Functional gene profiling through metaRNAseq approach reveals diet-dependent variation in rumen microbiota of buffalo (Bubalus bubalis).

Recent advances in next generation sequencing technology have enabled analysis of complex microbial community from genome to transcriptome level. In the present study, metatranscriptomic approach was applied to elucidate functionally active bacteria and their biological processes in rumen of buffalo (Bubalus bubalis) adapted to different dietary treatments. Buffaloes were adapted to a diet containing 50:50, 75:25 and 100:0 forage to concentrate ratio, each for 6 weeks, before ruminal content sample collection. Metatranscriptomes from rumen fiber adherent and fiber-free active bacteria were sequenced using Ion Torrent PGM platform followed by annotation using MG-RAST server and CAZYmes (Carbohydrate active enzymes) analysis toolkit. In all the samples Bacteroidetes was the most abundant phylum followed by Firmicutes. Functional analysis using KEGG Orthology database revealed Metabolism as the most abundant category at level 1 within which Carbohydrate metabolism was dominating. Diet treatments also exerted significant differences in proportion of enzymes involved in metabolic pathways for VFA production. Carbohydrate Active Enzyme(CAZy) analysis revealed the abundance of genes encoding glycoside hydrolases with the highest representation of GH13 CAZy family in all the samples. The findings provide an overview of the activities occurring in the rumen as well as active bacterial population and the changes occurring through different dietary treatments.

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.

The effect of a high-roughage diet on the metabolism of aromatic compounds by rumen microbes: a metagenomic study using Mehsani buffalo (Bubalus bubalis).

In developing countries, livestock are often fed a high-lignin, low-nutrient diet that is rich in aromatic compounds. It is therefore important to understand the structure of the microbial community responsible for the metabolism of these substances. A metagenomic analysis was therefore carried out to assess the microbial communities associated with the liquid and solid fractions of rumen biomaterial from domestic Mehsani buffalo (Bubalus bubalis) fed with varying proportions of roughage. The experimental design consisted of three feeding regimes (50, 75 and 100 % roughage) and two roughage types (green and dry). Genes associated with aromatic compound degradation were assessed via high-throughput DNA sequencing. A total of 3914.94 Mb data were generated from all treatment groups. Genes coding for functional responses associated with aromatic compound metabolism were more prevalent in the liquid fraction of rumen samples than solid fractions. Statistically significant differences (p < 0.05) were also observed between treatment groups. These differences were dependent on the proportion of roughage fed to the animal, with the type of roughage having little effect. The genes present in the highest abundance in all treatment groups were those related to aromatic compound catabolism. At the phylum level, Bacteroidetes were dominant in all treatments closely followed by the Firmicutes. This study demonstrates the use of feed type to selectively enrich microbial communities capable of metabolizing aromatic compounds in the rumen of domestic buffalo. The results may help to improve nutrient utilization efficiency in livestock and are thus of interest to farming industries, particularly in developing countries, worldwide.

Identification of putative SNPs in progressive retinal atrophy affected Canis lupus familiaris using exome sequencing.

Progressive retinal atrophy (PRA) is one of the major causes of retinal photoreceptor cell degeneration in canines. The inheritance pattern of PRA is autosomal recessive and genetically heterogeneous. Here, using targeted sequencing technology, we have performed exome sequencing of 10 PRA-affected (Spitz=7, Cocker Spaniel=1, Lhasa Aphso=1 and Spitz-Labrador cross breed=1) and 6 normal (Spitz=5, Cocker Spaniel=1) dogs. The high-throughput sequencing using 454-Roche Titanium sequencer generated about 2.16 Giga bases of raw data. Initially, we have successfully identified 25,619 single nucleotide polymorphisms (SNPs) that passed the stringent SNP calling parameters. Further, we performed association study on the cohort, and the highly significant (0.001) associations were short-listed and investigated in-depth. Out of the 171 significant SNPs, 113 were previously unreported. Interestingly, six among them were non-synonymous coding (NSC) SNPs, which includes CPPED1 A>G (p.M307V), PITRM1 T>G (p.S715A), APP G>A (p.T266M), RNF213 A>G (p.V1482A), C>A (p.V1456L), and SLC46A3 G>A (p.R168Q). On the other hand, 35 out of 113 unreported SNPs were falling in regulatory regions such as 3'-UTR, 5'-UTR, etc. In-depth bioinformatics analysis revealed that majority of NSC SNPs have damaging effect and alter protein stability. This study highlighted the genetic markers associated with PRA, which will help to develop genetic assay-based screening in effective breeding.

Characterization of the rumen microbiome of Indian Kankrej cattle (Bos indicus) adapted to different forage diet.

Present study described rumen microbiome of Indian cattle (Kankrej breed) to better understand the microbial diversity and largely unknown functional capacity of the rumen microbiome under different dietary treatments. Kankrej cattle were gradually adapted to a high-forage diet (four animals with dry forage and four with green forage) containing 50 % (K1), 75 % (K2) to 100 % (K3) forage, and remaining concentrate diet, each for 6 weeks followed by analysis of rumen fiber adherent and fiber-free metagenomic community by shotgun sequencing using ion torrent PGM platform and EBI-metagenomics annotation pipeline. Taxonomic analysis indicated that rumen microbiome was dominated by Bacteroidetes followed by Firmicutes, Fibrobacter, Proteobacteria, and Tenericutes. Functional analysis based on gene ontology classified all reads in total 157 categories based on their functional role in biological, molecular, and cellular component with abundance of genes associated with hydrolase activity, membrane, transport, transferase, and different metabolism (such as carbohydrate and protein). Statistical analysis using STAMP revealed significant differences (P < 0.05) between solid and liquid fraction of rumen (in 65 categories), between all three treatments (in 56 categories), and between green and dry roughage (17 categories). Diet treatment also exerted significant difference in environmental gene tags (EGTs) involved in metabolic pathways for production of volatile fatty acids. EGTs for butyrate production were abundant in K2, whereas EGTs for propionate production was abundant during K1. Principal component analysis also demonstrated that diet proportion, fraction of rumen, and type of forage affected rumen microbiome at taxonomic as well as functional level.

