Dharamshibhai N. Rank

Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells.

Myostatin (MSTN), a member of transforming growth factor-β (TGF-β) superfamily, is a negative regulator of the skeletal muscle growth, and suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or genetic manipulation (knockout or knockdown) has been reported to interrupt its proper function and to increase the muscle mass in many mammalian species. RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. In the present study transient silencing of MSTN gene in chicken embryo fibroblast cells was evaluated using five different shRNA expression constructs. We report here up to 68% silencing of myostatin mRNA using these shRNA constructs in transiently transfected fibroblasts (p<0.05). This was, however, associated with induction of interferon responsive genes (OAS1, IFN-β) (3.7-64 folds; p<0.05). Further work on stable expression of antimyostatin shRNA with minimum interferon induction will be of immense value to increase the muscle mass in the transgenic animals.

Detection of virulence determinants by real time PCR in E. coli isolated from mutton.

Ninety three Escherichia coli isolates belonging to 35 serotypes isolated from market mutton were tested to find out the prevalence of virulence determinants, Verotoxin 1 (VT1), Verotoxin 2 (VT2), Intimin (eae) genes and enterohemolysin production. Real Time PCR based detection was carried out for virulence genes using SYBR green format and amplicons were confirmed by melt curve analysis. Prevalence of VT1 gene in these isolates was much higher (38.70%) on the other hand, that of VT2 gene was nil (0%) while that of eae was very low (3.22%). Enterohemolysin production was found in 31.18% isolates when tested on washed sheep blood agar supplemented with CaCl(2). All enterohemolysin producing isolates were also positive for the VT1 gene.

Evaluation of genetic diversity and population structure of west-central Indian cattle breeds

Evaluations of genetic diversity in domestic livestock populations are necessary to implement region-specific conservation measures. We determined the genetic diversity and evolutionary relationships among eight geographically and phenotypically diverse cattle breeds indigenous to west-central India by genotyping these animals for 22 microsatellite loci. A total of 326 alleles were detected, and the expected heterozygosity ranged from 0.614 (Kenkatha) to 0.701 (Dangi). The mean number of alleles among the cattle breeds ranged from 7.182 (Khillar) to 9.409 (Gaolao). There were abundant genetic variations displayed within breeds, and the genetic differentiation was also high between the Indian cattle breeds, which displayed 15.9% of the total genetic differentiation among the different breeds. The genetic differentiation (pairwise FST ) among the eight Indian breeds varied from 0.0126 for the Kankrej-Malvi pair to 0.2667 for Khillar-Kenkatha pair. The phylogeny, principal components analysis, and structure analysis further supported close grouping of Kankrej, Malvi, Nimari and Gir; Gaolao and Kenkatha, whereas Dangi and Khillar remained at distance from other breeds.

Tissue-specific temporal exome capture revealed muscle-specific genes and SNPs in Indian buffalo (Bubalus bubalis).

Whole genome sequencing of buffalo is yet to be completed, and in the near future it may not be possible to identify an exome (coding region of genome) through bioinformatics for designing probes to capture it. In the present study, we employed in solution hybridization to sequence tissue specific temporal exomes (TST exome) in buffalo. We utilized cDNA prepared from buffalo muscle tissue as a probe to capture TST exomes from the buffalo genome. This resulted in a prominent reduction of repeat sequences (up to 40%) and an enrichment of coding sequences (up to 60%). Enriched targets were sequenced on a 454 pyro-sequencing platform, generating 101,244 reads containing 24,127,779 high quality bases. The data revealed 40,100 variations, of which 403 were indels and 39,218 SNPs containing 195 nonsynonymous candidate SNPs in protein-coding regions. The study has indicated that 80% of the total genes identified from capture data were expressed in muscle tissue. The present study is the first of its kind to sequence TST exomes captured by use of cDNA molecules for SNPs found in the coding region without any prior sequence information of targeted molecules.

