A genome-wide approach to screen for genetic variants in broilers (Gallus gallus) with divergent feed conversion ratio.

Feed conversion ratio (FCR) is an economically important trait in broilers and feed accounts for a significant proportion of the costs involved in broiler production. To explore the contribution of functional variants to FCR trait, we analyzed coding and non-coding single-nucleotide variants (SNVs) across the genome by exome sequencing in seven pairs of full-sibs broilers with divergent FCR and with a sequence coverage at an average depth of fourfold. We identified 192,119 high-quality SNVs, including 30,380 coding SNVs (cSNVs) in the experimental population. We discovered missense SNVs in PGM2, NOX4, TGFBR3, and TMX4, and synonymous SNVs in TSNAX, ITA, HSP90B1, and COL18A1 associated with FCR. Haplotype analyses of genome-wide significant SNVs in PGM2, PHKG1, DGKZ, and SOD2 were also observed with suggestive evidence of haplotype association with FCR. Single-variant and FCR QTL-related genes-based association analyses of SNVs identified newly associated genes for FCR in the regions subjected to targeted exome sequencing. The top seven SNVs were next evaluated in independent replication data sets where SNV chr. 3: 13,990,160 (c. 961G>C) at TMX4 was replicated (p < 0.05). Collectively, we have detected SNVs associated with FCR in broiler as well as identification of SNVs in known FCR QTL region. These findings should facilitate the discovery of causative variants for FCR and contribute to marker-assisted selection.

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.

Human leukocyte antigen alleles, genotypes and haplotypes frequencies in renal transplant donors and recipients from West Central India.

BACKGROUND: Human leukocyte antigen (HLA) is comprised of a highly polymorphic set of genes which determines the histocompatibility of organ transplantation. The present study was undertaken to identify HLA class I and class II allele, genotype and haplotype frequencies in renal transplant recipients and donors from West Central India. MATERIALS AND METHODS: HLA typing was carried out using Polymerase Chain Reaction-Sequence Specific Primer in 552 live related and unrelated renal transplant recipients and donors. RESULTS: The most frequent HLA class I and class II alleles and their frequencies in recipients were HLA-AFNx0101 (0.1685) and AFNx0102 (0.1649), HLA-BFNx0135 (0.1322), and HLA-DR beta 1 (DRB 1)FNx0115 (0.2192), whereas in donors, these were HLA-AFNx0102 (0.1848) and AFNx0101 (0.1667), HLA-BFNx0135 (0.1359), and HLA-DRB1FNx0115 (0.2409). The two-locus haplotype statistical analysis revealed HLA-AFNx0102-B61 as the most common haplotype with the frequency of 0.0487 and 0.0510 in recipients and donors, respectively. Further, among the three locus haplotypes HLA-AFNx0133-BFNx0144-DRB1FNx0107 and HLA-AFNx0102-BFNx0161-DRB1FNx0115 were the most common haplotypes with frequencies 0.0362 and 0.0326, respectively in recipients and 0.0236 and 0.0323, respectively in donors. Genotype frequency revealed a high prevalence of genotype HLA-AFNx0102/AFNx0124 in recipients (0.058) compared to donors (0.0109) whereas low prevalence of HLA-AFNx0101/AFNx0102 in recipients (0.0435) than in donors (0.0797). The phylogenetic and principal component analysis of HLA allele and haplotype frequency distribution revealed genetic similarities of various ethnic groups. Further, case control analysis provides preliminary evidence of association of HLA-A genotype (P < 0.05) with renal failure. CONCLUSION: This study will be helpful in suitable donor search besides providing valuable information for population genetics and HLA disease association analysis.

High-throughput genotype-based population structure analysis of selected buffalo breeds.

India is considered as the home tract of some of the best buffalo breeds. However, the genetic structure of the Indian river buffalo is poorly understood. Hence, there is a need to characterize the populations and understand the genetic structure of various buffalo breeds for selection and to design breeding strategies. In this study, we have analyzed genetic variability and population structure of seven buffalo breeds from their respective geographical regions using Axiom Buffalo Genotyping Array. Diversity, as measured by expected heterozygosity, ranged from 0.364 in Surti to 0.384 in Murrah breed, and pair-wise F ST values revealed the lowest genetic distance between Murrah and Nili-Ravi (0.0022), while the highest between Surti and Pandharpuri (0.030). Principal component analysis and structure analysis unveiled the differentiation of Surti, Pandharpuri, and Jaffarabadi in first two principal components and at K = 4, respectively, while remaining breeds were grouped together as a separate single cluster and admixed. Murrah and Mehsana showed early linkage disequilibrium (LD) decay, while Surti breed showed late decay. In LD blocks to quantitative trait locis (QTLs) concordance analysis, 4.65% of concordance was observed with 873 LD blocks overlapped with 2,330 QTLs. Overall, total 4,090 markers were identified from all LD blocks for six types of traits. Results of this study indicated that these single-nucleotide polymorphism (SNP) markers could differentiate phenotypically distinct breeds like Surti, Pandharpuri, and Jaffarabadi but not others. So, there is a need to develop SNP chip based on SNP markers identified by sequence information of local breeds.

