PCR-SCCP and sequence analysis of leptin gene reveals novel polymorphism in intron 1 and allele fixation in exon 2 of Indian buffaloes (Bubalus bubalis)

The present study was undertaken to delineate single nucleotide polymorphisms within exon 2 and its flanking regions of bubaline leptin gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. A total of 118 buffaloes from seven different breeds were studied, revealing three distinct SSCP variants, viz., A, B and C, with a frequency of 0.703, 0.195 and 0.102, respectively. Sequence analysis of the region revealed 9 nucleotide variations including 8 transitions and one transversion as compared to cattle. Two A-G mutations within the coding DNA region were non-synonymous, resulting in change of amino acids. Basic local alignment search tool (BLAST) analysis of bubaline leptin gene revealed a sequence homology of 95-96% with bovines, caprines and ovines, while it varied from 86% to 93% with other farm animal species. Sequence analysis of SSCP variants showed a single nucleotide polymorphism (SNP) within intron 1 with the variant ‘A’ having homozygous ‘T’ allele and variant ‘B’ having homozygous ‘G’ allele, while variant ‘C’ having heterozygous ‘T/G’ alleles. The identified SNP locus was found to deviate from Hardy-Weinberg equilibrium with less than expected frequency of heterozygotes. The study also found no variation within exon 2 and complete fixation of unfavorable allele ‘C’, which has been earlier reported to be associated with lower milk and protein yield in cattle.