Development Of SNP Markers And Research Of Related Gene In Turbot (Scophthalmus Maximus)based On High Throughput Transcriptome Sequencing Data

Turbot (Scophthalmus maximus L.) is economically important in wild fisheries and hasstimulated the development of aquaculture for several other species. Since turbot was introducedto China in1992, breeding and culture technology has been greatly advanced andcommercialized.However, the flatfish farming industry remains limited to certain areas byenvironmentconstraint.With the rapid development of second-generation sequencing technology,high-throughput transcriptome sequencing cost reductions for large-scale screening SNP markersprovided an opportunity. Based on Scophthalmus maximus transcriptome sequencing data, turbotSNP markers were screened, initially explored gender-related SNP markers and cloned thefull-length cDNA of ATP synthase-α subunit.The main results are as follows:1.This study is based on Scophthalmus maximus transcriptome sequencing data,45SNPswere selected, and63pairs of primer were designed, of which21(46.7%) loci using highresolution melting (HRM) technology with small amplicons genotyping success.21sites all havetwo haplotypes, were proved to be polymorphic. Observed heterozygosity (Ho) estimates rangedfrom0.256to1.000, Expected heterozygosity（He） estimates ranged from0.276to0.518and19loci were in Hardy-Weinberg equilibrium after Bonferroni correction.14SNPs were located inthe coding region,3non-synonymous changes. Most SNP-containing genes were related tosignal transduction mechanisms and transcription.2. In this study, using five SNP markers developed in the last chapter, genetic differencesbetween male and female turbot traits were analyzed, and the effective number of alleles of SNPloci, observed heterozygosity, expected heterozygosity and polymorphism information contentand the minor allele frequency were calculated, two gender-related SNP markers wereidentified.Correlation coefficient of S10is-0.288(p <0.05), correlation coefficient of S11is0.304(p <0.05).3. In the experiment, using RT-PCR and RACE technology, according to the sequenceobtained from transcriptome sequencing, primers were designed to amplify RACE fragment, afull-length cDNA of ATPase α subunit gene from turbot spleen was got,2034bp. The sequenceswere analyzed by bioinformatics, the amino acid sequence encoded by this gene was predicted,the largest ORF of1656bp encoding551amino acids, with a molecular weight of59.75kDa andan isoelectric point (pI) of9.36; four kinds of secondary structure were predicted, threeconserved domains were got.Phylogenetic analysis demostated that ATPase α subunit has themost highest homology to those of other vertebrates, respectively.