Comprehensive analyses of 723 transcriptomes enhance genetic and biological interpretations for complex traits in cattle.

By uniformly analyzing 723 RNA-seq data from 91 tissues and cell types, we built a comprehensive gene atlas and studied tissue specificity of genes in cattle. We demonstrated that tissue-specific genes significantly reflected the tissue-relevant biology, showing distinct promoter methylation and evolution patterns (e.g., brain-specific genes evolve slowest, whereas testis-specific genes evolve fastest). Through integrative analyses of those tissue-specific genes with large-scale genome-wide association studies, we detected relevant tissues/cell types and candidate genes for 45 economically important traits in cattle, including blood/immune system (e.g., CCDC88C) for male fertility, brain (e.g., TRIM46 and RAB6A) for milk production, and multiple growth-related tissues (e.g., FGF6 and CCND2) for body conformation. We validated these findings by using epigenomic data across major somatic tissues and sperm. Collectively, our findings provided novel insights into the genetic and biological mechanisms underlying complex traits in cattle, and our transcriptome atlas can serve as a primary source for biological interpretation, functional validation, studies of adaptive evolution, and genomic improvement in livestock.

Reduced representation bisulphite sequencing of ten bovine somatic tissues reveals DNA methylation patterns and their impacts on gene expression.

BACKGROUND: As a major epigenetic component, DNA methylation plays important functions in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in economically important animals like cattle. RESULTS: Using reduced representation bisulphite sequencing (RRBS), we obtained single-base-resolution maps of bovine DNA methylation from ten somatic tissues. In total, we evaluated 1,868,049 cytosines in CG-enriched regions. While we found slightly low methylation levels (29.87 to 38.06 %) in cattle, the methylation contexts (CGs and non-CGs) of cattle showed similar methylation patterns to other species. Non-CG methylation was detected but methylation levels in somatic tissues were significantly lower than in pluripotent cells. To study the potential function of the methylation, we detected 10,794 differentially methylated cytosines (DMCs) and 836 differentially methylated CG islands (DMIs). Further analyses in the same tissues revealed many DMCs (including non-CGs) and DMIs, which were highly correlated with the expression of genes involved in tissue development. CONCLUSIONS: In summary, our study provides a baseline dataset and essential information for DNA methylation profiles of cattle.

What's in your next-generation sequence data? An exploration of unmapped DNA and RNA sequence reads from the bovine reference individual.

BACKGROUND: Next-generation sequencing projects commonly commence by aligning reads to a reference genome assembly. While improvements in alignment algorithms and computational hardware have greatly enhanced the efficiency and accuracy of alignments, a significant percentage of reads often remain unmapped. RESULTS: We generated de novo assemblies of unmapped reads from the DNA and RNA sequencing of the Bos taurus reference individual and identified the closest matching sequence to each contig by alignment to the NCBI non-redundant nucleotide database using BLAST. As expected, many of these contigs represent vertebrate sequence that is absent, incomplete, or misassembled in the UMD3.1 reference assembly. However, numerous additional contigs represent invertebrate species. Most prominent were several species of Spirurid nematodes and a blood-borne parasite, Babesia bigemina. These species are either not present in the US or are not known to infect taurine cattle and the reference animal appears to have been host to unsequenced sister species. CONCLUSIONS: We demonstrate the importance of exploring unmapped reads to ascertain sequences that are either absent or misassembled in the reference assembly and for detecting sequences indicative of parasitic or commensal organisms.

Analysis of copy number variations among diverse cattle breeds.

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or approximately 1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

Gene number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes.

