Genotyping of MHC class II DRB3 gene using PCR-RFLP and DNA sequencing in small ruminant breeds of Western Himalayan state of Himachal Pradesh, India

Abstract

Sheep and goat farming especially under migratory system is an important activity in state of Himachal Pradesh. DRB locus of major histocompatibility complex is widely studied for its association with disease resistance. Present study was planned to analyse genetic diversity of DRB3 locus in small ruminant breeds of Himachal Pradesh using PCR-RFLP and DNA sequencing. 63, 68, 80 and 73 true to the breed animals belonging to Gaddi sheep, Rampur-Bushair sheep, Gaddi goats and Chegu goats, respectively were utilized. Amplification of exon 2 of DRB3 gene yielded 285bp amplified product in different breeds. Three different digestion patterns corresponding to 2 alleles and seven digestion patterns corresponding to 4 alleles were observed with PstI and HaeIII restriction digestion. For Pst 1 digestion frequency of AA, AB and BB genotypes ranged from 0.09 to 0.22, 0.40 to 0.53 and 0.28 to 0.46 in different breeds studied. For HaeIII digestion frequency of five genotypes (AA, BB, CC, AB and AC) which were detected in all breeds varied from 0.18 to 0.32, 0.11 to 0.13, 0.07 to 0.17, 0.18 to 0.30 and 0.09 to 0.3 in different breeds, while frequency of CD and AD genotype unique to Rampur-Bushair sheep population was 0.09 and 0.06, respectively. The observed allele number (No) ranged from 5 to 6 in different breeds while effective allele number (Ne) ranged from 4.46 to 5.15 in different populations. Hobs and Hexp values ranged 0.42 to 0.49 and 0.55 to 0.59, respectively in different breeds. The nucleotide variability was detected using sequences from different breeds which was found at 36 places. In the present study the specific amplification of the exon 2 of DRB3 gene of native sheep and goat populations demonstrated marked polymorphism. Further association studies need to be carried out to investigate the association of these SNPs of DRB region with parasitic diseases.