Background: Myostatin (MSTN) gene is a major inhibitor of skeletal muscle growth, and variants within its promoter region may affect transcriptional activity and musclerelated traits. Mongolian native horses have adapted to endurance and harsh environmental conditions, whereas Halla horses exhibit enhanced musculature owing to crossbreeding with Thoroughbreds. MSTN promoter variations in these breeds have not been sufficiently investigated, highlighting a critical research gap that motivated the present study. Methods: Two single nucleotide polymorphisms (SNPs) in the MSTN promoter region were examined in 19 Mongolian and 4 Halla horses using PCR amplification and Sanger sequencing. Additionally, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway annotations were used to characterize gene functions. Results: Two promoter region SNPs were identified in MSTN gene. These distribution patterns represent preliminary observations within the sampled populations, providing a valuable foundation for future investigations. Functional annotation further supported the regulatory relevance of MSTN through its involvement in muscle growth inhibition and the TGF-β/ACVR2B/SMAD pathway. Conclusions: This study provides the first baseline characterization of MSTN promoter polymorphisms in Mongolian and Halla horses. The g.26T>C variant was identified in Mongolian horses, whereas the shared g.156T>C SNP represents a regulatory variant observed in both breeds, pending confirmation in broader populations. However, due to the limited sample size, we consider these findings to be preliminary observations rather than definitive evidence of breed-specific adaptation. The results of this study are expected to serve as fundamental data for establishing future genetic information– based breeding and conservation strategies for Mongolian horses.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Sample collection and DNA extraction
    Primer design
    PCR amplification and sequencing
    In-silico functional annotation of the MSTN gene
RESULTS
    MSTN SNP markers and sanger sequencing analysis
    In-silico functional annotation of the MSTN gene
DISCUSSION
CONCLUSION
REFERENCES

• Zultsetseg Byambasuren(Department of Biotechnology, Hankyong National University, Anseong 17579, Korea)
• Khaliunaa Tseveen(Department of Biotechnology, Hankyong National University, Anseong 17579, Korea)
• Nyambazar Amarsaikhan(Department of Biotechnology, Hankyong National University, Anseong 17579, Korea)
• Gwang Hyeon Lee(Department of Biotechnology, Hankyong National University, Anseong 17579, Korea, Gyeonggi Regional Research Center, Hankyong National University, Anseong 17579, Korea, Genomic Information Center, Hankyong National University, Anseong 17579, Korea, Hankyong and Genetics, Anseong 17579, Korea)
• Hong Sik Kon(Department of Biotechnology, Hankyong National University, Anseong 17579, Korea, Gyeonggi Regional Research Center, Hankyong National University, Anseong 17579, Korea, Genomic Information Center, Hankyong National University, Anseong 17579, Korea, Hankyong and Genetics, Anseong 17579, Korea) Corresponding author