Molecular Characterization of Osmanabadi breed of goat at FecG loci

Abstract

The Osmanabadi breed of goat is one of the most prolific breeds of goat in Maharashtra. These goats are considered helpful for quality chevon and are known for better Prolificacy. Several mutations that increase ovulation rate in ewes have been discovered in the genes BMPR-1B (Bone Morphogenetic Protein Receptor 1B) known as FecB, BMP-15 (Bone Morphogenetic Protein 15) known as FecX, and GDF-9 (Growth Differentiation Factor 9) known as FecG. These fecundity genes have posed a unique and exciting opportunity to attain a high level of Prolificacy in sheep and goats. These genes have proved to be trackable and persistent in a breed. Fecundity traits offer a new way to allow certain breeds to obtain a high level of Prolificacy. Since there is a high degree of homogeneity between ovine and caprine genomes, an attempt is made to study the known polymorphic points of one of the important fecundity genes, GDF-9, in Osmanabadi goats. Considering the variation in the fecundity of Osmanabadi goats, the present research work was planned to genotype Osmanabadi goats at FecG loci. The known eight DNA variants in ovine GDF-9, including G1, G4, G6, G7, G8 (FecGH), FecTT, and G1189A were studied in the present investigation. These mutations result in amino acid changes in ovine GDF-9 viz., G1 (G260A; R87H), G4 (G721A; E241K), G6 (G994A; V332I), G7 (G1111A; V371M), FecGE (T1034G; F345C) FecTT (A1279C; S109R) and G1189A (V397I). In the present study, 165 Osmanabadi goat breed blood samples (162 females and three males) were collected from the Sheep and Goat farm maintained at Mahud, Dist. Solapur (M.S.) by Punyashlok Aahilyadevi Maharashtra Sheep and Goat Development Corporation Ltd., Pune, were genotyped at FecG loci following DNA isolation. PCR amplification of FecG loci of the GDF-9 gene was carried out using the genomic DNA template. The PCR was performed using tetra-primer sets specific to each locus. A simple tetra primer amplification refractory mutation system (T-ARMS) PCR was adopted to screen known ovine fecundity gene GDF-9 polymorphism in the caprine GDF-9. On several attempts to use different annealing temperatures and/ or various reaction mixtures, T-ARMS-PCR of the G7 locus could amplify only the control fragment at 343 bp. Therefore, the control PCR product of 20 representative individuals was sent for direct sequencing using G7 OF primer. The sequence analysis of the G7 T-ARMS-PCR control fragment also enabled us to analyze the other FecG mutations, viz. FecGE; T1034G (FecGSI) and FecTT; A1279C. The G1189A mutation in the caprine GDF-9 gene was also studied based on sequencing results of the G7 control fragment. The observed bands of T-ARMS-PCR on agarose gel electrophoresis at the G1 locus represented a wild-type fragment at 247 bp and a control fragment at 396 bp. Similar results were found for other loci G4 (261 bp and 417 bp), G6 (223 bp and 362 bp), and G8 (108 bp and 198 bp) corresponding to wild-type and control fragments representing normal non-carrier animals at respective loci. The sequenced Osmanabadi goats revealed wild-type alleles at G7, FecGE, FecTT and G1189A loci representing non-carrier goats. In the present investigation, Osmanabadi goats are observed as monomorphic, carrying wild-type alleles at the G1, G4, G6, G7, G8 (FecGH), FecGE, FecTT and G1189A loci in the GDF-9 gene.