De Novo assembly of the Japanese flounder (Paralichthys olivaceus) spleen transcriptome to identify putative genes involved in immunity

Abstract

Background: Japanese flounder (Paralichthys olivaceus) is an economically important marine fish in Asia and has suffered from disease outbreaks caused by various pathogens, which requires more information for immune relevant genes on genome background. However, genomic and transcriptomic data for Japanese flounder remain scarce, which limits studies on the immune system of this species. In this study, we characterized the Japanese flounder spleen transcriptome using an Illumina paired-end sequencing platform to identify putative genes involved in immunity.

Methodology/principal findings: A cDNA library from the spleen of P. olivaceus was constructed and randomly sequenced using an Illumina technique. The removal of low quality reads generated 12,196,968 trimmed reads, which assembled into 96,627 unigenes. A total of 21,391 unigenes (22.14%) were annotated in the NCBI Nr database, and only 1.1% of the BLASTx top-hits matched P. olivaceus protein sequences. Approximately 12,503 (58.45%) unigenes were categorized into three Gene Ontology groups, 19,547 (91.38%) were classified into 26 Cluster of Orthologous Groups, and 10,649 (49.78%) were assigned to six Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, 40,928 putative simple sequence repeats and 47, 362 putative single nucleotide polymorphisms were identified. Importantly, we identified 1,563 putative immune-associated unigenes that mapped to 15 immune signaling pathways.

Conclusions/significance: The P. olivaceus transciptome data provides a rich source to discover and identify new genes, and the immune-relevant sequences identified here will facilitate our understanding of the mechanisms involved in the immune response. Furthermore, the plentiful potential SSRs and SNPs found in this study are important resources with respect to future development of a linkage map or marker assisted breeding programs for the flounder.

Fig 1. Length distribution of the assembled (blue) and the annotated unigenes (red) of P. olivaceus.

Fig 2. Venn diagram showing the comparison among P. olivaceus transcriptomic sequences with the known sequences from D. rerio and P. olivaceus EST deposited in the NCBI database.

Fig 3. Distribution of top-hit species.

Fig 4. Gene ontology (GO) annotations of the annotated unigenes.

12,503 unigenes were assigned to three GO categories containing 52 functional subcategories.

Fig 5. Cluster of Orthologous Groups (COG) annotations of annotated unigenes.

19,547 unigenes were classified into 26 COG categories.

Fig 6. Quantitative Real-time PCR (qRT—PCR) validation of the expressed genes in transcriptome sequencing.

IRAK4: interleukin-1 receptor-associated kinase 4; IRF7: interferon regulatory factor 7; INFAR: interferon receptor 1; NFKB1: nuclear factor NF-kappa-B p105 subunit; TRAF6: TNF receptor-associated factor 6; AKT: RAC serine/threonine-protein kinase; IRAK1: interleukin-1 receptor-associated kinase 1; F2: coagulation factorII; TNFA: tumor necrosis factor superfamily. Values are presented as means ± standard deviation（n = 5）and the error bars indicate the standard deviation.