Approaches to Thoroughbred Horse Identification in Diverse Sample Types Using Multiplexed SNP-PCR Sequencing Panels

Area of Science:

• Animal Genetics
• Forensic Science
• Molecular Biology

Background:

• Accurate identification of equine samples is crucial for various applications, including gene editing detection and forensic analysis in doping cases.
• Existing methods may lack the discriminatory power or efficiency required for unambiguous sample source determination in Thoroughbred horses.

Purpose of the Study:

• To develop and validate a single-reaction, 93-marker SNP-PCR sequencing panel for individual identification of Thoroughbred horses.
• To assess the performance, sensitivity, and reliability of the SNP panel across different sample types and DNA concentrations.

Main Methods:

• A panel of 93 single nucleotide polymorphism (SNP) markers was designed for PCR amplification and sequencing.
• Marker amplification performance and discriminating power were evaluated using 264 Thoroughbred blood samples.
• Sensitivity was tested using DNA dilution series and various biological matrices (blood, hair, feces).

Main Results:

• The SNP panel demonstrated high amplification performance and discriminating power in Thoroughbred horses.
• No genotyping errors were observed down to 0.25 ng/μL DNA concentration in dilution series.
• The panel performed well on forensic samples like blood on labels, though variability was noted with low DNA concentrations.

Conclusions:

• The developed SNP-PCR sequencing panel is a sensitive and specific tool for individual identification of Thoroughbred horses.
• This equine DNA identification method is suitable for forensic applications, gene editing controls, and doping control.
• The panel offers a robust solution for unambiguous equine sample source determination.