Development Of SNP And SSR Markers And Establishment Of Parentage Analysis Technique In Scylla Paramamosain

The mud crab Scylla paramamosain is an important marine aquaculture crab inChina and it is mainly distributed along the Indo-West Pacific regions and thesoutheast of China, including coastal region of Zhejiang, Fujian, Taiwan, Guangdong,Guangxi and Hainan. With the development of market demands and aquaculture scale,the demand of the seed is also growing. Therefore, the breeding of excellent varietiesand molecular markers assisted breeding has become an important research subject.Hoewer, clear pedigree information is of great importantance for effective selection ofparents and for parental management, and molecular genetic markers can effectivelyidentify family and obtain the pedigree information. So for species that lack ofmolecular markers relatively, the development of molecular genetic markers becomesparticularly important. This study explored37gene related candidate SNPs using thedirect sequencing method based on wild Scylla paramamosain populations of Ningdeand Qing Lan, respectively. SNaPshot method was used for genotyping, and24SNPswere successful classified finally. We also developed63polymorphic microsatellitemarkers based on majority of microsatellite DNA sequences from transcriptome deepsequencing of Scylla paramamosain and10polymorphic microsatellite markers wereused for pedigree analysis and identification research. The results obtained are asfollows:1.Single nucleotide polymorphisms (SNPs) are thought to be well suitable forgenetic and evolutionary studies. In this study, we reported the first set of SNPmarkers in a commercially important crab species, Scylla paramamosain. A total of12,500base pairs high quality DNA sequences were obtained from15genes, andthirty-seven SNPs were identified, representing one SNP every338base pairs.Twenty-four SNPs were successfully genotyped in a single population. All loci hadtwo alleles and the minor allele frequency ranged from0.02to0.44. The observed andexpected heterozygosity ranged from0.04to0.59and from0.04to0.50, respectively. No significant departures from Hardy-Weinberg equilibrium at each locus was found.The linkage disequilibrium was detected in six loci pairs, but absent after sequentialBonferroni correction. These SNP markers will provide a useful addition to thegenetic tools for genetic and evolutionary studies for S. paramamosain.2. Totally300pairs of primers were designed bsed on the majority ofmicrosatellite sequences obtained from Scylla paramamosain transcriptome deepsequencing, and63polymorphic microsatellite markers were developed. Theobserved number of alleles were from2to14, with an average of5.6every loci. Theeffective number of alleles were from1.4to9.1, witn an average of3.8every loci.The observed heterozygosity and expected heterozygosity were from0.19to1(mean0.67)and from0.27to0.90(mean0.66), respectively. The polymorphism informationcontent (PIC) was ranged from0.25to0.89, with the average of0.62. There are11loci deviate significantly from Hardy Weinberg equilibrium after Bonferronicorrection(PH-W<0.0008), and no linkage disequilibrium was found between locipairs.3.10polymorphic microsatellite markers were used for genealogicalidentification and phylogenetic relationship analysis for4full-sib families. The resultsshowed that, the non-exclusion probability were ranged from59.3%to88%withunknown the parent pair(NE-1P), from41.2%to72.6%with known one parentalgenotypes (NE-2P), respectively. The cumulative probability of non-exclusionunder the two cases were97.02%and99.83%, respectively. The cumulativeprobability of non-exclusion under the other3cases were all greater than99.9%. Theresults also revealed that the more the polymorphic microsatellite markers were used,the higher the non-exclusion reached. In the case of unknown parental genotypes,setting a confidence level of95%, the success rate can reach100%, when7polymorphic microsatellite markers were used. Then paternity analysis was workedout by introducing a double-blind test, the results showed that95%of the individualscan accurately find their real parents. This result proved the feasibility ofmicrosatellite markers used in paternity analysis in Scylla paramamosain.