Large-scale Development Of SNP Marker And Its Potential Application In Genetic Breeding Of Crassostrea Gigas

Crassostrea gigas belongs to mollusks, widely distributed across the world inAsia, America, Europe and Australia. Crassostrea gigas started to be cultured inChina since1980s, and now become one of the important cultured mollusk of China.Summer mortality and the degenerationof production traits impact the aquacultureseriously in the past decades, which bring new requirements to the Crassostrea gigasbreeding. At the same times, the oyster is established to be the model species by thepromotion of whole genome sequencing, which lay the foundation to the molecularbreeding of oyster.The development of molecular biology and genomics promote the transitionfrom the trational breeding to the molecular breeding, which focused on thegenotyping and phenotyping.. Whole genomic sequencing of the C. gigas make itpossible to screen and validate the SNPs.Three transcriptome were used to obtain theSNPs in the gene region, and a platform based on HRM was established to distinguishthe genotypes of the SNP. A total of1329SNP markers were validated based on thisplatform, accounting for65.2%of the total slected SNPs. And84%of the developedSNP markers are transition which contains32.5%R;35.8%Y;7%K and9%M, theremaining are transversion which contains10%W and6%S. The primers sequencewere used to locate the the SNPs to the genome and the1329SNP markers distributein653Scaffolds, accounted for38.1%of the whole genome..The obtained SNPmarkers are distributed in864genes of197KEGG path ways, and these path ways involve metabolism, stress, immune, resistance, adjustment, reproduction and bodycomposition of Crassostrea gigas. These developed SNP markers provide a goodfoundation for validation of the gene function and the phenotyping–genotyping of theeconomico traitss of the oyster..The HSP70gene family is expanded in Crassostrea gigas compared to otherspecies, which may have a relationship to the adaption of intertidal zone life, andthere are studies showed a correlation between the summer mortality and the HSPgenes. A total of43SNP markers were developed from the88HSP70genes, and theSNP markers are genotyped in the high temperature-sensitive and high temperature-resistant group. Allele frequency and genotype frequency of all the markers in thetwo groups are counted and correlation analysis is performed to calculate thecorrelation between the alleles frequency and genotype frequency to the hightemperature resistance. Two SNP markers are found significant correlated to thetemperature resistance (p<0.05) and one SNP allele (SNP863) shows extremelysignificant correlation to the temperature resistance (p<0.01). This indicats that thegenes contained the markers play a very important role in high temperature resistanceof Crassostrea gigas. Both of the two SNP markers are Y, and the frequency of alleleT and genotype TT of the SNP809marker are high in the high temperature resistantgroups, while the frequency of allele C and genotype CC of SNP863marker are highin the high temperature resistance groups. The two markers provide a good startingpoint for the further mechanism research of high temperature resistance. And establishclear goals for the high temperature resisting new varieties of breeding.A genetic map is established with SNP tags obtained by resequencing of areduced representation library in a F1family of Crassostrea gigas. The geneticlinkage map is composed of10linkage groups with total of1584SNP markers. Andthe total length of the genetic map is737.34cM. There are336markers in the largestlinkage group while there are61markers in the smallest one, and the average distancebetween markers on this map is0.47cM.78.41%(1242) SNP markers are mapped to654Scaffolds with the overall length over282M, which accounted for half of the whole genome. The establishment of the genetic map laid the foundation for QTLanalysis and chromosome mapping of scaffold.Species identification are also studied in this research, a species identificationmethod based on the SNP genotyping platform for oyster is established. And a pair ofprimer from the DNA barcoding (COI) sequence is obtained with which can identifythe5species in Crassostrea at the same times. This method shows more simple, moreclearly, more precise and more effectively against the usual methods, and this is anopen system for species identification, all the shellfish species which can be identifiedby DNA barcoding can be identified by this method.