Two novel SNPs identified in STAT1 gene adjoining a QTL for milk production in Holstein Friesian crossbreds of Kerala.

We analyzed the effect of a single nucleotide polymorphism, g. C3141T in the 3' UTR of Signal transducer and activator of transcription-1 gene (STAT1) on milk production traits in the Holstein Friesian crossbred cattle of Kerala (n = 144) by association analysis and expression study. The population was genotyped by restriction fragment length polymorphism using Pag1. Association study using the General Linear Model-Analysis of Variance revealed that none of the yield or composition traits analyzed were significantly differed. The expression profile of STAT1 gene in leucocytes of animals bearing homozygous genotypes was compared by quantitative real time PCR using SYBR green chemistry with and relative expression was not found to be significantly differed. The second stage of the study, the STAT1 mRNA spanning 3213 bp was amplified from leucocytes and sequenced (GenBank: MT459802.1). Two novel SNPs were identified; one synonymous mutation in the coding region (g.A1212G) and the other in the 3'UTR (g.T3042C). The novel SNPs might contribute to STAT1 gene regulation mediated by alternate spicing or binding sites for regulatory molecules. The results reiterate the importance of extensive studies of STAT1 gene variants to substantiate the presence of a quantitative trait loci for dairy traits in the vicinity of STAT1 gene.

Sirtuin3 (SIRT3) gene molecular characterization and SNP detection in prolific and low prolific goat breeds.

Sirtuin3 (SIRT3) is a member of the Sirtuin family of NAD+-dependent deacetylase. They have evolved as a vital protein in preventing the activation of reactive oxygen species (ROS) in oocytes. A novel study on caprine SIRT3 was conducted, to characterize caprine SIRT3, to detect potential polymorphisms in SIRT3 and to analyze their association with litter size in the two indigenous goat breeds of India viz., the prolific Malabari and low prolific Attappady Black goats. A 1070 bp mRNA sequence of SIRT3 cDNA comprised of an ORF of 1002 bp encoding 333 amino acids, having 96% identity with bovine SIRT3. The genomic DNAs from the goats (n = 222) were subjected to PCR and single strand conformation polymorphism (SSCP) of exon 5 fragment (213 bp) of caprine SIRT3. On analysis, two genotypes viz., DD and DE were observed with frequencies of 0.63 and 0.37 respectively. Further sequencing of the PCR products of the respective genotypes revealed a novel synonymous SNP (MF176159:c.691C > T). Genotypes of this fragment had a significant influence on number of kids born (P < 0.05) with DD genotype being superior to DE genotype. These results highlight the role of SIRT3 in reproduction traits and the detected novel SNP would aid in the Marker Assisted Selection programmes and thus SIRT3 can considered as a potential candidate gene for reproduction traits in goats.

Evaluation of the association of SLC11A1 gene polymorphism with incidence of paratuberculosis in goats.

Paratuberculosis is one of the chronic granulomatous enteritis that predominantly affects ruminants world wide, caused by Mycobacterium avium ssp. paratuberculosis (MAP). In ruminants, microsatellite polymorphisms of the 3' untranslated region (3'UTR) of the solute carrier family 11 member A1 (SLC11A1) gene were associated with resistance to intracellular pathogen infections. This research was carried out to detect the polymorphisms in A and B regions of the 3'UTR of SLC11A1 gene and to evaluate the potential association between these polymorphisms and MAP infection in goats. MAP-specific antibodies were detected by ELISA and MAP infection was confirmed by IS900 PCR in 150 adult goats from different regions of Kerala, India. The polymorphism of microsatellite regions A and B at 3'UTR of the SLC11A1 gene was analysed in goats by an automated technique, fragment analysis, using fluorescent-tagged forward primers. Eight alleles with sizes ranging from 221 to 239 bp were found in region A. Region B revealed two alleles, 117 bp (B7) and 119 bp (B8). Animals with B8 alleles were found to have higher incidence of paratuberculosis than animals with B7 alleles (P < 0.01). There was no statistically significant association found between region A genotypes and paratuberculosis incidence. These results suggest that caprine SLC11A1 gene has significant role in paratuberculosis resistance in goats and further studies might help in development of a PCR-based genotyping test for paratuberculosis resistance and selection of superior animals for future goat breeding programmes.

cDNA cloning, structural analysis, SNP detection and tissue expression profile of the IGF1 gene in Malabari and Attappady Black goats of India.

Insulin-like growth factor 1 (IGF1) plays an important role in growth, reproduction, foetal development and cell proliferation. The present study was conducted to clone and sequence the full-length coding sequence of the caprine IGF1 gene from Attappady Black and Malabari breeds, two indigenous goat breeds of south India, to analyse its structure, and to ascertain the relative abundance of IGF1 mRNA in different tissues. The caprine IGF1 cDNA (GenBank accession nos: KJ549851 and KJ549852) contained a 465-bp open reading frame encoding IGF1 protein with 154 amino acid residues. A novel SNP was detected in the 3'UTR region, g.931A>G. Genotyping was performed in 277 goats from the two genetic groups using the PCR-single strand conformational polymorphism (SSCP) and two genotypes, AA and AG were observed at this locus. IGF1 is a secretary pathway protein with 49 amino acid-long signal peptide with 19 phosphorylation sites. Caprine IGF1 amino acid sequence was 83-99% identical to other species with highest identity with the ruminants. Relative expression of IGF1 was highest in uterus and liver (P < 0.05), followed by oviduct and muscle. This work provided an important experimental basis for further research on the functions of IGF1 in goats.

Association of a Cac8I polymorphism in the IGF1 gene with growth traits in Indian goats.

The Insulin-like Growth Factor 1 (IGF1) gene is a member of somatotropic axis and plays a key role in proliferation of cells, mitosis, myogenesis, meiosis, differentiation in foetal development and post natal growth. The objectives of this study were to verify the single nucleotide polymorphisms (SNPs) in IGF1 gene and their association with growth traits in two indigenous native goat genetic groups of Kerala, viz., Malabari and Attappady Black. A total of 277 goats were genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using the restriction enzyme Cac8I. One SNP, A224G was detected in the 5' non-coding region of the IGF1 gene, and accordingly two genotypes were revealed, GG and AG. This SNP was significantly associated with growth traits in Attappady Black goats, which is maintained as meat breed in Kerala. Results from this study demonstrated higher performance of GG animals for growth traits. The association of IGF1 gene with these traits emphasizes the importance of caprine IGF1 as a candidate gene for growth traits in goat breeding.

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.

Evaluation and comparison of native and recombinant LipL21 protein-based ELISAs for diagnosis of bovine leptospirosis

A 21-kDa leptospiral lipoprotein (LipL21) was evaluated for its diagnostic potential to detect bovine leptospirosis by ELISA. Both native LipL21 (nLipL21) and recombinant LipL21 (rLipL21) proteins were tested and compared regarding diagnostic efficiency, and no statistically significant difference was observed. The sensitivity of rLipL21 ELISA for 62 microscopic agglutination test (MAT) positive sera was 100% and the specificity with 378 MAT negative sera was 97.09%. Thus, rLipL21 protein-based ELISA could be used as an alternative to MAT for the diagnosis of bovine leptospirosis.