Development Of Ssr Makers And MCH Class ⅠCloning And Expressing Of Siniperca Chuatsi By RNA-Seq

Siniperca chuatsi, belonging to Perciformes Sinipercinae Siniperca, commonly known as mandarin fish, osmanthus fish, fat salmon, season, etc., is a freshwater benthic carnivorous fish. It is one of the chief economic freshwater fishes in China due to its large size, fast growth and delcious flesh. In recent years, due to over-fishing rivers, the waters of the deterioration of the ecological environment and aquaculture production inbreeding and other reasons, resulting in decline of natural germplasm, breeding germplasm traits degradation, fish disease outbreaks continue the status quo, has affected Siniperca chuatsi germplasm continue its normal development of aquaculture.Therefore, it’s an urgent need to use the means of modern molecular biology research Siniperca chuatsi breeding germplasm resources protection and other aspects. Thus, with the methed of transcriptome sequencing technology,we developed Siniperca chuatsi growth associated with EST-SSR markers, then, genetic structure and genetic diversity of four breeding populations were studied; Cloned Siniperca chuatsi MHC class I gene expression analysis and provide a theoretical basis for Siniperca chuatsi breeding and cultivation of new varieties resistant, the main research is structured as follows:1 Development of EST-SSR markers in Siniperca chuatsi by RNA-Seq1 In this study, Transcriptome sequencing were performed using Illumina high-throughput plateform. In all, 35 285 sequences containing 14 094 SSRs were identified from 51 245 consensus sequences(non-redundant or unigenes), the frequency and the distribution density were 27.51% and 184 per Mb separately in Siniperca chuatsi transcriptom, and a total of 7 335 motifs were designed primers successfully. The selected 100 pairs of EST-SSRs associated with economic traits obtained in the four GO levels 2 terms were validated and consequently 20 polymorphicSSRs were obtained.2 There were 262 alleles were detected acrossed the 120 Siniperca chuatsi of 4populations and the number of effective alleles(Ne) were between 2-3, the average alleles(Na) and effective alleles(Ne) values of each loci were 2.15 and 1.80respectively; the average observed(Ho) and expected(He) heterozygosity values were and respectively, and the mean polymorphism information content(PIC) values was0.34 which has reached to the middle polymorphism lever(0.5>P>0.25).The level genetic diversity of four breeding population were Taihu population> Dongting population> Yangtze Delta population> Poyang lake population.3 The genetic distances between Poyang lake and Yangtze Delta populations was the largest, which reached to 0.1302,while the genetic similarity index of it was the smallest, which indicated that the genetic relationship between these two populations was more distant. Otherwise, the genetic distances values and genetic similarity index between Poyang and Dongting lake were 0.9453, respectively,suggesting that the genetic relationship between these two populations was more closer. The UPGMA dendrogram based on Nei’s genetic distance indicated that the Dongting and Poyang populations are grouped in one cluster, which is then clustered with Taihu populations,and joined with Yangtze Delta populations finally.2 Correlation analyses of growth traits of Siniperca chuatsi based on EST-SSR markers13 polymorphic microsatellites were selected to analyze the linkage analysis of four growth traits among 60 individuals, finding that 5 loci of which were associated with the growth traits in all. The result indicated that: the locus C10127 had a significant impact on body length(P<0.05)and extremely significant on body thick(P<0.01), and AB at C10127 was favorable genotype for body length and body thick while AC was disadvantage genotype; locus C19016 showed a significant impact on body weight( P<0.05) and AB at C19016 was favorable genotype for body length; locus C14847 had a extremely significant impact on body length(P<0. 01) and a significant impact on body height, body thick, body weight( P<0.05), in which BB was the dominant genotype of body thick; locus C12350 had a significant impact on bodyweight(P<0.05)and extremely significant on body length(P<0.01),BC at C12350 was favorable genotype for the four growth traits while AC and AC was disadvantage genotype for body length; locus C18691 showed a significant impact on body height(P<0.05)and extremely significant on body thick, body weight(P<0.01),BB was the dominant genotype of four growth traits while AC was the negative genotype.3 Molecular cloning and gene expressing analysis of MHC class I in Siniperca chuatsi1 The primers were designed according to Siniperca chuatsi MHC class I gene c DNA sequence to obtain the whole sequence of it through RACE(rapid amplication of c DNA end) method of 5’ and 3’ end. The full length of S.chuatsi MHC class I gene c DNA is 1925 bp, with 74 bp 5’-UTR(untranslated region), an open read frame(ORF) of1071 bp, encoding 357 amino acids probably, 3’-UTR with length of 780 bp, having obvious termination codon TGA, polyadenylation signals AATAAA and poly A tail. The protein of S.chuatsi MHC class I owns one transmembrane domain as well as a signal peptide. Finally, analyzed the sequences of MHC class I, fishes and human by using DNAMAN and MEGA. Phylogenetic analysis using Clustal W2 software showed that the similarity was more than 87% from Lithognathus mormyrus 、 Sparus aurata 、Lutjanus sanguineus、Dicentrarchus labrax and so on.2 With fluorescent quantitative RT-PCR method, we tested the relative expression quantity of MHC class I genes in S.chuatsi heart, liver, spleen, lung, kidney,gill, brain, intestine and muscle tissue. The result showed that, the expression of MHC class I gene differences from each other. Higher expression in liver and intestine, lower in gill, muscle, lung, lung, brain, merely in kidney.