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

The research presents the development and assessment of a new system that uses 13 distinct genetic markers, or short tandem repeats (STRs), to identify individual horses. This system aims to improve the accuracy, efficiency and cost-effectiveness of genetic tests used for horse identification and parentage checks.

Background

• Previously, identification of individual horses and parentage tests were carried out using methods that had some shortcomings. These methods either decreased the accuracy of results, did not cover all commonly used STRs, or were more expensive and time-consuming.
• The researchers in this study were looking to devise a more effective system using 13 commonly used STRs for horse identification that would overcome these disadvantages.

Methods

• They developed a 13-plex STR typing system, including twelve commonly used autosomal (non-sex chromosomal) di-nucleotide STRs and one Y-chromosomal STR, for specific identification of horse DNA.
• These STRs were selected for their high variability and thus, their capacity to uniquely identify individual horses.
• To build their new multiplex PCR system, the researchers designed new primers for eight of the STRs.
• The researchers also evaluated the system for species specificity, sensitivity, and repeatability to ensure it only worked on horse DNA and provided reliable, repeatable results.

Results

• The 13-plex STR genotyping system was successfully able to generate full profiles with just 1 ng of horse DNA input using a low-cost polymerase.
• This multiplex PCR based system was found to be sensitive enough to pick up single copies of each STR and specific only to horses, ruling out potential interference from human or other animal DNA.
• The team also created an allelic ladder (a kind of reference guide for identifying STR alleles) using data from 200 horses from 12 different breeds.
• An interesting observation was a nonspecific peak at 316 bp in locus HMS2, which ended up being an artifact and should be ignored in future analyses.

Conclusion

• The researchers concluded that the new 13-plex STR genotyping system is efficient, cost-effective, highly sensitive, and species-specific.
• They suggest it will be highly useful in forensic investigation and the horse industry for applications requiring individual horse identification and parentage testing.