Systematic Identification and Characterization of Long Non-Coding RNAs in the Silkworm, Bombyx mori.

Long noncoding RNAs (lncRNAs) are emerging as important regulators in various biological processes. However, to date, no systematic characterization of lncRNAs has been reported in the silkworm Bombyx mori. In the present study, we generated eighteen RNA-seq datasets with relatively high depth. Using an in-house designed lncRNA identification pipeline, 11,810 lncRNAs were identified for 5,556 loci. Among these lncRNAs, 474 transcripts were intronic lncRNAs (ilncRNAs), 6,250 transcripts were intergenic lncRNAs (lincRNAs), and 5,086 were natural antisense lncRNAs (lncNATs). Compared with protein-coding mRNAs, silkworm lncRNAs are shorter in terms of full length but longer in terms of exon and intron length. In addition, lncRNAs exhibit a lower level of sequence conservation, more repeat sequences overlapped and higher tissue specificity than protein-coding mRNAs in the silkworm. We found that 69 lncRNA transcripts from 33 gene loci may function as miRNA precursors, and 104 lncRNA transcripts from 72 gene loci may act as competing endogenous RNAs (ceRNAs). In total, 49.47% of all gene loci (2,749/5,556) for which lncRNAs were identified showed sex-biased expression. Co-expression network analysis resulted in 19 modules, 12 of which revealed relatively high tissue specificity. The highlighted darkgoldenrod module was specifically associated with middle and posterior silk glands, and the hub lncRNAs within this module were co-expressed with proteins involved in translation, translocation, and secretory processes, suggesting that these hub lncRNAs may function as regulators of the biosynthesis, translocation, and secretion of silk proteins. This study presents the first comprehensive genome-wide analysis of silkworm lncRNAs and provides an invaluable resource for genetic, evolutionary, and genomic studies of B. mori

Summary of transcripts for <i>Bombyx mandarina</i>.

<p>Summary of transcripts for <i>Bombyx mandarina</i>.</p

Transcriptome Sequencing and Positive Selected Genes Analysis of <i>Bombyx mandarina</i>

<div><p>The wild silkworm <i>Bombyx mandarina</i> is widely believed to be an ancestor of the domesticated silkworm, <i>Bombyx mori</i>. Silkworms are often used as a model for studying the mechanism of species domestication. Here, we performed transcriptome sequencing of the wild silkworm using an Illumina HiSeq2000 platform. We produced 100,004,078 high-quality reads and assembled them into 50,773 contigs with an N50 length of 1764 bp and a mean length of 941.62 bp. A total of 33,759 unigenes were identified, with 12,805 annotated in the Nr database, 8273 in the Pfam database, and 9093 in the Swiss-Prot database. Expression profile analysis found significant differential expression of 1308 unigenes between the middle silk gland (MSG) and posterior silk gland (PSG). Three sericin genes (<i>sericin 1</i>, <i>sericin 2</i>, and <i>sericin 3</i>) were expressed specifically in the MSG and three fibroin genes (<i>fibroin-H</i>, <i>fibroin-L</i>, and <i>fibroin/P25</i>) were expressed specifically in the PSG. In addition, 32,297 Single-nucleotide polymorphisms (SNPs) and 361 insertion-deletions (INDELs) were detected. Comparison with the domesticated silkworm p50/Dazao identified 5,295 orthologous genes, among which 400 might have experienced or to be experiencing positive selection by Ka/Ks analysis. These data and analyses presented here provide insights into silkworm domestication and an invaluable resource for wild silkworm genomics research.</p></div

Transcriptomic Analysis of the Anterior Silk Gland in the Domestic Silkworm (Bombyx mori) - Insight into the Mechanism of Silk Formation and Spinning.

Silk proteins are synthesized in the middle and posterior silk glands of silkworms, then transit into the anterior of the silk gland, where the silk fibers are produced, stored and processed. The mechanism of formation and spinning of the silk fibers has not been fully elucidated, and transcriptome analyses specific to the anterior silk gland have not been reported. In the present study, we explored gene expression profiles in five regions of silk gland samples using the RNA-Seq method. As a result, there were 959,979,570 raw reads obtained, of which 583,068,172 reads were mapped to the silkworm genome. A total of 7419 genes were found to be expressed in terms of reads per kilobase of exon model per million mapped reads ≥ 5 in at least one sample. The gene numbers and expression levels of the expressed genes differed between these regions. The differentially expressed genes were analyzed, and 282 genes were detected as up-regulated in the anterior silk gland, compared with the other parts. Functions of these genes were addressed using the gene ontology and Kyoto Encyclopedia of Genes and Genomes databases, and seven key pathways were enriched. It suggested that the ion transportation, energy metabolism, protease inhibitors and cuticle proteins played essential roles in the process of silk formation and spinning in the anterior silk gland. In addition, 210 genes were found differently expressed between males and females, which should help to elucidate the mechanism of the quality difference in silk fibers from male and female silkworms

Summary of SNPs and Indels for <i>Bombyx mandarina</i>.

<p>*Total number/ synonymous site number / nonsynonymous number</p><p>Summary of SNPs and Indels for <i>Bombyx mandarina</i>.</p

Gene ontology (GO) annotation.

<p>Gene ontology (GO) annotation.</p

Clusters of orthologous group (COG) annotation.

<p>Clusters of orthologous group (COG) annotation.</p

Functional enrichment of protein-coding genes in network modules and module-tissue correlation, and the corresponding <i>p</i>-values.

<p>(A) Functional enrichment of protein-coding genes in network modules. For each module, representative enrichment GO terms are shown, with bar plot of -log10 (p.adjust). Light to dark blue represent increasing enrichment factors (from 0 to 1). (B) Module-tissue correlations and corresponding <i>p</i>-values. Boxes contain Pearson correlation coefficients and their associated <i>p</i>-values. Positive correlation (red) indicates that the module is positively correlated with the specific tissue, whereas negative correlation (green) indicates the reverse. MpT, Malpighian tubule; ASG, anterior silk gland; AMSG, anterior-middle silk gland; MMSG, middle-middle silk gland; PMSG, posterior middle silk gland; PSG, posterior silk gland; Br, brain.</p

Tissue specificity of lncRNAs and protein-coding genes.

<p>(A) Heatmaps of 5,514 lncRNA loci (red; left) and 16,201 protein-coding loci (blue; right) based on normalized expression values (the sum of expression values across all tissues per locus is set to one, using the method described by Cabili, M. N., et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147147#pone.0147147.ref044" target="_blank">44</a>]). (B) The distribution of maximal tissue specificity scores for each transcript across 21 tissues.</p

Venn diagram of the up-regulated genes between ASG and other parts of the silk gland.

<p>(A) Respective overlaps of up-regulated genes in the ASG vs other parts of the silk glands from female silkworms. (B) Respective overlaps of up-regulated genes in the ASG vs other parts of the silk glands from male silkworms. (C) Up-regulated genes in the ASG and other parts of silk glands from females and males.</p