| The blood clam, Tegillarca granosa, is an economically-important bivalve speciesextensively farming in Asia. Because of its status as a nutrient-rich species with afavorable taste, cultivation of T. granosa has become extensive along the southeast coastof China. In a previous study, we sequenced the diverse transcriptome of T. granosa(SRA052081) using Roche454sequencing technology. Basing on the transcriptome, wedeveloped139EST-SSRs and78EST-SNPs. In the meantime, we used9polymorphicSSR loci to detect genetic diversity of four different geographical populations. Thisstudy would be particularly useful for further investigations into population variation,conservation genetics, genetic linkage map construction and quantitative trait locus(QTL) mapping in T. granosa.1. Development of EST-SSR in T. granosa454transcriptome library includes contig sequences and singleton sequences.Using the SSRHunter software searches both the nucleic acid sequence repeats2-6andthe length is not less than12bp. In the singleton sequences,50pairs of primers weredesigned successfully by using132candidates of SSR, It was revealed from theinvestigation of polymorphism that24SSR loci were polymorphic. The number ofalleles of the24EST-SSR loci varied from2to6, and the mean value is about3.5. Therange of the observed heterozygosity, the expected heterozygosity and the polymorphicinformation content was0.000to0.591,0.045to0.727and0.043to0.696. There are9high polymorphic SSR loci,4moderate polymorphic SSR loci, and11highpolymorphism SSR locus.17SSR locus are significant deviations from Hardy-Weinbergequilibrium after Bonferroni correction.In the contig sequences, a total of246primer pairs were successfully designed.After optimization of experimental conditions,157primer pairs showed clear andconsistent amplification patterns. Among these EST-SSRs,115were found to bepolymorphic.The number of alleles observed for the115loci varied from2to10withan average locus of3.78alleles. The observed heterozygosity ranged from0.000to1.000, while the expected heterozygosity varied from0.040to0.799. The average observed heterozygosity (0.3885) across all loci was lower than the expectedheterozygosity (0.4338). There are29highly polymorphic loci and42moderatepolymorphic loci, which are suitable for population genetic analysis. Thirty-eight of115loci significantly deviated from HWE after Bonferroni’s correction.2. Development of78SNP markers based on transcriptome in T. granosaIn total,275predicted SNPs were randomly chosen for detection. Among them,147loci were successfully amplified, of which78loci were polymorphic.The observedheterozygosity varied from0.000to0.9000, while the expected heterozygosity rangedfrom0.1997to0.5088. In addition, the minor allele frequency varied from0.0167to0.5000.32loci had significantly departed from Hardy–Weinberg equilibrium afterBonferroni’s correction.54SNPs are the type of transition including23A-T and31T-C.24SNPs are the type of transversion which containing13A-C,4A-T,2G-C and5G-T。3. Analysis on population genetic variationNine highly heterozygosity SSRs were used to analysis on four differentgeographical population. Nine SSRs loci got76alleles of160individuals in four groups,the average number of alleles was8.44, the number of alleles between3to12.Population genetic differentiation between Jiangsu and Zhejiang was in weak level,Guangdong and Fujian also belongs to the weak of differentiation. The highest degreeof differentiation was between Zhejiang and Fujian population, its Fstwas0.0766,which belongs to the medium degree of differentiation. Genetic distance minimum of0.1151in Jiangsu and Zhejiang, Guangdong and Fujian population genetic distance isalso a smaller0.2563, including genetic distance the furthest as Fujian and Zhejiang to0.5757. Between Jiangsu and Zhejiang had the minimum genetic distance with the0.1151, Guangdong and Fujian population genetic distance is also smaller. One of thebiggest genetic distance was between Fujian and Zhejiang with0.5757. |
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