Characterization of BoLA class II DQA and DQB by PCR-RFLP, cloning, and sequencing reveals sequence diversity in crossbred cattle

The BoLA class II DQA and DQB genes in crossbred cattle were studied using PCR-RFLP, cloning, and sequencing techniques. Seventy-two crossbred cattle (Vrindavani) were used in the current study. HaeIII and XbaI restriction enzymes digested DQA exon 2-3, revealing seven (HaeIII-A-G) and three (XbaI A-C) motifs, respectively. The BoLA-DQB gene was analyzed using PCR-RFLP with PstI and TaqI restriction enzymes, yielding five restriction motifs for each restriction enzyme (PstI-A-E and TaqI-A-E). In crossbred cattle, addition, deletion, and substitutions were observed in distinct sequences, resulting in variations in overall gene length. Changes in nucleotides at positions 64-80, 110-200, and 207-264 were largely responsible for polymorphism in DQA exon 2. The phylogenetic analysis predicted a high degree of nucleotide and amino acid changes in DQA exon 2-3 and DQB exon 2. DQA genes had a nucleotide dissimilarity of 0.3-25.4 percent, while DQB genes had a nucleotide dissimilarity of 1.5-14.3 percent. We cloned and sequenced 20 genotypes based on PCR-RFLP of the DQA and DQB genes. The current study observed variation in the DQA and DQB genes and will serve as a foundation for future research on the BoLA DQA and DQB genes.