DMRT3 Gene Variations in Horse Breeds Selected for Gaited Movement –   Established Research and Novel Findings
Background and objectives: The DMRT3 gene, often referred to as the "gait keeper," plays a key role in controlling alternative gaits in horses, such as tölt and pace. This study aimed to determine the frequency of known and to identify novel polymorphisms within the second exon of the DMRT3 gene across six horse breeds: gaited (Icelandic Horse, French Trotter), non-gaited (Arabian Horse, Malopolski Horse), and pony breeds (Welsh Pony, Icelandic Pony, Shetland Pony). Data collection: A total of 244 blood or hair samples were collected from representative individuals of the seven breeds (French Trotter; Icelandic Horse; Welsh Pony; Icelandic pony horse; Shetland Pony; Arabian and Malopolski horses). Research methods: The second exon of the DMRT3 gene analyzed using Sanger sequencing. Detected polymorphisms were annotated and classified using Ensembl and NCBI databases. Allele frequencies and genotype distributions were statistically compared between breeds and breeding populations. Results: Six polymorphisms were identified, including three novel variants. The known stop-gain variant c.902C>A (DMRT3_Ser301STOP) was confirmed in Icelandic Horses and French Trotters, with significant genotype differences between two French Trotter studs (p < 0.05). A novel 18-nucleotide in-frame insertion was found exclusively in Shetland Ponies. Additionally, a missense variant (c.967T>A; p.Tyr323Asn) and a synonymous change (c.855T>C) were detected in non-gaited breeds. Contributions: This study confirms the distribution of the DMRT3_Ser301STOP allele in gaited breeds and reveals novel polymorphisms with potential functional relevance. It expands current knowledge by identifying population- and breed-specific variation in DMRT3, suggesting a broader genetic influence on locomotor traits beyond gaited horses. The findings support the continued exploration of DMRT3 as a molecular marker in equine locomotion research and selective breeding.   Keywords: horses; gait type; genetic marker; DMRT3; novel polymorphisms  Highlights  • Three novel polymorphisms in the DMRT3 gene identified across horse breeds.  • DMRT3_Ser301STOP allele confirmed in Icelandic Horses and French Trotters.  • A unique 18-nt in-frame insertion found exclusively in Shetland Ponies.  • Missense and synonymous variants detected in non-gaited Arabian and Malopolski horses.  • Findings suggest broader locomotor role of DMRT3 beyond gaited breeds.