Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F-108 tri-block copolymer and capillary electrophoresis.

The research study describes a method for identifying variations in horse coat color genes to provide a quick method for equine identification. The study suggests the presented method could potentially be useful for forensic investigations.

Background and Objective

• The research focuses on identifying single nucleotide polymorphisms (SNPs), a type of genetic variation, in specific genes associated with horse coat color. Due to these genetic changes can significantly impact the phenotype or physical characteristics (like coat color), their detection can provide valuable insights into the equine individuals’ specific traits.
• Current techniques for identifying these mutations – such as sequencing, hybridization assays, or mini-sequencing – can be costly and time-consuming. Therefore, the research aims to expand the detection of these mutations using a simpler and quicker method.

Methods

• The research utilized a polymer called F-108 in a process known as CE-SSCP (capillary electrophoresis single-strand conformation polymorphism) to differentiate between SNPs in specific genes related to horse coat color. These genes included the melanocortin-1 receptor (mc1r), the solute carrier family member 2 (slc45a2/matp), and type III receptor protein-tyrosine kinase (kit).
• The CE-SSCP with F-108 method was compared with mini-sequencing using SNaPshot kits, a traditional approach for SNP detection.

Findings

• Results showed that the F-108 polymer method could accurately distinguish between homozygous (same alleles) and heterozygous (different alleles) individuals for the mc1r and kit genes, but it had trouble resolving heterozygous individuals for the slc45a2 gene at a specific temperature (38ºC).
• The constraints for the method arose due to the need for lower temperatures, the SNP position being close to the start (5′-end) of the gene, and similar free energy values leading to indistinguishable secondary structures.

Implication

• Although the approach faced some limitations, the study claims the method could still serve as a fast and reliable way of generating phenotypic descriptions, which could be invaluable in the field of equine forensics.