Nucleotide sequencing and DNA polymorphism studies of BMP 15 gene in Corriedale and local Kashmir valley sheep (Ovis aries)

The families of TGF-β proteins are the most important growth factors in the ovary for growth and differentiation of early ovarian follicles. Three related oocyte-derived members of the transforming growth factor-β superfamily, namely GDF9, BMP15 and BMPR-IB have been shown to be essential for follicular growth and ovulation. The objective of the present study was to detect the incidence of mutation in intronic portion of BMP 15 gene in Corriedale and local Kashmir valley sheep breeds. Blood samples were collected from 85 ewes and genomic DNA was extracted using the modified phenol chloroform method. The quantity and quality of extracted DNA was examined using spectrophotometry and gel electrophoresis, respectively. A fragment with the size of 356 bp was amplified using polymerase chain reaction (PCR) with a pair of specific primers. The amplified PCR products were digested with Mph11031 restriction enzyme. In the presence of mutation at this locus, the Mph11031 enzyme cannot recognize the restriction site. However, here in the absence of mutations, the enzyme recognizes one restriction site and divides the amplified fragment into two fragments of 152 and 204 bp. The 356 bp fragment was also analyzed for polymorphism by SSCP technique. The results indicated two different banding patterns AA and AB for this fragment. Later on two different allelic forms A and B were confirmed by nucleotide sequencing. The 356 bp nucleotide sequence was subjected to alignment analysis and it was observed that sequence similarity of this fragment with that of other sheep and Jining grey goat was more than 97.8%. Phylogenetic analysis revealed that both designated A and B alleles as well as published sequence of sheep form a common cluster indicating their evolutionary closeness. The origin of Jining grey goat was located some distance away from the sheep. The overall frequencies of AA and AB genotypes were 0.79 and 0.21. The breed wise frequencies were 0.78 and 0.22 in Corriedale sheep and the frequencies in Kashmir valley sheep were 0.80 and 0.20 for AA and AB genotypes, respectively. The overall allelic frequencies of A and B alleles were 0.89 and 0.11 whereas allelic frequencies Corriedale sheep was 0.89 and 0.11 and that of Kashmir valley sheep were 0.90 and 0.10.