Dissecting Genomic Factors of Stallion Fertility

Abstract

Stallion fertility is a complex trait, affected by a considerable genetic component. Knowledge about the genetic factors contributing to reduced fertility in stallions is currently limited. The goal of this Dissertation was to investigate select autosomal and Y-linked factors of stallion fertility to enhance knowledge about the genetic component of stallion fertility.

For the select autosomal factors, we validated the association of the FKBP6 gene with stallion subfertility due to impaired acrosome reaction (IAR). We developed two TaqMan genotyping assays for the IAR-associated SNPs in the FKBP6 gene and confirmed significant association (P < 0.0001) between low per-cycle pregnancy rate and the IAR-susceptibility FKBP6 genotype A/A-A/A in Thoroughbreds. Through whole genome sequencing, we identified a 171 Kbp haplotype block specific to subfertile Thoroughbreds with the FKBP6 A/A-A/A genotype. Testis transcriptome analysis of FKBP6 in select Thoroughbreds revealed that the gene is expressed from both alleles and not monoallelically as thought previously.

Regarding the male-specific Y chromosome (MSY), we developed droplet-digital PCR assays for copy number (CN) analysis of 7 multi-copy genes and SRY to determine CN variation (CNV) of these genes in a global equine population and compare CNV with MSY haplotypes (HTs). We show that TSPY is the most variable gene among individuals and breeds. The SRY gene is a single-copy gene in most horses but may have additional copies in indigenous breeds. Comparison of MSY gene CNV with MSY HTs, revealed no correlation between the two forms of MSY variation. Additionally, we conducted CN analysis of these genes in horses with disorders of sex development or subfertility. We observed significantly lower CNs in TSPY and ETSTY2 within cryptorchid males, though no significant CNVs were observed in subfertile males. CN analysis of these genes indicated that the ampliconic assembly in eMSYv3 needs improvement. Thus, we re-sequenced and re-assembled this region utilizing Nanopore technology. We generated an improved 1.53 Mbp assembly of the ampliconic MSY and closed one of the three gaps in single-copy Y. Finally, we conducted a detailed molecular cytogenetic analysis of a reciprocal translocation between horse chromosomes Y and 13 in a Friesian stallion with complete azoospermia.