Cloning, molecular modeling and characterization of acidic cellulase from buffalo rumen and its applicability in saccharification of lignocellulosic biomass

Cellulase hydrolyses the cellulose by cleaving the β-1,4-linkages to produce mono-, oligo- and shorter polysaccharide units. These enzymes have applications in various industries such as pulp and paper, laundry, food and feed, textile, brewing industry and in biofuel production. In the present study we have cloned acid-cellulase gene (Cel-1) from the fosmid library of buffalo rumen metagenomic DNA and functionally expressed it in Escherichia coli. The ORF encoding cellulase consisted of 1176-bp, corresponding to protein of 391 amino acid and has catalytic domain belonging to glycosyl hydrolase family 5. The purified protein has a molecular weight of 43-kDa on SDS-PAGE and its expression was confirmed by western blotting. The tertiary structure of the cellulase (Cel-1) showed a classical (α/β) TIM-like barrel motif. Model surface charge of Cel-1 predicted that surface near active site was mostly negative which might be responsible for the stability of enzyme at lower pH. The pH and temperature for maximum enzyme activity were 4.5 and 45°C respectively. Various metal ions enhanced the enzyme activity and in presence of Mn+2 activity was significantly increased. Cel-1 hydrolyzed pre-treated wheat straw and released reducing sugars (62.60%). These desirable properties of Cel-1 make it attractive for the bioconversion of biomass.

Microbial diversity and community composition of caecal microbiota in commercial and indigenous Indian chickens determined using 16s rDNA amplicon sequencing

BackgroundThe caecal microbiota plays a key role in chicken health and performance, influencing digestion and absorption of nutrients, and contributing to defence against colonisation by invading pathogens. Measures of productivity and resistance to pathogen colonisation are directly influenced by chicken genotype, but host driven variation in microbiome structure is also likely to exert a considerable indirect influence.MethodsHere, we define the caecal microbiome of indigenous Indian Aseel and Kadaknath chicken breeds and compare them with the global commercial broiler Cobb400 and Ross 308 lines using 16S rDNA V3-V4 hypervariable amplicon sequencing.ResultsEach caecal microbiome was dominated by the genera Bacteroides, unclassified bacteria, unclassified Clostridiales, Clostridium, Alistipes, Faecalibacterium, Eubacterium and Blautia. Geographic location (a measure recognised to include variation in environmental and climatic factors, but also likely to feature varied management practices) and chicken line/breed were both found to exert significant impacts (p < 0.05) on caecal microbiome composition. Linear discriminant analysis effect size (LEfSe) revealed 42 breed-specific biomarkers in the chicken lines reared under controlled conditions at two different locations.ConclusionChicken breed-specific variation in bacterial occurrence, correlation between genera and clustering of operational taxonomic units indicate scope for quantitative genetic analysis and the possibility of selective breeding of chickens for defined enteric microbiota.

Single nucleotide variant detection in Jaffrabadi buffalo (Bubalus bubalis) using high-throughput targeted sequencing

The water buffalo is among the most important livestock species of southern Asia, contributing greatly to the ecosystem and rural livelihood of the region. The identification of large-scale single nucleotide polymorphisms in this species would greatly facilitate our understanding of the genetic basis of economically important traits such as milk production, fertility traits and general health traits. The present study investigated the cost-effective method of exome capture and single nucleotide variant (SNV) identification from genomic DNA of Jaffrabadi buffalo using biotin-labelled cDNA as probes. Sequencing of enriched fragments generated 608 Mb of data, which was mapped to a Bos taurus genome assembly followed by variant calling and annotation. Furthermore, 393 coding SNVs were identified, leading to 143 non-synonymous substitutions (nsSNVs) in 75 genes. Of the 75 nsSNV-containing genes, four matched the genes that have previously been reported to be potentially associated with economically important traits such as milk production and meat production. Furthermore, functional annotation using gene ontology (GO) enrichment identified categories such as glutamate receptor activity (GO: 0008066) enriched in the fertility trait samples. These results provide a framework for the application of cost-effective methods of target capture in SNV detection from non-model organisms such as the water buffalo.