Allelic variant analysis of the DRB3 gene by PCR-RFLP in mediterranean buffaloes breed

Abstract

The DRB3 gene is a member of the mammalian major histocompatibility complex (MHC) class II cluster. Allelic variants of this gene have been associated with bovine leukemia virus infection and mastitis in cattle. In this study we searched for allelic variants in the exon 2 of the DRB3 gene using the method PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). DNA samples from 24 Mediterranean buffaloes were extracted from follicle bulb. For PCR amplification of the exon 2 we used one cattle derived primer pair previously described in the literature and successfully used to generate PCR products with buffalo DNA, including the breeds Murrah, Nili-Ravi, Surti, Jafarabadi and Carabao. The expected PCR product size with buffalo DNA was 304 bp, which was digested with three restriction enzymes, RsaI (restriction site: GT↓AC), PstI (restriction site: CTGCA↓G) and HaeIII (restriction site: GG↓CC). The results showed six polymorphic patterns with the enzyme RsaI: RsaI-A (237+67 bp), RsaI-B (183+121 bp), RsaI-C (114+93+67+30 bp), RsaI-D (144+93+67 bp), RsaI-E (144+67+54+39 bp) and RsaI-F (114+67+54+39+30 bp); four polymorphic patterns with the enzyme HaeIII: HaeIII-A (222+82 bp), HaeIII-B (170+82+52 bp), HaeIII-C (193+82+29 bp) and HaeIII-D (170+134 bp) and only one polymorphic pattern with the enzyme PstI: PstI-A (230+68+6 bp). The digestion with the enzyme HaeIII generated eight genotypes within the population, where 17 animals were classified as heterozygotes, with the following genotypes: HaeIII-A/HaeIII-D in five animals, HaeIIIB/ HaeIII-D in five animals, HaeIII-A/HaeIII-B in three animals, HaeIII-A/HaeIII-C in two animals and HaeIII-C/HaeIII-D observed in two animals. The RsaI restriction enzyme also generated eight genotypes, where 15 animals were heterozygotes. The most frequent genotype observed was RsaI-A /RsaI-B found in six animals. The PstI restriction enzyme generated one heterozygote genotype - PstI-A/304 bp - observed in three animals, and one homozygote genotype (304/304 bp) observed in 21 animals. This study provides some insight about the allelic variants observed in one population of Mediterranean buffalo breed in Brazil. The variant analysis showed low genetic variability among the individuals, possibly due to a high degree of inbreeding in the population. These facts are concerning, since the MHC cluster genes are the most polymorphic genes of the mammalian genome.