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<p>Gray is a dominant coat color phenotype in horses caused by a ~ 4.6&#xa0;kb tandem triplication within intron 6 of syntaxin 17 (<i>STX17</i>). The copy number variation (CNV) of the duplicated segment influences the graying rate. The rare <i>G2</i> allele (CNV = 2) is associated with a slower graying rate compared to the common <i>G3</i> 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 <i>G3</i>/<i>g</i> and <i>G2</i>/<i>G2</i>. 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&#xa0;years, of which 4374 were classified as non-gray and 222 as gray. Based on age and coat appearance, 23&#xa0;Gy candidates were prioritized for slow-gray evaluation and analyzed by ddPCR; one was classified as <i>G2</i>/<i>g</i>, and 22 as <i>G3</i>/<i>g</i> or <i>G2</i>/<i>G2</i>. LA-PCR detected a g-derived band in all 22 cases, confirming that they were <i>G3</i>/<i>g</i>. Pedigree analysis suggested that the <i>G2</i> 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 <i>G2</i> alleles in the Japanese Thoroughbred population.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 发表日期：

    2026年7月11日

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  • Koki Kawate,
  • Risako Furukawa,
  • Mio Kikuchi,
  • Taichiro Ishige,
  • Kazuhiro Seki,
  • Teruaki Tozaki,
  • Hironaga Kakoi

摘要

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.

Graphical Abstract