Development And Characterization Of SNP Markers And Their Application In Family Analysis And Species Identification For Pacific Oyster Crassostrea Gigas

Crassostrea gigas (Pacific oyster) belongs to mollusks, widely distributed alongthe seashore of China, Korea, and Japan, is a bivalve with wide tolerance oftemperature and salinity. It is the top-drawer species in the world shellfish industry,and become one of the most important cultured species in China. Owning to thecharacteristics of high-density genome-wide distribution, genetic stability,co-dominance, and allowing high-throughput assays, single nucleotidepolymorphisms (SNP) is a kind of ideal molecular marker for the genomics andgenetics research. Now, redundant sequences found in EST database can be a usefulway for mining SNP markers or other mutation sites. High resolution melting curve(HRM) has been developed to detect and genotype SNPs because it is a simple, rapidand cost-effective technology. This research is to develop and characterize SNPmarkers of C. gigas by HRM method, and apply SNPs in family analysis andCrassostrea species identification. The results provide new insight of SNPs in thefields of high-density genetic mapping，biodiversity conservation，population structureanalysis and marker-assisted breeding.1. The development and characterization of SNPsThough mining EST database and manual quality filtering, a total of48769putative SNPs were detected from46171ESTs.55gene-derived SNPs were isolatedand characterized by means of HRM analysis. The observed and expectedheterozygosities ranged from0.063to0.563and0.091to0.448, with an average of0.284and0.279, respectively. The PIC ranged from0.059to0.371with an average of0.231. The combined power of exclusion was greater than99.99%across all theseSNPs.2. The application of SNPs in family analysisAll the55polymorphic SNPs were analyzed in six C. gigas single-pair matingfamilies. No null allele was detected in these families.48loci were polymorphic andsegregated in at least one of the six families:26in family A,21in family B,27infamily C,20in family D,19in family E,19in family F. As indicated by the Chi-square test,22(84.6%),19(90.5%),16(59.3%),15(75.0%),15(78.9%), and15(79.0%) SNPs segregated in expected Mendelian inheritance ratios in these familyrespectively, while the other showed significant distortion (P <0.05).The power of polymorphic SNPs in parentage assignment was evaluated, and thereal data demonstrated that17%of all real offspring were unambiguously assignedparents with30SNPs, and100%of the offspring were correctly allocated to theirparents when40or more SNPs were used. Simulation results show that theassignment success was obviously decreased with the increase number of parents.Parentage assignment results suggested that more marker sets were needed tocounteract low polymorphism so as to achieve stable assignment.3. The application of SNP marker in the Crassostrea species identification.Small amplicon and species-specific primer were used to genotype themitochondrial16S rRNA genes of five different Crassostrea oysters. Resultsdemonstrate that HRM is a powerful genetic tool for species classification and allowsthe unambiguous detection of the sequences difference caused by SNPs.