Comparative analysis of SNP candidates in disparate milk yielding river buffaloes using targeted sequencing.

River buffalo (Bubalus bubalis) milk plays an important role in economy and nutritious diet in several developing countries. However, reliable milk-yield genomic markers and their functional insights remain unexposed. Here, we have used a target capture sequencing approach in three economically important buffalo breeds namely: Banni, Jafrabadi and Mehsani, belonging to either high or low milk-yield group. Blood samples were collected from the milk-yield/breed balanced group of 12 buffaloes, and whole exome sequencing was performed using Roche 454 GS-FLX Titanium sequencer. Using an innovative approach namely, MultiCom; we have identified high-quality SNPs specific for high and low-milk yield buffaloes. Almost 70% of the reported genes in QTL regions of milk-yield and milk-fat in cattle were present among the buffalo milk-yield gene candidates. Functional analysis highlighted transcriptional regulation category in the low milk-yield group, and several new pathways in the two groups. Further, the discovered SNP candidates may account for more than half of mammary transcriptome changes in high versus low-milk yielding cattle. Thus, starting from the design of a reliable strategy, we identified reliable genomic markers specific for high and low-milk yield buffalo breeds and addressed possible downstream effects.

Bacterial diversity dynamics associated with different diets and different primer pairs in the rumen of Kankrej cattle.

The ruminal microbiome in herbivores plays a dominant role in the digestion of lignocellulose and has potential to improve animal productivity. Kankrej cattle, a popular native breed of the Indian subcontinent, were used to investigate the effect of different dietary treatments on the bacterial diversity in ruminal fractions using different primer pairs. Two groups of four cows were assigned to two primary diets of either dry or green forages. Each group was fed one of three dietary treatments for six weeks each. Dietary treatments were; K1 (50% dry/green roughage: 50% concentrate), K2 (75% dry/green roughage: 25% concentrate) and K3 (100% dry/green roughage). Rumen samples were collected using stomach tube at the end of each dietary period and separated into solid and liquid fractions. The DNA was extracted and amplified for V1-V3, V4-V5 and V6-V8 hypervariable regions using P1, P2 and P3 primer pairs, sequenced on a 454 Roche platform and analyzed using QIIME. Community compositions and the abundance of most bacterial lineages were driven by interactions between primer pair, dietary treatment and fraction. The most abundant bacterial phyla identified were Bacteroidetes and Firmicutes however, the abundance of these phyla varied between different primer pairs; in each primer pair the abundance was dependent on the dietary treatment and fraction. The abundance of Bacteroidetes in cattle receiving K1 treatment indicate their diverse functional capabilities in the digestion of both carbohydrate and protein while the predominance of Firmicutes in the K2 and K3 treatments signifies their metabolic role in fibre digestion. It is apparent that both liquid and solid fractions had distinct bacterial community patterns (P<0.001) congruent to changes in the dietary treatments. It can be concluded that the P1 primer pair flanking the V1-V3 hyper-variable region provided greater species richness and diversity of bacterial populations in the rumen of Kankrej cattle.

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.

Transcriptome analysis and SNP identification in SCC of horn in (Bos indicus) Indian cattle.

Single Nucleotide Polymorphisms (SNPs) have become the marker of choice for genome wide association studies. In order to provide the best genome coverage for the analysis of disease, production and performance traits, a large number of relatively evenly distributed SNPs are needed. The main objective of present work was to identify large numbers of gene-associated SNPs using high-throughput sequencing in squamous cell carcinoma of horn. RNA-seq analysis was conducted on 2 tissues viz. Horn Cancer (HC) and Horn Normal (HN) in Kankrej breed of cattle. A total of 909,362 reads with average read length of 405 bp for HC and 583,491 reads with average read length of 411 bp for HN were obtained. We found 9532 and 7065 SNPs as well as 1771 and 1172 Indels in HC and HN, respectively, from which, 7889 SNPs and 1736 Indels were uniquely present in HC, 5886 SNPs and 1146 Indels were uniquely present in HN and reported first time in Bos indicus, whereas the rest are already reported in Bos taurus dbSNP database. The gene-associated SNPs and Indels were high in upregulated genes of HC as compared to HN. Analysis of differentially expressed genes was identified, these genes are involved in regulation of cell proliferation, apoptosis, gene transcription, cell survival and metabolism through various metabolic pathways. The result of transcriptome expression profiling was validated using Real Time quantitative PCR in nine randomly selected genes. We identified numbers aberrant signaling pathways responsible for carcinogenesis in HC which are also commonly altered in squamous cell carcinoma (SCC) of lung in human being. We conclude that a large number of altered genes and dysfunction of multiple pathways are involved in the development of Horn Cancer. The present findings contribute to theoretical information for further screening of genes and identification of markers for early diagnosis of HC as well as SNPs identified in this report provide a much needed resource for genetic studies in B. indicus and shall contribute to the development of a high density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for gene associated SNPs in HC.