Mutation rate and spectrum of germline de novo mutations in a closed population of Thoroughbred horses.

Background: Thoroughbreds have been maintained as a closed breed for over 300 years since the crossbreeding between Arabian stallions and English mares. Despite interest in germline de novo mutations across species, their frequency in horses, particularly in Thoroughbreds, remains largely unexplored.

Aims/objective: This study aimed to identify de novo mutations in Thoroughbreds and estimate their frequency within a genetically closed population.

Methods: We performed deep whole-genome sequencing (≥230× depth, 150 bp paired-end reads) and Sanger validation in a Thoroughbred trio (sire, dam, and foal). Reads were aligned to the EquCab3.0 reference genome, and variants in regions with sequencing depth ≥20 (covering 99.6 % of the genome) were analyzed for Mendelian inconsistencies.

Results: A total of 48 de novo mutations were identified, comprising 46 single nucleotide substitutions and 2 deletions, none of which were found in public variant databases. Of these, 18 were of paternal origin and 6 were of maternal origin (P < 0.05). The estimated mutation rate was 9.59 × 10⁻⁹, which is in a comparable range to that reported in humans. One mutation (chr7:g.2084761G>A) introduced a nonsense variant in the zinc finger protein 77 gene, which encodes a putative transcriptional regulator.

Conclusions: De novo mutations were identified in the Thoroughbred genome, and the mutation rate is consistent with estimates from other mammalian studies. A significant bias toward paternal origin was observed. Our findings suggest that germline de novo mutations are a source of novel genetic variation in Thoroughbreds, even within a genetically closed population.