The genome of walking catfish Clarias magur (Hamilton, 1822) unveils the genetic basis that may have facilitated the development of environmental and terrestrial adaptation systems in air-breathing catfishes.

The walking catfish Clarias magur (Hamilton, 1822) (magur) is an important catfish species inhabiting the Indian subcontinent. It is considered as a highly nutritious food fish and has the capability to walk to some distance, and survive a considerable period without water. Assembly, scaffolding and several rounds of iterations resulted in 3,484 scaffolds covering ∼94% of estimated genome with 9.88 Mb largest scaffold, and N50 1.31 Mb. The genome possessed 23,748 predicted protein encoding genes with annotation of 19,279 orthologous genes. A total of 166 orthologous groups represented by 222 genes were found to be unique for this species. The Computational Analysis of gene Family Evolution (CAFE) analysis revealed expansion of 207 gene families and 100 gene families have rapidly evolved. Genes specific to important environmental and terrestrial adaptation, viz. urea cycle, vision, locomotion, olfactory and vomeronasal receptors, immune system, anti-microbial properties, mucus, thermoregulation, osmoregulation, air-breathing, detoxification, etc. were identified and critically analysed. The analysis clearly indicated that C. magur genome possessed several unique and duplicate genes similar to that of terrestrial or amphibians' counterparts in comparison to other teleostean species. The genome information will be useful in conservation genetics, not only for this species but will also be very helpful in such studies in other catfishes.

Effect of the pH in the enrichment of X or Y sex chromosome-bearing sperm in bovine.

BACKGROUND AND AIM: Studies have shown that the pH of the vagina during the course of fertilization may influence the migration of X- and Y-bearing spermatozoa and thus leading to skewness in the sex of the offspring. Hence, this study was carried out to check the effect of the pH in the enrichment of X or Y sex chromosome-bearing sperm in bovine (Bos indicus). MATERIALS AND METHODS: To check the effect of pH in the enrichment of X or Y sex chromosome-bearing sperm in bovine, we used buffers of various pH ranging from 5.5 to 9.0 for swim-up procedure of sperm sample and collected upper and bottom fraction from the same buffer and checked the abundance of X- and Y-bearing spermatozoa by droplet digital polymerase chain reaction using X- and Y-chromosome-specific DNA probe. RESULTS: The abundance of X- and Y-bearing spermatozoa was not differed significantly in either of the fraction collected. CONCLUSION: Thus, it appears to be unlikely that an immediate impact of pH on sperm can be a solitary impact on the sex of offspring in bovine.

Insight into bovine (Bos indicus) spermatozoal whole transcriptome profile.

Mature spermatozoa harbor both coding and non-coding type of RNAs which regulates spermatogenesis, fertilization and early development. Characterization of bovine sperm transcriptome can provide more insight into the molecular mechanisms involved in these processes. Here, we have analyzed whole transcriptome profile of Bos indicus spermatozoa to access the global RNA expression. RNA-Seq analysis identified 14,306 genes expressed with FPKM >0, while 405 genes expressed when threshold increased to FPKM >5. Functional annotations showed that sperm transcripts were associated with molecular processes (translation, ribosomal small and large subunit assembly) and cellular components (cytosolic small and large ribosomal subunit and membranes) related to known sperm functions at fertilization and spermatogenesis. The RNA-Seq data was validated using droplet digital PCR where both highly abundant gene viz. RN7SL1 and less abundant gene viz. ZFP280B were validated. This study may provide future directions in reproductive biology of Bos indicus.

Host transcriptome and microbiome interaction modulates physiology of full-sibs broilers with divergent feed conversion ratio.

