Optimization of Autosomal STR Markers for Equine Genotyping Using Multiplex PCR

The investigation of horse lineage was of paramount importance in the registration of different breeds, trade, and formulation of studbooks. The pioneering technique of DNA fingerprinting emerged as the first highly responsive method reliant on DNA for individual identification and the examination of genetic affiliations. Microsatellites were a valuable tool for analyzing the genetic variations present among different horse breeds. The International Society for Animal Genetics (ISAG) has endorsed a set of 17 specific Short Tandem Repeats (STRs) for the equine identification, although these can be quite expensive to obtain through commercially available multiplex kits. Objective: To determine five autosomal STR markers (HMS6, HMS7, ASB23, VHL20, and LEX14) were optimized using multiplex PCR for equine genotyping. Methods: DNA was extracted from a Thoroughbred horse blood sample via an organic extraction method. Sensitivity analysis determined the optimal PCR concentration. Genotyping was performed on the ABI PRISM® 3100XL, and data were processed with Gene Mapper ID 3.2v software. Results: The optimal conditions for multiplex PCR of HMS6, HMS7, ASB23, VHL20, and LEX14 primers were 60°C annealing temperature, 3ng DNA concentration and 6μM primer concentration. A 12.5μL PCR reaction volume was recommended for cost efficiency. Conclusions: The results of this research have the potential to create a cost-effective, regionally produced multiplex PCR kit. This kit would be designed for analyzing parentage lineage within the Equine family in Pakistan, incorporating ISAG-recommended markers: VHL20, HMS6, HMS7, ASB23, and additionally LEX14. It could significantly streamline the import and export of horses in Pakistan.