Genotypic analysis of the CSN2 gene in cattle: Validation of a1/a2 polymorphism by Sanger sequencing

Milk β-casein variants, particularly A1 and A2, have gained attention due to their impact on digestibility and potential health effects. The CSN2 gene, located on bovine chromosome 6, encodes β-casein, and a single nucleotide polymorphism (SNP) in exon 7 differentiates the A1 (histidine) and A2 (proline) alleles. The A1 variant is associated with the release of β-casomorphin-7 (BCM-7), which has been controversially linked to health concerns such as type 1 diabetes and cardiovascular disease, while A2 milk is considered easier to digest and more suitable for sensitive consumers. This study aimed to genotype selected cattle breeds for CSN2 variants using PCR and Sanger sequencing. Blood samples were collected, and genomic DNA was extracted via the phenol- chloroform method. A 121 bp region of exon 7 was amplified using gene-specific primers, followed by electrophoresis and Sanger sequencing for SNP detection. Chromatogram analysis revealed three genotypes: A1A1, A1A2, and A2A2, with heterozygotes being most prevalent. Sanger sequencing proved to be a precise, reliable tool for confirming genotypes, especially in distinguishing heterozygous conditions and avoiding ambiguities common in conventional PCR methods. The allele distribution observed aligns with previous findings in both indigenous and exotic cattle breeds and supports the growing emphasis on A2 milk in breeding programs. Future prospects include expanding genotyping across larger herds, studying correlations between β-casein variants and milk production traits, and applying marker-assisted selection (MAS) to establish A2A2-dominant lines, which would enhance both dairy quality and market value for health-focused milk production.