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Identification of a Novel Slow-Gray STX17 Lineage in Japanese Thoroughbreds via a Multi-tiered Copy Number Analysis Workflow.
Abstract: 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.
© 2026. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
Publication Date: 2026-07-11 PubMed ID: 42435135PubMed Central: 2989891DOI: 10.1007/s10528-026-11425-yGoogle Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This study identifies and characterizes a new slow-gray genetic variant affecting coat color in Japanese Thoroughbred horses using a novel multi-step genetic testing method.
- The researchers developed and applied a detailed workflow combining several genetic assays to accurately distinguish between common and rare alleles influencing the graying rate in horses.
Background
- Gray coat color in horses is caused by a duplication event in the STX17 gene, specifically a tandem triplication of a 4.6 kb segment within intron 6.
- The number of copies in this duplication (copy number variation or CNV) affects how quickly horses gray with age:
- The common G3 allele has three copies and leads to a normal graying rate.
- The rare G2 allele has two copies and is associated with a slower graying process and a different risk for melanoma, a type of skin cancer in horses.
- Identifying and differentiating these alleles accurately is important for better understanding coat color inheritance and health risks in Thoroughbreds.
Limitations of Prior Methods
- Long and accurate PCR (LA-PCR) can only determine if duplications exist or not; it cannot quantify copy numbers precisely.
- Droplet digital PCR (ddPCR) can estimate copy numbers but struggles to reliably distinguish certain genotypes, such as differentiating between one G3/g (3 copies and 1 normal allele) and two G2/G2 alleles.
- Therefore, neither method alone is sufficient to fully genotype individuals, especially ambiguous cases.
New Workflow Development
- The researchers developed a three-tiered workflow combining multiple genetic tests for enhanced accuracy:
- Multiplex real-time PCR targeting the duplication junction to quickly classify horses into gray or non-gray categories.
- Droplet digital PCR (ddPCR) for estimating the exact copy number variants in gray horses.
- Long and accurate PCR (LA-PCR) as a confirmatory test to resolve ambiguous genotypes detected by ddPCR.
- This stepwise process allows precise discrimination between non-gray horses, common gray (G3) genotype horses, and rare slow-gray (G2) genotypes.
Application and Results
- The workflow was applied to a large sample of 4596 Japanese Thoroughbred horses aged between 2 and 7 years.
- Using real-time PCR screening, 4374 horses were classified as non-gray and 222 as gray.
- From the gray group, 23 horses suspected of carrying slow-gray alleles (based on age and appearance) were selected for ddPCR analysis.
- Results from ddPCR indicated:
- 1 horse had G2/g genotype (slow-gray allele present).
- 22 horses seemed to have either G3/g or G2/G2 genotype; these were ambiguous cases.
- LA-PCR was then used for these 22 ambiguous cases, which all displayed a ‘g’-derived band indicating the G3/g genotype, effectively ruling out double G2 alleles.
- Pedigree analysis revealed the G2 slow-gray allele is maternally transmitted and represents a lineage distinct from previously identified Japanese slow-gray families, indicating a novel genetic origin.
Significance and Implications
- The newly established workflow provides a practical and reliable molecular approach to identify and monitor rare slow-gray alleles in Japanese Thoroughbreds.
- This enables breeders and researchers to better understand genetic influences on coat color and associated health risks like melanoma in horses.
- The identification of a novel G2 lineage helps clarify the genetic diversity and inheritance patterns of gray coat color in this horse population.
- The method offers improvements over previous assays by combining rapid screening and precise genotype confirmation, overcoming limitations of existing single-method approaches.
Cite This Article
APA
Kawate K, Furukawa R, Kikuchi M, Ishige T, Seki K, Tozaki T, Kakoi H.
(2026).
Identification of a Novel Slow-Gray STX17 Lineage in Japanese Thoroughbreds via a Multi-tiered Copy Number Analysis Workflow.
Biochem Genet.
https://doi.org/10.1007/s10528-026-11425-y Publication
Researcher Affiliations
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, 320-0851, Japan. k-kawate@lrc.or.jp.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, 320-0851, Japan.
- Equine Department, Japan Racing Association, Minato, Tokyo, 105-0003, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, 320-0851, Japan. ttozaki@lrc.or.jp.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, 320-0851, Japan.
Conflict of Interest Statement
Declarations. Conflict of interest: The authors declare no conflicts of interest. Ethical Approval: All the experimental protocols were approved by the Animal Care Committee of the Laboratory of Racing Chemistry (Approval Number: 20-4) and this study was conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
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