Screening Of Molecular Markers And Genetic Analysis Based On Transcriptome In Mud Crab, Scylla Paramamosain

The mud crab Scylla paramamosain is an important marineaquaculture crab in China and it is mainly distributed along the southeast ofChina. With the development of market demands and aquaculture scale, itis important to develop a new variety with high yield. Therefore, thebreeding of excellent varieties and molecular markers assisted breeding hasbecome an important subject. Yet, the development of molecular markers isthe foundation for research in Scylla paramamosain. In this study,microsatellites and single nucleotide polymorphisms (SNPs) wereidentified via454transcriptome high-throughput sequencing data,respectively, and SNPs were genotyped through Tm-shift assay in the mudcrab Scylla paramamosain. Then we used these microsatellite loci toanalyze genetic rule in a family of Scylla paramamosain. The resultsobtained are as follows:1. In this study,263pairs of primers were designed by Primer Premier5.0based on microsatellite sequences obtained from454transcriptomehigh-throughput sequencing data, and67polymorphic microsatellitemarkers were identified. A total of556alleles were detected in a singlepopulation of32individuals of S paramamosain. The number of alleles perlocus ranged from2to27, with the average of8.3. The polymorphisminformation content (PIC), observed (Ho) and expected heterozygosity (He)ranged from0.215to0.946, from0.1888to1.000, from0.230to0.963, respectively, with an average of0.899,0.703and0.736per locus. AfterBonferroni correction, ten loci deviated from Hardy-Weinberg equilibrium(HWE). The results showed that these markers have high polymorphicinformation content and has a certain reliability and credibility for theanalysis of genetic diversity of S. paramamosain.2. In this study, single nucleotide polymorphisms (SNPs) were identified,confirmed and genotyped in the mud crab Scylla paramamosain through454transcriptome high-throughput sequencing data and Tm-shift assay. Atotal of13,311bp high quality sequences were obtained by re-sequencingwhich contained91SNPs loci, with the density being one SNP every146bp. Of all91SNPs,40were successfully genotyped and characterizedusing30wild specimens by Tm-shift assay. The minor allele frequency perlocus ranged from0.017to0.500. The observed and expectedheterozygosity ranged from0.000to0.600and from0.033to0.509,respectively, with an average of0.142and0.239per locus. SeventeenSNPs loci were tested deviating from Hardy-Weinberg equilibrium. Nosignificant linkage disequilibrium between pairs of loci was detected aftersequential Bonferroni correction (P>0.00125). Seventeen SNPs loci wererelated with the known function genes. This study provided new molecularmarkers for the studies on investigation of population genetic diversity,construction of genetic linkage maps and molecular marker-assisted selectin this important crustacean species.3. In this study,155microsatellite markers were chosen for genetic ruleanalysis in a family including95individuals of S. paramamosain. And49microsatellites appear separation and genotyped in the offspring. Theresults showed that11microsatellites emergy separation in the progeny andno polymorphism in parents, but these loci can not be used to analyze marker-linkage. The separation tags of the female parent has34locus, eightof which deviated from the Mendelian low (P<0.05). Which31loci in themale parent, nine tags diverged from the Mendelian low (P<0.05). Thereare27separated markers in the parent. By using JoinMap software, onlysix markers appear chain phenomenon in linkage analysis of female, andnine in male. The purpose of the study lay the foundation on furtherconstruction of genetic linkage map, QTL mapping and marker-assistedbreeding in mud crab, Scylla paramamosain.