Efficient livestock production relies on effective conversion of feed into body weight gain (BWG). High levels of feed conversion are especially important in production of broiler chickens, birds reared for meat, where economic margins are tight. Traits associated with improved broiler growth and feed efficiency have been subjected to intense genetic selection, but measures such as feed conversion ratio (FCR) remain variable, even between full siblings (sibs). Non-genetic factors such as the composition and function of microbial populations within different enteric compartments have been recognized to influence FCR, although the extent of interplay between hosts and their microbiomes is unclear. To examine host-microbiome interactions we investigated variation in the composition and functions of host intestinal-hepatic transcriptomes and the intestinal microbiota of full-sib broilers with divergent FCR. Progeny from 300 broiler families were assessed for divergent FCR set against shared genetic backgrounds and exposure to the same environmental factors. The seven most divergent full-sib pairs were chosen for analysis, exhibiting marked variation in transcription of genes as well as gut microbial diversity. Examination of enteric microbiota in low FCR sibs revealed variation in microbial community structure and function with no difference in feed intake compared to high FCR sibs. Gene transcription in low and high FCR sibs was significantly associated with the abundance of specific microbial taxa. Highly intertwined interactions between host transcriptomes and enteric microbiota are likely to modulate complex traits like FCR and may be amenable to selective modification with relevance to improving intestinal homeostasis and health.

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

BACKGROUND: The 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. METHODS: Here, 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. RESULTS: Each 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. CONCLUSION: Chicken 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.

Analysis of consequences of non-synonymous SNP in feed conversion ratio associated TGF-ß receptor type 3 gene in chicken.

The recent advances in high throughput sequencing technology accelerate possible ways for the study of genome wide variation in several organisms and associated consequences. In the present study, mutations in TGFBR3 showing significant association with FCR trait in chicken during exome sequencing were further analyzed. Out of four SNPs, one nsSNP p.Val451Leu was found in the coding region of TGFBR3. In silico tools such as SnpSift and PANTHER predicted it as deleterious (0.04) and to be tolerated, respectively, while I-Mutant revealed that protein stability decreased. The TGFBR3 I-TASSER model has a C-score of 0.85, which was validated using PROCHECK. Based on MD simulation, mutant protein structure deviated from native with RMSD 0.08 Å due to change in the H-bonding distances of mutant residue. The docking of TGFBR3 with interacting TGFBR2 inferred that mutant required more global energy. Therefore, the present study will provide useful information about functional SNPs that have an impact on FCR traits.

Transcriptome analysis of the adult rumen fluke Paramphistomum cervi following next generation sequencing.

Rumen flukes are parasitic trematodes (Platyhelminthes: Digenea) of major socioeconomic importance in many countries. Key representatives, such as Paramphistomum cervi, can cause "Rumen fluke disease" or paramphistomosis and undermine economic animal productivity and welfare. P. cervi is primarily a problem in sheep, goat and buffalo production as a consequence of reduced weight gain and milk production, clinical disease or death. Recent technological advances in genomics and bioinformatics now provide unique opportunities for the identification and pre-validation of drug targets and vaccines through improved understanding of the biology of pathogens such as P. cervi and their relationship with their hosts at the molecular level. Here, we report next generation transcriptome sequencing analysis for P. cervi. RNAseq libraries were generated from RNA extracted from 15 adult P. cervi parasites sampled from each of three different host species (sheep, goat and buffalo) and a reference transcriptome was generated by assembly of all Ion Torrent PGM sequencing data. Raw reads (7,433,721 in total) were initially filtered for host nucleotide contamination and ribosomal RNAs and the remaining reads were assembled into 43,753 high confidence transcript contigs. In excess of 50% of the assembled transcripts were annotated with domain- or protein sequence similarity derived functional information. The reference adult P. cervi transcriptome will serve as a basis for future work on the biology of this important parasite. Using the widely investigated trematode virulence factor and vaccine candidate Cathepsin L as an example, the epitope GPISIAINA was found to be conserved in P. cervi isolated from three different host species supporting its candidacy for vaccine development and illustrating the utility of the adult P. cervi transcriptome.

Evaluation of a topical herbal drug for its in-vivo immunomodulatory effect on cytokines production and antibacterial activity in bovine subclinical mastitis.