BACKGROUND: WC1 co-receptors belong to the scavenger receptor cysteine-rich (SRCR) superfamily and are encoded by a multi-gene family. Expression of particular WC1 genes defines functional subpopulations of WC1(+) γδ T cells. We have previously identified partial or complete genomic sequences for thirteen different WC1 genes through annotation of the bovine genome Btau_3.1 build. We also identified two WC1 cDNA sequences from other cattle that did not correspond to sequences in the Btau_3.1 build. Their absence in the Btau_3.1 build may have reflected gaps in the genome assembly or polymorphisms among animals. Since the response of γδ T cells to bacterial challenge is determined by WC1 gene expression, it was critical to understand whether individual cattle or breeds differ in the number of WC1 genes or display polymorphisms. RESULTS: Real-time quantitative PCR using DNA from the animal whose genome was sequenced ("Dominette") and sixteen other animals representing ten breeds of cattle, showed that the number of genes coding for WC1 co-receptors is thirteen. The complete coding sequences of those thirteen WC1 genes is presented, including the correction of an error in the WC1-2 gene due to mis-assembly in the Btau_3.1 build. All other cDNA sequences were found to agree with the previous annotation of complete or partial WC1 genes. PCR amplification and sequencing of the most variable N-terminal SRCR domain (domain 1 which has the SRCR "a" pattern) of each of the thirteen WC1 genes showed that the sequences are highly conserved among individuals and breeds. Of 160 sequences of domain 1 from three breeds of cattle, no additional sequences beyond the thirteen described WC1 genes were found. Analysis of the complete WC1 cDNA sequences indicated that the thirteen WC1 genes code for three distinct WC1 molecular forms. CONCLUSION: The bovine WC1 multi-gene family is composed of thirteen genes coding for three structural forms whose sequences are highly conserved among individual cattle and breeds. The sequence diversity necessary for WC1 genes to function as a multi-genic pattern recognition receptor array is encoded in the genome, rather than generated by recombinatorial diversity or hypermutation.

Effect of reduced heifer nutrition during in utero and post-weaning development on glucose and acetate kinetics.

Energetic efficiency was evaluated in composite bred heifers born from dams receiving 1·8 or 1·2 kg/d winter supplementation for approximately 80 d before parturition. Heifers were then developed post-weaning and randomly assigned to heifer development treatments of either control (100 %; ad libitum; n 8/year) or restricted (80 %; fed 80 % of supplementation fed to controls adjusted to a common body weight: n 8/year) in a 2-year study. A glucose tolerance test (GTT) and acetate irreversible loss test (AILT) were administered to heifers at the termination of a 140 d development period when the heifers were approximately 403 d of age and consumed a silage-based diet, and again at 940 d of age when pregnant with their second calf and grazing dormant forage. No differences were measured (P>0·08) for dam winter nutrition or heifer development treatment for baseline serum metabolites or measures in either the GTT or the AILT. However, changes in baseline serum concentrations (P>0·05) were different between metabolic challenges, which occurred at different stages of development. No difference in acetate disappearance (P = 0·18) and half-life (P = 0·66) was measured between the two metabolic challenges. A trend for glucose half-life to be shorter in heifers born from dams receiving in utero winter treatments that supplied 1·2 kg/d of winter supplementation was observed (P = 0·083). Heifers developed with lower total DM intake during a 140 d development period had similar glucose and acetate incorporation rates as ad libitum-fed heifers when evaluated at two different production stages.

Next generation sequencing to define prokaryotic and fungal diversity in the bovine rumen.

A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate microbial diversity in the bovine rumen of 12 cows consuming a forage diet. Observed bacterial species richness, based on the V1-V3 region of the 16S rRNA gene, was between 1,903 to 2,432 species-level operational taxonomic units (OTUs) when 5,520 reads were sampled per animal. Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7. Abundance of Prevotella species varied widely among the 12 animals. Archaeal species richness, also based on 16S rRNA, was between 8 and 13 OTUs, representing 5 genera. The majority of archaeal OTUs (84%) found in this study were previously observed in public databases with only two new OTUs discovered. Observed rumen fungal species richness, based on the 18S rRNA gene, was between 21 and 40 OTUs with 98.4-99.9% of OTUs represented by more than one read, using Good's coverage. Examination of the fungal community identified numerous novel groups. Prevotella and Tannerella were overrepresented in the liquid fraction of the rumen while Butyrivibrio and Blautia were significantly overrepresented in the solid fraction of the rumen. No statistical difference was observed between the liquid and solid fractions in biodiversity of archaea and fungi. The survey of microbial communities and analysis of cross-domain correlations suggested there is a far greater extent of microbial diversity in the bovine rumen than previously appreciated, and that next generation sequencing technologies promise to reveal novel species, interactions and pathways that can be studied further in order to better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and environmental impact.

