Identification of the locus causing skeletal atavism in Shetland ponies

Shetland ponies are used for horse shows, as mounts for children and adults, and carriage driving. Over the last decades, there have been several reports of congenital abnormalities in skeletal structures including limb, ulna, and fibula development. Fulldevelopment of fibula and ulna in the horse is an example of atavism; reappearance of a phenotype from an earlier development of fibula and ulna in the horse is an example of atavism; reappearance of a phenotype from an earlierevolutionary specimen. This condition causes lameness and severe movement disabilities, most often leading to euthanization of foals. Skeletal atavism in Shetland ponies has been characterized as an autosomal recessive trait. We used the Equine 50K SNP chip to genotype a group of Shetland ponies (five affected horses, 19 carriers, and 24 controls) together with a group of American samples collected in USA (eight cases, five carriers, and ten controls). All control individuals were stallions without any confirmed affected offspring. The genome­wide association analysis did not reveal any SNPs significantly associated with the disorder.Hence, we decided to perform whole­genome resequencing of six Swedish cases and a pool of 21 control stallions using Illumina Hi­seq technology (PE 2x100 bp) with resulting average depths of 7X (cases) and 55X (pool). All reads were aligned to the genome reference assembly by using BWA. We called variations and determined genotypes by GATK. From the sequencing data, we identified a locus where SA cases were fixed for a non­reference allele while the control pool was fixed or close to fixation for the reference allele. We genotyped the locus in Swedish and American cases, carriers, and controls. The genotyping analysis confirmed the inferred genotypes from sequencing and validated the association in the American samples. The reason why the GWAS failed was that the 50k SNP chip lacked informative markers for the genomic region harboring the disease locus. The genetic basis for skeletal atavism in Shetland horses will be presented.