BACKGROUND: Antibiotics have been in use in the treatment of bovine mastitis since decades; however, their use is associated with cost issues and human health concern. Use of herbal drugs does not generally carry these disadvantages. Many plants/herbs have been evaluated in the treatment of bovine mastitis with additional property of immunomodulation in affected mammary gland. AIM: To evaluate a topical herbal drug in two breeds of cattle for its in-vivo immunomodulatory effect on cytokines production and antibacterial activity in bovine subclinical mastitis. MATERIALS AND METHODS: The response to treatment was evaluated by enumerating somatic cell count (SCC), determining total bacterial load, and studying the expression of different cytokines (interleukin [IL]-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor, interferon (IFN)-γ and tumor necrosis factor [TNF]-α). RESULTS: The pre- and post-treatment SCC in mastitic quarters statistically did not differ significantly, however, total bacterial load declined significantly from day 0 onwards in both the breeds. Highly significant differences (P < 0.01) were observed in all the cytokines on day 0, 5, and 21 postlast treatment in both the breeds. The expression level of all the cytokines showed a significant increase on day 5, while a decrease was noticed on day 21 in both the breeds of cattle. The comparison of cytokine expression profiles between crossbred and Gir cattle revealed a significant difference in expression of IL-6 and TNF-α. However, other cytokines exhibited a similar pattern of expression in both breeds, which was non-significant. CONCLUSION: The topical herbal drug exhibited antibacterial and immunomodulatory activities in subclinical mastitis and thus the work supports its use as alternative herbal therapy against subclinical udder infection in bovines.

Metagenome of Mehsani buffalo rumen microbiota: an assessment of variation in feed-dependent phylogenetic and functional classification.

AIM: To gain a greater understanding of the ecology and metabolic potential of the rumen microbiome with the changes in the animal diet. METHODS: Diet composed of varying proportion of green and dry roughages along with grains was given to 8 Mehsani buffaloes, and rumen metagenome was sketched using shotgun semiconductor sequencing. RESULTS: In the present study, the Bacteroidetes were found to be dominant at the phyla level and Prevotella at the genus level. The ratio of Firmicutes to Bacteroidetes was found to be higher in the solid fraction as compared to the liquid fraction. In the solid fraction of the dry roughage group, the significant increment (p < 0.05) in Bacteroidetes abundance was observed with increment of roughage concentration. At the genus level, Clostridium significantly increased with the increment in roughage concentration. A comparison of glycoside hydrolase and cellulosome functional genes revealed more glycoside hydrolase 3 encoding genes with higher fiber diet and significant difference in carbohydrate-active enzymes family composition between green and dry roughage groups of the liquid fraction. CONCLUSION: The present study provides a base to understand the modulating behavior of microbiota which can be manipulated to improve livestock nutrient utilization efficiency and for targeting the efficient catabolism of complex carbohydrate molecules as well.

Transcriptomic dissection of myogenic differentiation signature in caprine by RNA-Seq.

Muscle growth and development from the embryonic to the adult stage of an organism consists of a series of exquisitely regulated and orchestrated changes in expression of genes leading to muscle maturation. In this study, we performed whole transcriptome profiling of adult caprine skeletal muscle derived myoblast and fused myotubes. Using Ion Torrent PGM sequencing platform, a total of 948,776 and 799,976 reads were generated in myoblasts and fused myotubes, respectively. The sequence reads were analyzed on CLC Genomics Workbench using Bos taurus RNA database to study the gene expression in both stages to study different genes responsible for muscle development and regeneration. The up and down-regulated genes were analyzed for gene ontology (GO) and KEGG pathways by Database for Annotation, Visualization and Integrated Discovery (DAVID) database. We found many genes exclusive to multinuclear fused myotubes and contractile nature of skeletal muscle, whereas up-regulated genes in myoblast stage were related to cell division and transcriptional regulation. Out of 27 genes selected for expression validation by RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), 19 genes showed the expression pattern comparable with CLC Genomics Workbench findings. Further, mRNA originated muscle specific microRNAs (miRNA-1 and miRNA-133b) were also observed in the fused myotubes along with other miRNAs with possible importance in muscle development. This study highlights important genes responsible for muscle development and differentiation in adult skeletal muscle system.

Microbial and Carbohydrate Active Enzyme profile of buffalo rumen metagenome and their alteration in response to variation in the diet.

Rumen microbiome represents rich source of enzymes degrading complex plant polysaccharides. We describe here analysis of Carbohydrate Active Enzymes (CAZymes) from 3.5 gigabase sequences of metagenomic data from rumen samples of Mehsani buffaloes fed on different proportions of green or dry roughages to concentrate ration. A total of 2597 contigs encoding putative CAZymes were identified by CAZyme Analysis Toolkit (CAT). The phylogenetic analysis of these contigs by MG-RAST revealed predominance of Bacteroidetes, followed by Firmicutes, Proteobacteria, and Actinobacteria phyla. Moreover, a higher abundance of oligosaccharide degrading and debranching enzymes in buffalo rumen metagenome and that of cellulases and hemicellulases in termite hindgut was observed when we compared glycoside hydrolase (GH) profile of buffalo rumen metagenome with cow rumen, termite hindgut and chicken caecum metagenome. Further, comparison of microbial profile of green or dry roughage fed animals showed significantly higher abundance (p-value<0.05) of various polysaccharide degrading bacterial genera like Fibrobacter, Prevotella, Bacteroides, Clostridium and Ruminococcus in green roughage fed animals. In addition, we found a significantly higher abundance (p-value<0.05) of enzymes associated with pectin digestion such as pectin lyase (PL) 1, PL10 and GH28 in green roughage fed animals. Our study outlines CAZyme profile of buffalo rumen metagenome and provides a scope to study the role of abundant enzyme families (oligosaccharide degrading and debranching enzymes) in digestion of coarse feed.

