Relative abundance of tissue mRNA and association of the single nucleotide polymorphism of the goat NGF gene with prolificacy.

Nerve Growth Factor (NGF) promotes the development of pre-antral ovarian follicles through ovarian innervations and regulation of ovarian response to gonadotropins. The present study was conducted to study the tissue gene expression profile, to characterize the genetic variants, find associations of the NGF gene with prolificacy in the prolific Malabari and less prolific Attappady Black goats because NGF has an important role in reproduction by augmenting ovarian folliculogenesis. Relative abundance of NGF mRNA was greatest in reproductive tissues signifying its role in reproduction. The PCR-SSCP analysis of a 251bp fragment of Exon 3 of the NGF gene from the 277 goats revealed four diplotypes (EE, EF, FF and EG) with respective frequencies of 0.76, 0.22, 0.01 and 0.01. Sequencing of the representative samples revealed one synonymous and one novel non synonymous mutations (g.705G>A and g.715C>T). Statistical analysis indicated that the SNP g.705G>A was associated with litter size in Attappady Black goats (P<0.05) and a PCR-RFLP was designed using the restriction enzyme, BpiI, for rapid screening of the SNP. The results of the present study suggest that the NGF gene is a primary candidate gene affecting prolificacy in goats and may be used for Marker Assisted Selection (MAS) in goats, especially in lowly prolific Attappady Black goats.

Nerve Growth Factor gene ovarian expression, polymorphism identification, and association with litter size in goats.

The Nerve Growth Factor (NGF) plays an important role in reproduction by augmenting folliculogenesis. In this study, the coding regions of caprine NGF gene were analyzed to detect single-nucleotide polymorphisms (SNPs), their association with litter size, and the relative ovarian expression of NGF gene in the two indigenous goat breeds of South India viz., the prolific Malabari and less-prolific Attappady Black. The sequence analysis of the third exon containing the entire open reading frame of NGF gene was observed to be of 808 bp with one nonsynonymous mutation at 217th position. Later, polymerase chain reaction (PCR) was performed to amplify a region of 188 bp covering the region carrying the detected mutation. The genomic DNAs from the goats under study (n = 277) were subjected to PCR and single strand conformation polymorphism (SSCP). On analysis, four diplotypes viz., AA, AB, AC, and AD were observed with respective frequencies of 0.50, 0.22, 0.27, and 0.01. Sequencing of the representative samples revealed an additional synonymous mutation, i.e., g.291C>A. Statistical analysis indicated that NGF diplotypes and the SNP g.217G>A were associated with litter size in goats (P < 0.05). Relative expression of NGF gene was significantly higher in the ovaries of goats with history of multiple than single births (P < 0.05). The results of the present study suggest the significant effect of the NGF gene on litter size in goats and identified SNPs would benefit the selection of prolific animals in future marker-assisted breeding programs. The two novel PCR-restriction fragment length polymorphisms designed, based on the detected SNPs, would help in the rapid screening of large number of animals in a breeding population for identifying individual animals with desired genetic characteristics.

Molecular cloning, SNP detection and association analysis of 5' flanking region of the goat IGF1 gene with prolificacy.

The insulin-like growth factor 1 has an important role in reproduction, foetal development and growth. It regulates the secretion of gonadotrophin releasing hormone, stimulates ovarian function and steroidogenesis. The present study was conducted to characterise the 5' flanking region of goat IGF 1 gene, ascertain ovarian expression of the IGF1 gene, detect SNPs and assess the association with prolificacy in the two indigenous goat breeds of South India viz., low prolific Attappady Black and high prolific Malabari. The 5' flanking region of IGF1 gene was PCR amplified, cloned and sequenced from both breeds. Genotyping was performed in 277 goats from the two genetic groups using the PCR-Single Strand Conformational Polymorphism (SSCP) and the expression of the IGF1 gene in the ovary was analysed by quantitative real time PCR. The 5' flanking region of the IGF1 gene was 601 bp long and located at 450 bp upstream of the start codon. Sequence exhibited 97-99% similarity with that of the sheep, cattle and sika deer IGF1 genes. Three genotypes, PP, PQ and QR were observed at this locus with the frequency of 0.62, 0.30 and 0.08, respectively. Sequencing of the representative PCR products from each genotype revealed two SNPs, g.224A>G and g.227C>T. The population was found to be in Hardy-Weinberg disequilibrium at both loci. Statistical results indicated that these loci were associated with litter size (P ≤ 0.05). However, no significant difference was found in the expression of the IGF1 gene in the ovaries of the two goat breeds. These results suggest the significant influence of the IGF1 gene on prolificacy in goats and identified SNPs would benefit the selection of prolific animals in future breeding programs.