Short Tandem Repeat Analysis of Genetic Diversity Metrics in American Standardbreds and an Investigation on the Cause of the Rabicano Coat Color Phenotype Utilizing Short and Long Read Sequencing Methods.
- Esdaile, Elizabeth Susan
- Advisor(s): Bellone, Rebecca R
Abstract
The governing body for American Standardbreds, the United States Trotting Association (USTA), closed the studbook in 1973, halting new geneflow into the population. In 2009, the USTA capped the studbook of new sires at 140 mares bred per year to maintain genetic diversity within the breed. To investigate the state of genetic diversity within the American Standardbred during this shift in breeding practices, genotypes for sixteen STRs from American Standardbred horses foaled from 2010 to 2015 and their sires and dams (n=50,621) were investigated. Allelic richness (Ar), expected heterozygosity (HE), observed heterozygosity (HO), inbreeding coefficient (FIS), and fixation index (FST) were calculated. These analyses found that trotting and pacing sires were less genetically diverse than dams (HE pbonferroni=.030 and <.001, respectively) and their offspring (Ar pbonferroni =.03 and <.001, respectively), and only pacing offspring were significantly less diverse than their dams (HE pbonferroni =.002). Inbreeding coefficients for trotters (FIS=-0.014) and pacers (FIS=-0.012) suggest that breeding practices have maintained diversity within the breed. Moderate levels of genetic differentiation (.066
In addition to genetic diversity, horse breeders are concerned with selecting for traits that are of economic importance and prevent detrimental conditions. One such trait of economic importance is coat color, which can impact the value of a horse. One popular equine coat color is rabicano, a phenotype with an unknown genetic cause that can be found in many breeds of horses, including Quarter Horses and Arabians. Rabicano can be recognized by white ticking in the flank, sometimes expanding forward up the barrel, and white banding on the tailhead. Using a candidate gene approach, Illumina short read sequencing data from four rabicano horses, 17 non-rabicano controls, and one indeterminate phenotype were utilized to identify and prioritize coding variants from 659 pigmentation-related genes for further investigation. Long read sequencing data from one of the rabicano horses were also evaluated to identify and investigate structural variants in the same set of candidate genes as the potential genetic cause of this phenotype. Six SNPs in ANKRD27, CEP290, CRB1, FMN1, KIF13A, and OCA2 were investigated in a larger sample set comprised of 61 rabicano cases and 36 non-rabicano controls. While not perfectly concordant, the variant in CEP290 (ENSECAT00000052715.2:c.538A>G) was the most concordant with phenotype, with a p-value of 2.24x10-07. This suggests that either none of these variants are the cause of rabicano across breeds or a more complex mode of inheritance best explains this phenotype. Given that the variant in CEP290 was most concordant with the rabicano phenotype, is located proximally to KITLG, and the phenotypic similarities of KITLG variants in other species, putative regulatory and structural variants flanking KITLG were also investigated. This analysis identified a potential 1.7Mb haplotype on ECA28 surrounding KITLG associated with the rabicano phenotype. The most concordant SNP was ECA28:rs397240012 (NC_009171.3:g.15967332G>A) (p=7.36x10-09). This SNP is located within a H3K4me1 annotated peak (indicative of a gene enhancer) in skin and is 160kb from the start of transcription for KITLG. Additional analyses are required to refine the haplotype across breeds and identify the causal variant of rabicano in horses.