RNA-Seq reveals differentially expressed isoforms and novel splice variants in buccal mucosal cancer.

Buccal mucosal cancer (BMC) is a multifactorial disease with poorly defined genetic profile and prognosis due to late detection stage and unavailability of reliable prognostic markers. To identify aberrant transcriptional events, we employed high throughput RNA-Seq analysis of BMC and normal tissue. Comparative transcriptome analysis with Cufflinks revealed 260 up and 328 down regulated genes whereas, 350 up and 397 down regulated isoforms by at least two folds over buccal normal in BMC. Study revealed 46 splice variants in normal and 106 in cancer, out of which 10 variants were validated with end point RT-PCR. Expression of two isoforms of CD74 was validated using RT-qPCR and found in accordance with RNA-Seq. Further extensive follow up analysis of modulator genes, isoforms and splice variants found in this study, might be useful in deep understanding of pathological changes in BMC and development of prospective intervention strategies.

The landscape of alternative splicing in buccal mucosa squamous cell carcinoma.

OBJECTIVES: Alternative splicing (AS) is a key regulatory mechanism in the process of protein synthesis generating transcriptome and proteome diversity. In this study, we attempted to identify alternative splicing in a pair of BMSCC cancer and adjacent normal tissue using RNAseq datasets and also assessed the potential of these datasets to provide quantitative measurements for alternative splicing levels. MATERIALS AND METHODS: We performed high-throughput sequencing of buccal mucosal cancer and healthy tissue cDNA library which resulted in a transcriptome map of BMSCC cancer. RNAseq analysis was performed to assess alternative splicing complexity in cancer tissue and to search splice junction sequences that represent candidate 'new' splicing events. The splice junctions were predicted by SpliceMap software and putative assembled transcripts validated using the RT-PCR. We also analyzed the coding potential of alternative spliced candidate by HMMER. RESULTS: We detected a total of 11 novel splice junctions derived mostly from alternate 5' splice site; including two of them which contained new translation initiation sites (TISs). We have identified novel IgG pseudogene and a fusion transcript of MEMO1 and RPS9, which were further confirmed by PCR from genomic DNA. We also found novel putative long non-coding RNA (lncRNA), which is antisense to SPINK5 gene. The coding potential of these AS variants revealed that alternative splicing caused premature termination, insertion/deletion of amino acid (s) or formation of novel N-terminus. CONCLUSIONS: Differential splicing of these novel AS variants between cancer and adjacent normal tissue suggests their involvement in BMSCC cancer development and progression.

Identification of novel transcripts deregulated in buccal cancer by RNA-seq.

The differential transcriptome analysis provides better understanding of molecular pathways leading to cancer, which in turn allows designing the effective strategies for diagnosis, therapeutic intervention and prediction of therapeutic outcome. This study describes the transcriptome analysis of buccal cancer and normal tissue by CLC Genomics Workbench from the data generated by Roche's 454 sequencing platform, which identified total of 1797 and 2655 genes uniquely expressed in normal and cancer tissues, respectively with 2466 genes expressed in both tissues. Among the genes expressed in both tissues, 1842 were up-regulated whereas 624 were down-regulated in cancer tissue. Besides transcripts known to be involved in cancer, this study led to the identification of novel transcripts, with significantly altered expression in buccal cancer tissue, providing potential targets for diagnosis and cancer therapeutics. The functional categorization by the KEGG pathway and gene ontology analysis revealed enrichment of differentially expressed transcripts to various pathways leading to cancer, including the p53 signaling pathway. Moreover, the gene ontology analysis unfolded suppression of transcripts involved in actin mediated cell contraction process. The down-regulation of four of these transcripts MYL1, ACTA1, TCAP and DESMIN in buccal cancer were further supported by quantitative PCR signifying its possible implication in the cancer progression.