Transcriptomics Analysis of <i>Crassostrea hongkongensis</i> for the Discovery of Reproduction-Related Genes

<div><p>Background</p><p>The reproductive mechanisms of mollusk species have been interesting targets in biological research because of the diverse reproductive strategies observed in this phylum. These species have also been studied for the development of fishery technologies in molluscan aquaculture. Although the molecular mechanisms underlying the reproductive process have been well studied in animal models, the relevant information from mollusks remains limited, particularly in species of great commercial interest. <i>Crassostrea hongkongensis</i> is the dominant oyster species that is distributed along the coast of the South China Sea and little genomic information on this species is available. Currently, high-throughput sequencing techniques have been widely used for investigating the basis of physiological processes and facilitating the establishment of adequate genetic selection programs.</p><p>Results</p><p>The <i>C</i>.<i>hongkongensis</i> transcriptome included a total of 1,595,855 reads, which were generated by 454 sequencing and were assembled into 41,472 contigs using <i>de novo</i> methods. Contigs were clustered into 33,920 isotigs and further grouped into 22,829 isogroups. Approximately 77.6% of the isogroups were successfully annotated by the Nr database. More than 1,910 genes were identified as being related to reproduction. Some key genes involved in germline development, sex determination and differentiation were identified for the first time in <i>C</i>.<i>hongkongensis</i> (<i>nanos</i>, <i>piwi</i>, <i>ATRX</i>, <i>FoxL2</i>, <i>β-catenin</i>, etc.). Gene expression analysis indicated that <i>vasa</i>, <i>nanos</i>, <i>piwi</i>, <i>ATRX</i>, <i>FoxL2</i>, <i>β-catenin</i> and <i>SRD5A1</i> were highly or specifically expressed in <i>C</i>.<i>hongkongensis</i> gonads. Additionally, 94,056 single nucleotide polymorphisms (SNPs) and 1,699 simple sequence repeats (SSRs) were compiled.</p><p>Conclusions</p><p>Our study significantly increased <i>C</i>.<i>hongkongensis</i> genomic information based on transcriptomics analysis. The group of reproduction-related genes identified in the present study constitutes a new tool for research on bivalve reproduction processes. The large group of molecular markers discovered in this study will be useful for population screening and marker assisted selection programs in <i>C</i>.<i>hongkongensis</i> aquaculture.</p></div

Amino acid sequence alignment of the ADD domain from <i>C</i>.<i>hongkongensis ATRX</i> ortholog with vertebrate ATRX.

<p>The highly conserved C2-C2 zinc finger and C4-C4 zinc finger are indicated by asterisks.</p

Presence of sex determination/differentiation genes from <i>Caenorhabditis elegans</i>, <i>Drosophila melanogaster</i>, <i>Danio rerio</i>, <i>Mus musculus</i> and <i>Crassostrea hongkongensis</i>.

<p>C, <i>Caenorhabditis elegans</i> (nematode); D, <i>Drosophila melanogaster</i> (fruit fly); Dr, <i>Danio rerio</i> (zebrafish); M, <i>Mus musculus</i> (mouse);Ch, <i>Crassostrea hongkongensis</i> (Oyster)</p><p>Presence of sex determination/differentiation genes from <i>Caenorhabditis elegans</i>, <i>Drosophila melanogaster</i>, <i>Danio rerio</i>, <i>Mus musculus</i> and <i>Crassostrea hongkongensis</i>.</p

Classification of single nucleotide polymorphisms (SNPs) identified from the <i>C</i>.<i>hongkongensis</i> transcriptome.

<p>Transitions occurred more frequently than transversions. The overall frequency of all types of SNPs including indels was one per 392 bp.</p

Vasa gene identified in the <i>Crassostrea hongkongensis</i> transcriptome and its expression profile.

<p>(A) Domain structure of the <i>C</i>.<i>hongkongensis vasa</i> ortholog. (B) Molecular phylogenetic tree of Vasa subfamily and related proteins. Bootstrap values from 1000 trials are indicated at each branch node. The scale bar indicates 0.05 amino acid replacements per site. <i>C elegans</i> Ce-GLH-1 was used as an outgroup. The transcript encoding <i>Chvasa</i>, indicated by a red open box, is properly aligned to the clade of molluscan Vasa colored by green. The Vasa subfamily proteins are enclosed by the dotted lines. For the GenBank accession numbers of the reference sequences, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134280#pone.0134280.s006" target="_blank">S3 Table</a>. (C) Expression profile of <i>C</i>.<i>hongkongensis vasa</i> ortholog (<i>Chvasa</i>) in adult tissues with mean ± s.e.m. as error bars (n = 5).</p

Reproduction-related pathways identified in the <i>C</i>. <i>hongkongensis</i> transcriptome.

<p>Reproduction-related pathways identified in the <i>C</i>. <i>hongkongensis</i> transcriptome.</p

Analysis of Piwi genes identified in <i>C</i>.<i>hongkongensis</i> transcriptome.

<p>(A) Domain structure of the <i>C</i>.<i>hongkongensis Piwi</i> orthologs. (B) Phylogenetic tree constructed using the neighbor-joining method on the basis of the amino acid sequences alignment of <i>C</i>.<i>hongkongensis Piwi</i> ortholog with Piwi-like proteins of vertebrates. A total of 1000 bootstrap trials were run. Bootstrap values were indicated at each branch node. The scale bar represents an evolutionary distance of 0.1 amino acid substitutions per position. The transcript encoding <i>C</i>.<i>hongkongensis Piwi</i> ortholog, colored by red, was aligned to the clade of vertebrate Piwil1proteins. For the GenBank accession numbers of the reference sequences, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134280#pone.0134280.s006" target="_blank">S3 Table</a>. (C) Expression profile of <i>ChPiwil1</i> in adult tissues by qRT-PCR with mean±s.e.m. as error bars (n = 5). (D-L) Expression profile of <i>ChPiwil1</i>during <i>C</i>.<i>hongkongensis</i> oogenesis by ISH. Histological analysis of <i>C</i>.<i>hongkongensis</i> female gonad at early developing stage (D), late developing stage (E) and mature stage (F) stained with HE. Expression profile of <i>ChPiwil1</i>during <i>C</i>.<i>hongkongensis</i> oogenesis by ISH with antisense (G-I) and sense probe (J-L). Positive cells are stained with brown or purple. OG: oogonia; EO: early vitellogenic oocyte; MO: mature ova.</p

Gene Ontology(GO) analysis of the <i>Crassostrea hongkongensis</i> transcriptome on level 2.

<p>The percentage and distribution of top-level GO-terms were portrayed in the three categories: (A) Cellular component; (B) Molecular function and (C) Biological process.</p

Summary statistics of the transcriptome assembly for <i>Crassostrea hongkongensis</i>.

<p>Summary statistics of the transcriptome assembly for <i>Crassostrea hongkongensis</i>.</p

Heat map of sex determination/differentiation genes expressed at different adult tissues of <i>C</i>.<i>hongkongensis</i>.

<p>Genes showing similar expression profiles on all samples (columns) were clustered together. Color represents the normalized expression after variance-stabilizing transformation. Expression levels are depicted with a color scale, in which shades of red represent higher expression and shades of green represent lower expression.</p