High-resolution melting (HRM) method for genotyping Kappa-Casein (CSN3) alleles in buffaloes

The kappa-casein gene (CSN3) plays a crucial role in milk protein composition, with allelic variants A (X1) and B (X2) influencing important dairy traits in buffaloes. Given the superior nutritional and technological properties of buffalo milk, accurate and efficient identification of these variants is crucial for genetic improvement programs. However, existing genotyping methods frequently entail labor-intensive post-PCR manipulations, underscoring the need for efficient and cost-effective alternatives. This study aimed to develop and validate a novel and efficient High-Resolution Melting (HRM) analysis method for genotyping CSN3 alleles in buffaloes.

For method development, DNA from 100 buffaloes, sourced from six regions of São Paulo State, Brazil, was initially sequenced. This sequencing was performed to confirm the presence of A and B alleles and to establish reference genotypes. These three identified genotypes (AA, AB, and BB) served as reference standards for HRM optimization. Following initial validation, the method was then applied to genotype an additional 438 samples, accumulating a total of 538 genotyped animals.

The developed HRM method demonstrated high specificity and sensitivity, showing 100% concordance with sequencing data for reference samples and clearly differentiating all genotypes (AA, AB, and BB). Analysis of all 538 animals revealed overall genotype frequencies of 46.3% for AA, 44.8% for AB, and 8.9% for BB. Allele A predominated, with a frequency of 68.7%.

This novel HRM-based method offers a robust, rapid, and cost-effective tool for large-scale buffalo genotyping, presenting significant potential for genetic improvement programs aimed at enhancing milk quality.