Development And Application Of SSR Markers In Rhopilema Esculentum

Rhopilema esculentum belongs to the Cnidaria, Scyphozoa, Rhizostomeae,Rhizostomatidae and Rhopilema. It is an important edible jellyfish in fishery resourcesfor its internationally recognized quality and significantly economic value.R.esculentum has unique nutritional structure and medical efficiency, therefore it hasgreat market both domestic and international. In recent years, R.esculentum plays animportant role in China’s export. As a traditional fishing species, the national catch ofR.esculentum even reached700,000tons in1970s. However, over the last twenty years,its natural resources reduced sharply due to long-term overfishing and deterioration ofmarine ecosystem. According to the statistics, national catch of R.esculentum haddropped to100,000tons in1980s, the catch were even less than10,000tons in2011.Reasons for the gradually exhausted jellyfish resources are various. Knowing moreabout the genetic diversity and situation of germplasm resources of R.esculentum willprovide scientific foundation to the development of germplasm resources protection,marker assist breeding, artificial culture and genetic breeding. This research includes thefollowing contents:(1) Development of genomic-SSR in R.esculentum and its population analysis:Fifty-nine pairs of microsatellite DNA markers were isolated from R.esculentumgenomic library through magnetic beads enrichment. After being synthesized, primerswere tested in a population of R.esculentum collected from Shidao. Then we got15effective primers which could amplify the genetic polymorphism. A total of93alleleswere obtained, the number of alleles per locus ranged from2to12, each locus amplified6.2alleles in average. The observed and expected heterozygosities ranged from0.0417to0.8750and0.0086to0.7055, respectively. The observed genotypes deviated fromHardy-Weinberg expectations in1loci after Bonferroni correction (p<0.0033), resultingfrom heterozygote deficiency.(2) Development of EST-SSR in R.esculentum and its population analysis:Seventeen loci with polymorphism were developed in R.esculentum after testingeighty-three pairs of EST-SSR markers. Results show that the distribution of the numberof alleles was from2to11, with a total of69. The expected heterozygosity ranged from0.1180to0.5353, with an average of0.3568. The observed heterozygosity ranged from 0.0417to0.5833, with an average of0.3346.1loci deviated from the Hardy-WeinbergLaw (p<0.0029) after Bonferroni’s correction and1loci existed linkage disequilibrium.(3) Applications of R.esculentum EST-SSR molecular markers:a) Research on cross-genus transferability of EST-SSR molecular makers: Onehundred and twenty-seven microsatellite markers were selected from R.esculentumtranscriptome library. After being amplified in a group of48individuals, fourteenmicrosatellite markers were isolated and characterized for Nemopilema nomurai. Thenumbers of alleles per locus was from2to4, with a total of42. The observed andexpected heterozygosities ranged from0.2979to0.7692and0.1333to0.7025, withaverages of0.5919and0.3574, respectively.2loci significantly deviated fromHardy-Weinberg equilibrium after Bonferroni’s correction, linkage disequilibriumexisted between two loci pairs (RE5-RE11and RE5-RE49).b) Research on cross-order transferability of EST-SSR molecular makers: Onehundred and twenty-seven microsatellite markers were selected from R.esculentumtranscriptome library. After being amplified in a group of48individuals, thirty-fivemicrosatellite markers were isolated and characterized for A.aurita, which had4kindsof repeats. Among these sequences,10were dinucleotide repeats,23were trinucleotide.The most common dinucleotide and trinucleotide repeats were the motif (AT)n、(CTT)nand (GTT)n. The range of production was139-279bp, the optimum annealingtemperature was between48and62.c) Application of R.esculentum EST-SSR in two geographical groups: Using14polymorphic loci developed in this study, we analyzed the population genetic structurein two geographical group samples of N.nomurai. The results showed that the numberof observed and effective alleles was4.21and2.03, respectively. The observedheterozygosity value was0.2415and the unbiased expected heterozygosity was0.8405.Through analyzing the genetic differentiation coefficient (Fst), two sites showedmoderate genetic differentiation, ten sites were mild genetic differentiation. The averagegenetic differentiation coefficient of two groups was0.1060, which refered to10.60%ofgenetic differentiation among populations. The genetic similarity coefficient amonggroups (I) was0.8308, while the genetic distance (D) was0.1147. In conclusion, thegenetic difference degree between these two N. nomurai groups was extremely low, thusthey had high genetic similarity.d) For the first time, target fragments of β-catenin gene including EST-SSR andEST-SNP markers were cloned with genomic DNA templates of Rhopilema esculentumand Nemopilema nomurai by454GS-FLX transcriptome sequencing and PCRtechniques, respectively. Bioinformatics analysis showed that target fragmentsnucleotide sequences did not contain intron and the lengths of nucleotide sequences were166/169bp and157/160bp in R.esculentum and N. nomurai populations,respectively. There was only a base difference among individuals of R.esculentum,while there was no difference among individuals of N.nomurai, apart from SSRdifference. In the same site, the target fragments of β-catenin gene shared a distinctrepeat motif,(TGC)4-6(TGT)1-2(TGC)4-5in R.esculentum and (TGT)5-6in N. nomurai. Inaddition,14SNP loci from the target fragments of β-catenin gene were detected in twopopulations, including (T/C)1,(T/C)2,(C/T)3,(C/T)4,(C/T)5,(T/G)6,(G/C)7,(T/G)8,(A/G)9,(C/T)10,(G/A)11,(A/G)12,(C/T)13,(A/T)14.