Development of a 25-plex SNP assay for traceability in cattle

Area of Science:

• Animal Genetics
• Molecular Biology
• Forensic Science

Background:

• Single nucleotide polymorphisms (SNPs) are crucial genetic markers for DNA profiling due to their abundance and suitability for automation.
• The SNaPshot method offers a robust platform for multiplex SNP genotyping, enabling efficient analysis of multiple genetic loci simultaneously.

Purpose of the Study:

• To develop and validate a multiplex assay for genotyping 25 SNPs in cattle using the SNaPshot method.
• To assess the utility of this assay for DNA-based traceability, including parentage control and sample identification in commercial cattle breeds.

Main Methods:

• Utilized the SNaPshot primer extension method to multiplex 25 validated SNPs for cattle identity control.
• Employed four different fluorochromes and specifically designed extension primers for precise detection and length control of amplified products.
• Estimated allele frequencies and calculated genotype probabilities for Holstein, Simmental, Limousin, Angus, Charolais, and Tux Cattle breeds.

Main Results:

• Achieved high genotyping precision (>0.998) and a low probability of identical genotypes between unrelated individuals (10^-8 to 10^-10).
• Demonstrated a high exclusion power of 99.9% for parentage control when both putative parents' genotypes are known.
• Validated the assay's accuracy through traceability tests and analysis of parent-sib pairs and trio families.

Conclusions:

• The 25-plex SNaPshot assay is a reliable and efficient tool for DNA-based traceability in major commercial cattle breeds.
• The assay is adaptable for both low- and high-throughput applications, offering a valuable alternative for genetic identification and parentage verification.