High-throughput Transcriptome Analysis And Immune Signaling Gene Discovery Of The Chinese Mitten Crab Eriocheir Sinensis

The Chinese mitten crab Eriocheir sinensis is one of the important acquculturecrabs in China. Its production reaches300thousand tons per year and the annualoutput value can be more than20billion. However, with development of intensivecrab culture, frequent outbreaks of serious diseases and germplasm degradation leadto catastrophic economic losses in cultured E. sinensis stocks. They also largelyprevent the sustainable development of E. sinensis aquaculture. Using the nextgeneration high-throughput sequencing technology and transcriptome analysisplatform can help to identify disease-resistant SNPs ang immune signaling relatedgenes at the whole genome level. It will not only promote to improve our theroyknowledge deeply and systemly, but also has important practical significance in healthmanagement and disease control of the crab.E. sinensis genome is reported to be big and complicated which is about1.6Gb,but the available sequences are still not completely now. By comparison, the relatedtranscriptome data can be more easily and quickly sequenced by high-throughputsequenceing technique. In the present study, to fully induce various immune signalingpathways and obtain abundant immune related genes with high expression, we used amixture of three pathogen strains (Gram-positive bacteria Micrococcus luteus,Gram-negative bacteria Vibrio alginolyticus and fungi Pichia pastoris) to stimulate E.sinensis. Using the important immune tissue hepatopancreas of adult crab and thewhole body of larval crab after infectious as materials, we then sequenced transcriptomes of them through Illumina HiSeq2000paired sequencing. After denovo assembly with Trinity software, the assembled unigenes were annotated andfunctional classified by bioinformatics analysis. Functional gene identification,molecular marker discovery and differentially expressed analysis were then carriedout. Main results of the study were as follows:(1) Two cDNA (hepatopancreas and larvae) libraries were successfullyconstructed and sequenced on Illumina HiSeq2000platform. More than4Gbdatabase output was obtained from each transcriptome. Respectivelly,40776652and46099408raw reads were generated by sequencing, while39763094and44767566clean reads remained after screening. All the reads were about100bp in length. Theseclean reads were then assembled into70300and100252unigenes, with N50of1834and2095bp.(2) Approximately41.75%(21740of52074) and34.30%(22477of65535)non-redundant unigenes of the two transcriptomes were annotated to homologysequences of Nr, Nt, Swiss-Prot, GO, COG and KEGG databases. According to GOanalysis,18734and23188unigenes were assigned to three main categories includingbiological process, cellular component and molecular function, with the dominantsubcategories to be cellular process, cell and cell part, binding. Then,12243and23188unigenes were classified to26COG groups. We also found that the functiongroups related to immune signaling included signal transduction and defensemechanism. KEGG pathway analysis showed that11938and11454unigenes wereclaried to six kinds of pathways. Among them,1290and1402unigenes were relevantto immune system, signal transtruction, immune molecules and interaction.(3) Numerious immune genes associated with multiple pathways including Toll,IMD, Jak-Stat and MAPK pathways were fully and systematically identified. Manygenes such as Spatzle, MyD88, Pelle, TRAF6, IMD, IKK, JAK, STAT, FGF, PTP andJIP1were even first found of expression in crabs. These sequences will providevaluable data for immune study and disease control of the crab. (4) Our large-scale sequencing effort also revealed60517and49555putativeSNPs from hepatopancreas and larval transcriptomes, respectivelly. Among theseSNPs,39534and32085SNPs were transversion (Tv), while29083and17470weretransitions (Ts), with the mean ratio (Tv:Ts) of1.36and1.84across the twotranscriptomes.183and176candidant SNPs were further detected from35and38immune genes of the four mentioned pathways. Most genes contained only one SNP.They may offer important resources of candidate markers for future selective breedingof E. sinensis.(5) A total of3531differentially expressed genes (DEGs) were detected afterRPKM normalization and comparison between the two transcriptomes.2192geneswere up-regulated while1339were down-regulated in larval transcriptome comparedwith hepatopancreas transcriptome. GO enrichment analysis assigned2345,1878and1822DEGs to the three main GO categories. KEGG pathway enrichment analysisshowed that1531DEGs were classified to241specific pathways containing21statistically signaficant enriched pathways.Discovery of enormous immune genes and SNP markers related Toll, IMD,Jak-Stat and MAPK pathways contributes to researches on immune defense and dmolecular biology of E. sinensis, which will be helpful to health culture of the crab.