Development and validation of a novel 13‐plex PCR system for commonly used short tandem repeats in horses (Equus caballus)

Abstract

Summary Background Due to the thriving development of the modern horse industry and the occurrence of horse related crimes, the demand for methods of individual horse identification, parentage tests and other genetic analyses is increasing. Previous methods had disadvantages that decreased the accuracy of the results, lacked the inclusion of all commonly used short tandem repeats (STR) or increased the experimental cost and time. Objectives We aimed to develop a novel 13‐plex STR typing system to resolve the above issues. Study design Experimental study. Methods Twelve autosomal and most commonly used di‐nucleotide STRs (AHT4, AHT5, ASB2, ASB17, ASB23, HMS2, HMS3, HMS6, HMS7, HTG4, HTG10 and VHL20), and a Y‐chromosomal STR (YJ10) were included. We redesigned the primers of eight STRs to establish a novel multiplex PCR system and tested this system for species specificity, sensitivity and repeatability. Results Full profiles were easily generated in one fast PCR reaction using a low‐cost polymerase, as little as 1 ng of horse DNA template and 13 pairs of primers labelled with fluorescent dyes. No full profile was generated from DNA templates of humans or other commonly encountered animals. We also established an allelic ladder that contained 110 alleles based on 200 horses from 12 breeds and calculated standard population genetic parameters based on 150 Thoroughbreds. Stutter analysis showed that the averages of the stutter ratios were distinctly lower than those of lower allele ratios and the combined probability of paternity exclusion for this system were 0.994659935 (CPEduo) and 0.999854032 (CPEtrio). Main limitations A nonspecific and relatively low peak at 316 bp was frequently observed in locus HMS2, which is a nonexistent allele in all horses and should be ignored. Conclusions Our results indicate that this 13‐plex STR genotyping system is sensitive, species‐specific, cost‐effective and robust for applications in the horse industry and forensic investigation.

A novel 13‐plex STR typing system for individual identification and parentage testing of donkeys (Equus asinus)

“…This is the first reported STR typing system that is specific for donkeys. Similar to the horse STR multiplex typing system developed in 2018, the gender test for the donkey could be supplied in future experiment .…”

“…Since full genotype profiles were also observed in samples from the horse, mule and hinny, this system is also suitable for individual identification, and parentage testing, in the mule and hinny, animals that do not yet have a testing system. Since this typing system contains five STRs (HMS18, TKY297, TKY312, TKY337 and TKY343) that are not included in the horse’s new 13‐plex system or the previous 17‐plex system , this system can be used for the horse when extra loci are needed to obtain more accurate results. Our system showed good sensitivity, with full genotype profiles being obtained from as little as 5 ng of DNA template.…”

“…Primers for the 13 STR loci were previously designed to sequences from the horse . To establish the new 13‐plex STR typing system for the donkey, we redesigned primers for seven of the STRs (HMS7, HTG7, HTG10, TKY297, TKY312, TKY337 and TKY343), since nonspecific amplifications occur with the original horse primers or the product sizes were inappropriate for the 13‐plex system.…”

“…Stutters are the common phenomenon seen in genotyping analysis of dinucleotide STRs . The minus stutter peak is one repeat smaller than the true allele and the plus stutter peak is one repeat larger than the true allele.…”

“…The minus stutter peak is one repeat smaller than the true allele and the plus stutter peak is one repeat larger than the true allele. Analysis of the ratio of the intensity of the minus and plus stutter peaks can guide the experimenter to the correct genotype . Stutter analysis of the 13‐plex typing system was conducted on the genotyping results from the 150 donkeys.…”