Comparison of cattle BoLA-DRB3 typing by PCR-RFLP, direct sequencing, and high-resolution DNA melting curve analysis

Major histocompatibility complex (MHC) represents an important genetic marker for manipulation to improve the health and productivity of cattle. It is closely associated with numerous disease susceptibilities and immune responses. Bovine MHC, also called bovine leukocyte antigen (BoLA), is considered as a suitable marker for genetic diversity studies. In cattle, most of the polymorphisms are located in exon 2 of BoLA-DRB3, which encodes the peptide-binding cleft. In this study, the polymorphism of the BoLA-DRB3.2 gene in Holstein's calves was studied using high resolution melting curve analysis (HRM). Observed HRM results were compared to PCR-RFLP and direct sequencing techniques. Eight different HRM and seven different RFLP profiles were identified among the population studied. By comparing to sequencing data, HRM could completely discriminate all genotypes (eight profiles), while the RFLP failed to distinguish between the genotypes *1101/*1001 and *1104/*1501. According to the results, the HRM analysis method gave more accurate results than RFLP by differentiating between the BoLA-DRB3.2 genotypes. Due to the Co-dominant nature of the MHC alleles, HRM technique could be used for investigating the polymorphisms of genotypes and their associations with immune responses.