Transcriptome analysis of sex-related genes in the blood clam Tegillarca granosa.

Blood clams (Tegillarca granosa) are one of the most commercial shellfish in China and South Asia with wide distribution in Indo-Pacific tropical to temperate estuaries. However, recent data indicate a decline in the germplasm of this species. Furthermore, the molecular mechanisms underpinning reproductive regulation remain unclear and information regarding genetic diversity is limited. Understanding the reproductive biology of shellfish is important in interpreting their embryology development, reproduction and population structure. Transcriptome sequencing (RNA-seq) rapidly obtains genetic sequence information from almost all transcripts of a particular tissue and currently represents the most prevalent and effective method for constructing genetic expression profiles.Non-reference RNA-seq, an Illumina HiSeq2500 Solexa system, and de novo assembly were used to construct a gonadal expression profile of the blood clam. A total of 63.75 Gb of clean data, with at least 89.46% of Quality30 (Q30), were generated which was then combined into 214,440 transcripts and 125,673 unigenes with a mean length of 1,122.63 and 781.30 base pairs (bp). In total, 27,325 genes were annotated by comparison with public databases. Of these, 2,140 and 2,070 differentially expressed genes (DEGs) were obtained (T05 T08 vs T01 T02 T04, T06 T07 vs T01 T02 T04; in which T01-T04 and T05-T08 represent biological replicates of individual female and male clams, respectively) and classified into two groups according to the evaluation of biological replicates. Then 35 DEGs and 5 sex-related unigenes, in other similar species, were investigated using qRT-PCR, the results of which were confirmed to data arising from RNA-seq. Among the DEGs, sex-related genes were identified, including forkhead box L2 (Foxl2), sex determining region Y-box (Sox), beta-catenin (β-catenin), chromobox homolog (CBX) and Sex-lethal (Sxl). In addition, 6,283 simple sequence repeats (SSRs) and 614,710 single nucleotide polymorphisms (SNPs) were identified from the RNA-seq results.This study provided the first complete gonadal transcriptome data for the blood clam and allowed us to search many aspects of gene sequence information, not limited to gender. This data will improve our understanding of the transcriptomics and reproductive biology of the blood clam. Furthermore, molecular markers such as SSRs and SNPs will be useful in the analysis of genetic evolution, bulked segregant analysis (BSA) and genome-wide association studies (GWAS). Our transcriptome data will therefore provide important genetic information for the breeding and conservation of germplasm

Quality control analysis for RNA-seq data.

<p>Quality control analysis for RNA-seq data.</p

Partial Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of differentially expressed genes (DEGs) (T05 T08 <i>versus</i> T01 T02 T04).

<p>The x-axis shows 50 out of 104 pathways which contain more than one DEG while the y-axis shows the proportion of DEGs corresponding to each pathway.</p

Correlation analysis between two selected individual blood clams.

<p>Correlation analysis between two selected individual blood clams.</p

qRT-PCR validation of transcriptome sequencing.

<p>qRT-PCR validation of transcriptome sequencing.</p

Length distribution of unigenes showing assembly quality of the blood clam transcriptome.

<p>Length distribution of unigenes showing assembly quality of the blood clam transcriptome.</p

Venn diagrams for DEG relationships between two groups of blood clam showing the common differentially expressed genes (DEGs) and specific DEGs in each gene set.

<p>Venn diagrams for DEG relationships between two groups of blood clam showing the common differentially expressed genes (DEGs) and specific DEGs in each gene set.</p

Hierarchical cluster analysis of blood clams (T01 T02 T03 T04 <i>versus</i> T05 T06 T07 T08).

<p>Each column represents a sample, each row represents a gene, and each different color represents log₂ (Fragments per kilobase of transcript per million mapped reads (FPKM)) to indicate different expression levels. The clustering branch indicates similarity between genes or samples.</p

Singlenucleotidepolymorphism (SNP) types showing polymorphism of the sexual transcriptome sequence of the blood clam.

<p>Singlenucleotidepolymorphism (SNP) types showing polymorphism of the sexual transcriptome sequence of the blood clam.</p

Candidate genes for sex determination and differentiation in model organism.

<p>Candidate genes for sex determination and differentiation in model organism.</p