Transcriptome Sequencing And Positive Selected Genes Analysis Of Bombyx Mandarina

The wild silkworm Bombyx mandarina belongs to Insecta Lepidoptera Bombycidae. It is widely believed to be an ancestor of the domesticated silkworm, Bombyx mori. B. mandarina have been domesticated for at least 5000 years to increase and improve cocoon yield. Many traits are different between B. mandarina and B. mori including body size and color in the larval stage, size and silk quality of cocoons, immunocompetence, fight behavior and egg laying in the adult stage. Thus, B. mandarina and B. mori are good models for studying species domestication. With the development of next-generation sequencing technology, the next-generation sequencing-based RNA-Seq analysis provides opportunities for de novo assembly of genome reference-free species. Here, we performed transcriptome sequencing of the wild silkworm using an Illumina HiSeq2000 platform to attempt to explore the mechanism of silkworm domestication by Positive Selected Genes Analysis based transcriptome. These data and analyses presented here provide insights into silkworm domestication and an invaluable resource for wild silkworm genomics research. So, in the present study, we performed transcriptome sequencing for the middle silk gland (MSG) and the posterior silk gland (PSG) from B. mandarina and obtained the following results through the analysis:1. Transcriptome sequencing of B. mandarina and de novo assembly and SNP identificationIn the present study, we dissected and collected two tissues:middle silk glands (MSGs) and posterior silk glands (PSGs) from a single fifth-instar larva. We respectively extracted total RNAs from two MSG and PSG and constructed RNA libraries. Libraries were sequenced for 100 bp paired-end reads, and we obtained a total of 120,381,200 paired-end reads. After removing adapter, low-quality and rRNA sequences, we obtained a total of 100,004,078 high-quality clean reads with GC percentage 46.50%, the percentage of raw reads was 83.07%. A total of 50,773 transcripts with N50 length 1764 bp and mean length 941.62 bp were obtained by de novo assembly. Totally 33,759 unigenes with N50 length 1437 bp and mean length 762.20 bp were generated. Unigenes were annotated by aligning with different protein database, a total of 12,805 unigenes significantly matched in Nr,8273 matched in Pfam, and 9093 similar to proteins in the Swiss-Prot database. We compared unigenes with GO, KEGG and COG database for functional annotation. A total of 9571 unigenes had a GO annotation,5893 had a KEGG annotation and 6245 matched in COG. The annotation of unigenes showed that the results of assembly was good and it could lay the foundation of subsequent analysis. In addition,32,297 Single-nucleotide polymorphisms (SNPs) and 361 insertion-deletions (INDELs) were detected. It could reveal that heterozygosity of B. mandarina was high.2. Expression profile analysis of B. mandarinaIn our study, we performed expression profile analysis of MSG and PSG. A total of 1308 differentially expressed unigenes were identified. Totally 883 up-regulated expressed in MSG and 425 up-regulated in PSG. Three sericin genes (sericin 1, sericin 2, and sericin 3) were expressed specifically in the MSG and three fibroin genes (fibroin-H, fibroin-L, and fibroin/P25) were expressed specifically in the PSG. And the expression of other silk protein related genes were the same with B. mori. In addition, comparing with the sequences of silk protein gene between B. mandarina and B. mori, we found that the amino acid similarity of fibroin light chain was 99.2%, and fibroin/P25 was 99.0%, both have two amino acid substitutions; and in sericin protein, amino acid similarity of sericin 1 reached 98.0% after removing 52 amino acids gap, with 13 amino acid substitutions; sericin 2 had up to 477 amino acid gap, and its amino acid similarity was 98.7%, with 8 amino acid substitutions; sericin 3 protein of B. mandarina was more than B. mori 13 amino acids gap at the head, and its amino acid similarity was 91.8%, with a total of 12 amino acid substitutions. This shows that protein sequence of fibroin light chain and fibroin/P25 was conserved rather than sericin with amino acid insertions and deletions. And phylogenetic tree analysis of fibrohexamerin protein showed that the protein of B. mandarina may have several genes before domestication.3. Positive selected genes of B. mandarina identification and functional analysisIn the present study, we compared with the domesticated silkworm p50/Dazao, a total of 5295 pairs of putative orthologous genes were identified. After removing potential paralogs, totally 2806 pairs of orthologs with both nonsynonymous (Ka) and synonymous (Ks) were identified. Among which 400 might have experienced or to be experiencing positive selection by Ka/Ks analysis. A total of 83 pairs of orthologs might have experienced strong positive selection and 317 might be experiencing positive selection. KEGG pathway enrichment analysis of positive selection was performed to attempt to explore the pathway and function related to domestication. Among 400 unigenes considered as positively selected,168 were annotated with KEGG database which were in 126 KEGG pathways. A total of three pathways were enriched by KEGG pathway enrichment analysis, glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate, retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathway, and circadian rhythm. These pathways might be related to immunity and circadian rhythm to effect silkworm domestication.