Development of a 21-plex STR typing system for the identification of individuals and parentage testing in pig

Precise identification of individuals and accurate parentage verification is critical to livestock breeding programs, facilitating efficient genetic improvement and management practices. Short tandem repeat (STR)-based genotyping in pigs, current genotyping methods are often limited by inadequate resolution, suboptimal throughput and susceptibility to cross-species amplification. This study addresses these limitations by developing and validating a robust, species-specific 21-plex STR typing system. Integrating 13 core loci recommended by International Society for Animal Genetics/Food and Agriculture Organization with eight additional polymorphic markers identified from high-throughput sequencing, the multiplex assay was optimized for simultaneous amplification using fluorescence-labeled primers and capillary electrophoresis. Comprehensive primer optimization and thermal cycling adjustments established uniform amplification conditions, achieving balanced peak heights and distinct genotyping profiles with a sensitivity threshold of 0.5 ng DNA input per reaction. Species-specificity testing demonstrated no cross-reactivity with sheep, cattle and dogs and only weak amplification for the IGF1 locus in cats without compromising genotyping accuracy. Validation using diverse pig populations confirmed the assay's high discriminatory power and reproducibility. The resulting assay is technically rigorous, scalable and cost-effective, making it suitable for broad application in pig genetic improvement programs, pedigree verification, meat traceability and germplasm conservation.