Screening of Y‐chromosomal STR loci and development of a multiplex PCR system for paternal lineage identification in horses

BACKGROUND: With growing demand for pedigree verification and breed management in horses (Equus caballus), reliable paternal lineage tools are essential. Y-chromosomal STRs (Y-STRs) have advantages over autosomal STRs due to paternal inheritance and lack of recombination. However, few validated loci and no standardised efficient genotyping systems limit their use. Current methods often require multiple reactions, increasing cost and labour. Thus, identifying informative Y-STR loci and developing a multiplex PCR system for cost-effective paternal lineage analysis is urgently needed. OBJECTIVES: To identify informative horse Y-STR loci and develop a multiplex PCR system for paternal lineage tracing, breed conservation, pedigree verification, and forensic settings. STUDY DESIGN: Experimental study. METHODS: Y-STR loci were screened from the horse reference genome based on repeat motif, number, and location. Primers were designed and tested on stallions and mares to confirm Y-specificity. Loci with stable, male-specific amplification were combined with known markers into a multiplex PCR system. Sensitivity, repeatability, reproducibility, and performance were evaluated, and locus polymorphism was assessed in Debao ponies, Mongolian horses, Thoroughbreds, and Arabian horses. RESULTS: Twenty candidate loci were screened, and 15 of them showed clear, reproducible male-specific amplification. Three novel polymorphic loci were identified and combined with 5 known markers into 2-plex and 6-plex panels. Most loci were polymorphic in Debao ponies (PIC: 0.000-0.575) and Mongolian horses (PIC: 0.000-0.375), while less polymorphism was detected in Thoroughbreds (PIC: 0.000-0.099) and Arabian horses (PIC: 0.000-0.099). The optimised multiplex PCR system demonstrated high sensitivity, repeatability, and compatibility. MAIN LIMITATIONS: Reproducibility and stability across labs and platforms require further validation. CONCLUSIONS: Fifteen new Y-STR loci were identified, and 3 of them were polymorphic. Novel optimised multiplex PCR systems were developed for the identification of horse paternal lines, providing reliable tools for equine genetic study, breeding, and forensic applications.