Saddlebred (ASB) Horses for 48 165 single-nucleotide polymorphisms (SNPs). A statistically significant association was identified on ECA20 (corrected P = 0.017) for SNP BIEC2-532523. Of the 20 affected horses, 17 were homozygous for this SNP when compared to seven homozygotes among the unaffected horses, suggesting a major gene with a recessive mode of inheritance. The result was confirmed by testing an additional 13 affected horses and 166 unaffected horses using 35 SNPs in this region of ECA20 (corrected P = 0.036). Combined results for 33 affected horses and 287 non-affected horses allowed identification of a region of homozygosity defined by four SNPs in the region. Based on the haplotype defined by these SNPs, 80% of the 33 affected horses were homozygous, 21% heterozygous and 9% did not possess the haplotype. Among the non-affected horses, 15% were homozygous, 47% heterozygous and 38% did not possess the haplotype. The differences between the two groups were highly significant (P < 0.00001). The region defined by this haplotype includes 53 known and predicted genes. Exons from three candidate genes, TRERF1, RUNX2 and CNPY3 were sequenced without finding distinguishing SNPs. The mutation responsible for swayback may lie in other genes or in regulatory regions outside exons. This information can be used by breeders to reduce the occurrence of swayback among their livestock. This condition may serve as a model for investigation of congenital skeletal deformities in other species.

SummaryVariations in the SLC45A2 gene are responsible for the dilution phenotypes cream and pearl in domestic horses. Cream dilution is inherited in an incomplete dominant manner, diluting only red in the heterozygous state but both red and black pigments when two alleles are present. The pearl dilution is recessive and dilutes only the red and black pigment in the homozygous state or when paired with a cream allele. Horses that inherit one copy of pearl (Cprl) and one copy of the dominant cream allele (CCr) display a dilution phenotype similar to that of homozygous cream, suggesting that pearl is the result of a different variation in the same gene responsible for cream. We sequenced SLC45A2 in two ‘false double dilute’ horses that appeared phenotypically homozygous cream but tested as possessing only a single CCr allele. We also sequenced one known pearl carrier to screen for putative causal variants. The missense variant ECA21:SLC45A2:c.985G>A; p.Ala329Thr (Cprl) was present in one false double dilute and the pearl carrier and was also genotyped in an additional 126 horses for statistical evaluation. The genotype matched the expected phenotype in all horses (P‐value = 6.5 × 10−41) and is identical to a pearl variant found previously. The second false double dilute horse and one non‐dilute offspring genotyped as heterozygous for a novel missense variant ECA21:SLC45A2:c.568G>A (p.Gly190Arg), the proposed Csun variant (for the name of the horse). This variant produces a recessive dilution similar to pearl and indicates that multiple alleles of SLC45A2 result in dilution phenotypes in the domestic horse.