Analysis of bovine beta-casein A1 and A2 allele frequency in Holstein-Friesian cows by Real-time PCR with fluorescent hybridization probes

A2 milk popularity is increasing across the world and novel molecular techniques have been evaluated to develop reliable methods. This study aimed to genotype Holstein-Friesian cows concerning their A1/A2 status using Real-time PCR assay with the specifically designed FRET hybridization probes. In this context, DNA samples were obtained from 310 Holstein-Friesian milk samples. Concerning the Real-time PCR assay, the melting temperature of each amplicon was analyzed and the melting data was converted to a derivative plot using the LightCycler 480 System. The sensor probe was designed to match the wild-type sequence in the target DNA. In the Real-time PCR assay, the melting peaks obtained in the Real-time PCR assay were highly decisive and consistent for each genotype regarding CCT→CAT alteration. The results indicated a remarkably high frequency of the A2 allele (68%) and a considerable frequency of heterozygous animals (0.41). Population genetic analysis showed intermediate levels of genetic variability and biodiversity. The A2-herd conversion process is a complex process consisting of genetic testing of both cows and calves, evaluating replacement rates, and the conversion of heterozygotes by using A2-genotyped bull semen. In this sense, the key point is a reliable and rapid genotyping method to produce A1-free milk. This study suggests that Real-time PCR assay with the specifically designed FRET hybridization probes is a preferable method for A2 genotyping, and may be useful for further studies and instructive for companies or breeders who aim to produce A2 milk.