Research On The Mitochondrial Genome Of The Paraoncidium Reevessii And The Transcriptome Of Four Species In Onchidiidae

Onchidiidae are amphibious and hermaphroditic shellfish. They do not have theshell and free-living larval stage. They are widely distributed in India-Pacific coast andalso in the Yellow Sea and the East Sea and South Sea of China, are the transitionalspecies of marine and terrestrial. They are mainly found on rocks in seaside tide zone,beaches, sand and mangrove; also a small number of species living in brackish estuaries,inland freshwater and high altitude rainforest. Due to its characteristics of this kind oflife, they are considered by scholars to be the well representative of the radiant life fromthe ocean to land, therefore the researches on Onchidiidae have very importantscientific research value.In recent years, with the depth of research of Onchidiidae, Anatomy, ecology, andgeography of genetic research have been more thorough, but the molecular mechanismsand traits related genes almost never been reported in the papers. This adds theenormous difficulties to the later deep research. At the same time, the research of otherclose relatives is relative backwardness resulting that transcriptome research ofOnchidiidae is imperative. At present, due to lack of transcriptome sequencing, somany valuable experiments associated with Onchidiida can not be carried out,including gene phenotypes related breath, expression analysis of intraspecificdifferences, and the genomic comparisons with shellfish and molluscs. Despite the lackof a complete sequence of the genome, the results transcriptome sequencing can stillmakes a lot of experiments have been carried out.In this study, transcriptome cDNA libraries of the four species of Onchidiidae arebuilded by de novo RNA-seq sequencing. Based on the protein sequences of theUniProt database and the NCBI NR database, the splicing transcripts (contigs) werecompared with them to carry on the GO function annotation analysis and the Pathwayannotation analysis and help people to study thoroughly the macroscopic evolution pattern of the Onchidiidae. This will provide powerful evidences for the biologicalevolution.1. Using Illumina HiSeq-2000paired-end sequencing technology (PE), based onthe the transcriptome sequencing process and data analysis methods, the Chinese fourspecies of Onchidiidae are sequenced by transcriptome. After guaranteeing to obtainthe high-quality data, we obtained the effective sequence separately by using Trinityand the TGICL software, Platevindex mortoni:60,219,324Unigenes, Paraoncidiumreevesii:89,062,542Unigenes, Onchidium struma:62,624,204Unigenes, Peroniaverruculata:61,663,900Unigenes. Blasted with the Nr, Swissprot, GO and COGdatabases,13558,17929,16203and20610transcripts were annotated, respectively.Via Unigene N50for effectiveness evaluation of transcriptome splicing, we obtain theUnigene N50of the four spcies respectively:723bp,485bp,557bp and778bp.2. In difference expression analysis, the number of the Unigene of which fourspecies of Onchidiidae are marked at least by one data base (UniProt database or NCBINR database) respectively is: Platevindex mortoni14,897, Paraoncidium reevesii27,154, Onchidium struma45,346, Peronia verruculata23,717. Meanwhile, GOclassification and KEGG pathway positioning suggest that these genes covery variousbiological functions and metabolic pathways. We can obtain a lot of breathing, growth,reproduction and immune-related candidate genes.3. Make the SSR examination and SNP prediction to four species of Onchidiidaewith the bioinformatics. The SSR locus exist in the genome of most organisms, arewidely used in the field of genetic crossbreeding and drawing the chromosome geneticmap. Detection of SNPs in the samples will help to study the changes about proteinfunction which is caused by the single nucleotide variations. This can be as the teststandard of genotype and phenotype of the four species. This experiment systemexamined the SNP position spots of the four species of Onchidiidae epidermal tissues.Combined with the four expression profiles analysis, We use PAML software to analyseproteins of Protein homology groups (ortholog group) from different species forphylogenetic analysis (using NJ algorithm (bootstrap parameter=1000)) and withclustalx2mapping. The results are quite different with Chen Cheng’s phylogeography.The reason is possibly that protein variability of Onchidiidae is very big.The results of this study enrich the cDNA data resources of Onchidiidae, not onlyto explore the mechanism of Onchidiidae breathing and stress resistance-related genesand regulatory mechanisms to lay a solid foundation, but also for artificial breeding and genetics and breeding production practices of Onchidiidae to provide theoreticalguidance.