The horse (Equus caballus) holds major cultural and economic importance worldwide and can be involved in criminal investigations, such as cases of horse mutilation (“horse-ripping”) or human crimes where equine evidence links suspects, victims, and crime scenes. Reliable genetic tools are needed to identify individual animals from biological traces in these contexts. Despite this relevance, equine forensic genetics currently lacks a validated, court-admissible DNA marker system specifically designed for forensic applications.

To address this gap, STRide (Short Tandem Repeat Identification in Equids) aims to develop and forensically validate a novel tetranucleotide STR multiplex system for horses. Tetranucleotide STRs represent the gold standard in forensic genetics due to their allele resolution, reduced stutter artifacts, and high reproducibility. Using publicly available horse genomes, including a high-quality reference assembly, more than 2,000 candidate STR loci were identified through genome-wide screening and subsequently filtered to obtain high-quality markers suitable for forensic application. From these, approximately 15 highly informative loci, including a sex-determining marker, were selected to design two to three multiplex PCR assays. To ensure suitability for degraded forensic samples, all amplicons were designed to be shorter than 300 bp.

The STRide system will undergo comprehensive forensic validation in accordance with international guidelines, including assessment of sensitivity, specificity, precision, reproducibility, and robustness across diverse breeds and sample types. In addition, cross-species amplification will be evaluated in other equids, including donkeys and zebras.

Upon completion, STRide will provide a validated tetranucleotide STR multiplex system for equine forensic genetics, enabling reliable individual identification in cases of horse theft, animal cruelty, pedigree fraud, and human crimes involving equine biological evidence.

Authors

• Annika Mozer (Institute of Legal Medicine, University Hospital Bonn, Germany)
• Yvonne Tatjana Gerent (Federal Criminal Police Office, Germany)
• Martina Unterländer (Federal Criminal Police Office, Germany)
• Daniel Wittschieber (Institute of Legal Medicine, University Hospital Bonn, Germany)