Characterization of Transcriptional Complexity during Longissimus Muscle Development in Bovines Using High-Throughput Sequencing

<div><p>Background</p><p>Beef cattle are among the most economically important animals in the world because they are farmed for their meat and leather. However, a lack of genetic information remains an obstacle to understanding the mechanisms behind the development of this animal. Analysis of the beef cattle transcriptome and its expression profile data are essential to extending the genetic information resources for this species and would support studies on this animal.</p><p>Results</p><p>RNA sequencing of beef cattle was performed using the Illumina High-Seq2000 platform. A total of 25,605,140 and 26,214,800 reads were sequenced for embryonic and adult pooled samples, respectively. We identified 24,464–29,994 novel transcript units in two pooled samples. In addition, 8,533–10,144 genes showed evidence of alternative splicing, in agreement with the finding that alternative 3′ splicing is the most common type of alternative splicing event in cattle. We detected the expression levels of 16,174 genes, and 6,800 genes exhibited differential expression between the two pooled samples with a false discovery rate ≤0.001. Using GO enrichment and KEGG pathway analysis, multiple GO term and biological pathways were found to be significantly enriched for differentially expressed genes. In addition, we discovered that 30,618–31,334 putative single nucleotide polymorphisms were located in coding regions.</p><p>Conclusions</p><p>We obtained a high-quality beef cattle reference transcriptome using a high throughput sequencing approach, thereby providing a valuable resource for better understanding the beef cattle genome. The transcriptome data will facilitate future functional studies on the beef cattle genome and can be applied to breeding programs for cattle and closely related mammals.</p></div

Statistics of alternative splicing events and genes in two samples.

<p>(A): The red bars show the distribution of the number of genes for each type of alternative splicing mode in the pooled 30M sample. The green bars indicate the number of alternative splicing events that occurred in each gene. (B) is same as A except that the results for the Emb135 sample are presented.</p

Statistics of novel transcripts.

<p>The abscissa shows the samples 30M and Emb135, and the ordinate represents the number of novel transcripts occurred in each sample.</p

The top 20 Gene Ontology functional annotations for the differentially expressed genes.

<p>Bar graphs (A), (B) and (C) show three independent Gene Ontology (GO) information categories: cellular components, molecular functions and biological processes, respectively. In each bar graph, the abscissa represents the number of differentially expressed genes, and the ordinate is the ID number of the GO terms. All GO categories listed have enrichment <i>P</i> values <0.05.</p

Summary of sequence read alignments to the UMD 3.1 reference genome and gene.

<p>Total reads: total number of sequencing reads. Total base pairs: total number of base pairs. Total mapped reads: the reads that can aligned to reference sequence and the ratio of it. We not only classified statistic by mismatch number but also classified by uniqueness of alignment postion, following 4 ranks are result of classified statistic, perfect match: in total mapped reads, no mismatch; ≤3 bp mismatch: in total mapped reads, mismatch number less than 3 bp; unique match: in total mapped reads, reads aligned to only one postion; multi-position match: in total mapped reads, reads aligned to two or more places. Total unmapped reads: reads that don't aligned to reference sequence.</p

Pipeline of SNP Calling.

<p>The SNPs can be identified on the consensus sequence through the comparison with the reference.</p

Statistics of differentially expressed genes (DEGs).

<p>Red and green bars above the X-axis denote genes with up- and down-regulated expression in Emb135-VS-30M samples, respectively. The Y-axis denote number of differentially expressed genes with up- and down-regulated expression in Emb135-VS-30M samples, respectively.</p

Tissue expression profiles and transcriptional regulation of elongase of very long chain fatty acid 6 in bovine mammary epithelial cells

<div><p>In mammals, very long chain fatty acids (VLCFAs) perform pleiotropic roles in a wide range of biological processes, such as cell membrane formation, cell signal transduction, and endocrine regulation. Beef and milk are abundant of palmitic acid which can be further elongated into stearic acid for synthesizing VLCFAs. Elongase of very long chain fatty acid 6 (ELOVL6) is a rate-limiting enzyme for converting palmitic acid to stearic acid. Consequently, investigating the tissue expression patterns and transcriptional regulation of bovine <i>ELOVL6</i> can provide new insights into improving the composition of beneficial fats in cattle and expanding the knowledge of transcriptional regulation mechanism among domestic animals. In the current study, we found that bovine <i>ELOVL6</i> expressed ubiquitously. Dual-luciferase reporter assay identified that the core promoter region (-130/-41 bp) was located in the second CpG island. In addition, the deletion mutation of binding sites demonstrated that sterol regulatory element binding transcription factor 1 (SREBF1) and specific protein 1 (SP1) both were able to stimulate bovine <i>ELOVL6</i> promoter activity independently, while resulting the similar effect. To confirm these findings, further RNA interference assays were executed in bovine mammary epithelial cells (BMECs). In summary, these data suggest that bovine <i>ELOVL6</i> expressed ubiquitously and is activated by SREBF1 and SP1, via two binding sites present in the <i>ELOVL6</i> promoter region between -130 bp to -41bp.</p></div

Identified targets of novel miRNAs from <i>Arabidopsis</i> degradome data.

<p>Vertical arrows indicate the target cleavage positions. The number indicates the number of corresponding cleavage products.</p

The prediction and analyses of CpG islands in bovine <i>ELOVL6</i> promoter.

<p>(<b>a</b>) The predicted CpG islands in the bovine <i>ELOVL6</i> promoter (+1 to -1000 bp). The red vertical illustrated the GC-rich regions. The blue shading regions indicated the predicated CpG islands; (<b>b</b>) Multiple sequence alignment of the second CpG island. Seven predicted transcription factors binding sites were underlined. Black shaded sequences indicated that the base pair was identical in all sequences of the alignment. Dark grey shadow indicated conserved substitutions and light grey shadow illustrated semi-conserved substitutions.</p