Identification of a Novel Slow-Gray STX17 Lineage in Japanese Thoroughbreds via a Multi-tiered Copy Number Analysis Workflow.

Gray is a dominant coat color phenotype in horses caused by a ~ 4.6 kb tandem triplication within intron 6 of syntaxin 17 (STX17). The copy number variation (CNV) of the duplicated segment influences the graying rate. The rare G2 allele (CNV = 2) is associated with a slower graying rate compared to the common G3 allele (CNV = 3) and is also relevant to melanoma risk. Current assays are limited because long and accurate PCR (LA-PCR) detects only the presence or absence of duplications, while droplet digital PCR (ddPCR) cannot reliably distinguish certain genotypes such as G3/g and G2/G2. We constructed a stepwise workflow combining (i) multiplex real-time PCR targeting the duplication junction for rapid gray/non-gray screening, (ii) ddPCR for copy number estimation, and (iii) LA-PCR for confirmatory genotyping of ambiguous copy-number classes. Using real-time PCR, we screened 4596 Japanese Thoroughbreds aged 2-7 years, of which 4374 were classified as non-gray and 222 as gray. Based on age and coat appearance, 23 Gy candidates were prioritized for slow-gray evaluation and analyzed by ddPCR; one was classified as G2/g, and 22 as G3/g or G2/G2. LA-PCR detected a g-derived band in all 22 cases, confirming that they were G3/g. Pedigree analysis suggested that the G2 allele was transmitted through the maternal line and that this lineage was distinct from the previously reported Japanese slow-gray family. This workflow enables practical molecular discrimination among non-gray, common gray, and slow-gray genotypes, supporting the surveillance of rare G2 alleles in the Japanese Thoroughbred population.