An atlas of bovine gene expression reveals novel distinctive tissue characteristics and evidence for improving genome annotation.

BACKGROUND: A comprehensive transcriptome survey, or gene atlas, provides information essential for a complete understanding of the genomic biology of an organism. We present an atlas of RNA abundance for 92 adult, juvenile and fetal cattle tissues and three cattle cell lines. RESULTS: The Bovine Gene Atlas was generated from 7.2 million unique digital gene expression tag sequences (300.2 million total raw tag sequences), from which 1.59 million unique tag sequences were identified that mapped to the draft bovine genome accounting for 85% of the total raw tag abundance. Filtering these tags yielded 87,764 unique tag sequences that unambiguously mapped to 16,517 annotated protein-coding loci in the draft genome accounting for 45% of the total raw tag abundance. Clustering of tissues based on tag abundance profiles generally confirmed ontology classification based on anatomy. There were 5,429 constitutively expressed loci and 3,445 constitutively expressed unique tag sequences mapping outside annotated gene boundaries that represent a resource for enhancing current gene models. Physical measures such as inferred transcript length or antisense tag abundance identified tissues with atypical transcriptional tag profiles. We report for the first time the tissue-specific variation in the proportion of mitochondrial transcriptional tag abundance. CONCLUSIONS: The Bovine Gene Atlas is the deepest and broadest transcriptome survey of any livestock genome to date. Commonalities and variation in sense and antisense transcript tag profiles identified in different tissues facilitate the examination of the relationship between gene expression, tissue, and gene function.

Discovery of novel genetic networks associated with 19 economically important traits in beef cattle.

Quantitative or complex traits are determined by the combined effects of many loci, and are affected by genetic networks or molecular pathways. In the present study, we genotyped a total of 138 mutations, mainly single nucleotide polymorphisms derived from 71 functional genes on a Wagyu x Limousin reference population. Two hundred forty six F(2) animals were measured for 5 carcass, 6 eating quality and 8 fatty acid composition traits. A total of 2,280 single marker-trait association runs with 120 tagged mutations selected based on the HAPLOVIEW analysis revealed 144 significant associations (P < 0.05), but 50 of them were removed from the analysis due to the small number of animals (< or = 9) in one genotype group or absence of one genotype among three genotypes. The remaining 94 single-trait associations were then placed into three groups of quantitative trait modes (QTMs) with additive, dominant and overdominant effects. All significant markers and their QTMs associated with each of these 19 traits were involved in a linear regression model analysis, which confirmed single-gene associations for 4 traits, but revealed two-gene networks for 8 traits and three-gene networks for 5 traits. Such genetic networks involving both genotypes and QTMs resulted in high correlations between predicted and actual values of performance, thus providing evidence that the classical Mendelian principles of inheritance can be applied in understanding genetic complexity of complex phenotypes. Our present study also indicated that carcass, eating quality and fatty acid composition traits rarely share genetic networks. Therefore, marker-assisted selection for improvement of one category of these traits would not interfere with improvement of another.

A radiation hybrid map for the bovine Y Chromosome.

Screening a bovine Y Chromosome-specific DNA library resulted in 34 new microsatellites, six of which mapped to the pseudoautosomal region (PAR), and 28 localized to the Y-specific region. These microsatellites, together with 23 markers previously mapped to the bovine Y Chr, were scored on a 7000-rad cattle-hamster radiation hybrid (RH) panel. Retention frequency of individual markers ranged from 18.5% to 76.5% with an average of 48.4%. Markers with high retention frequency (>55%) were found to exist in multiple copies on the Y Chr. Thirteen markers were placed on the PAR RH map with the AmelY gene proximal to the pseudoautosomal boundary and 46 markers, including Sry and Tspy gene, on the Y-specific region of the RH map. The microsatellites developed and mapped in this work will be useful for comparative mapping of cattle, sheep, and goat, studying the origin, evolution, and migration of bovidae species and provide an initial platform to develop a high-resolution map of the Y Chr and positional cloning of Y-specific genes.