Development And Characterization Of SSR Markers In Paralichthys Olivaceus And Preliminary Analysis Of SNPs In Disease Resistance Genes Of Crassostrea Virginica

I.Isolation and characterization of SSR markers in Paralichthys olivaceusIn this study, microsatellite (or simple sequence repeat,SSR)markers of Japanese flounder (Paralichthys olivaceus Temminck et Schlegel)were isolated and characterized through construction of genomic genome library and colony hybridization, construction of microsatellite-enriched library, and mining of EST sequences in GenBank database.1.Library construction and colony hybridizationA genomic library of selected fragment sizes was constructed. Through colony hybridization using (AC)10 and (AG)10 as probes 20 SSRs were identified, of which 8 SSRs were detected to be polymorphic.A SSR-enriched library was constructed using (GA)20和(CA) 2o as probes.A total of 350 positive clones were screened by colony hybridization, and 299 SSRs were identified from 253 clones.Primers were designed for 173 SSRs,from which 131 pairs of primers could amplify fragments of the expected sizes.All the 8 polymorphic SSRs from the genomic library and 38 of the 131 polymorphic SSRs from the enriched library were selected for polymorphism analysis in 30 wild individuals.The results should that the amplified number of alleles of the 46 SSRs varied from 4 to 22, with average allele number of 14.3. The observed (Ho) and the expected heterozygocity(He) was 0.3285～0.9655 and 0.3854～0.9732, respectively,and the average polymorphism information content PIC was 0.79.2. Data mining from GenBank EST sequences EST data mining was also used for searching SSRs in Japanese flounder. There is a large amount of sequences in EST database of Japanese flounder. A total of 11111 EST sequences were found in the database, from which 882 SSRs were identified in 751 EST sequences that accounted for 6.76% of all sequences analyzed.Among these,dinucleotide SSRs,trinucleotide SSRs, tetranucleotide SSRs,pentanucleotide SSRs and hexanucleotide SSRs were 444, 25,53,95 and 40, respectively. Ten dinucleotide sequences were selected to design primers and to assay the polymorphism, of which 8 loci were proved to be polymorphic.The number of alleles ranged from 6 to 12, and averaged 8.88.The values of He and Ho ranged from 0.3285 to 0.9655 and 0.3854 to 0.9732, respectively, and the average PIC value was 0.79. Among these different methods identification, EST database mining is proven to be an efficient and low-cost method to obtaining new microsatellite markers.Furthermore, prior to the genome library method, the construction of microsatellite-enriched library was an optimal choice for its high efficiency and relative low cost.II Analysis of the SNPs in candidate genes associated with resistance of Crassostrea virginicaIn this study, the Big defensin gene (CvBD) and Serpin 1 gene in C. virginica(cvSerpin 1) were chosen as disease resistant genes for analysis of single nucleotide polymorphisms (SNP) and detection of their associations with the susceptibility or resistance to MSX (caused by the parasite Haplosporidium nelsoni) and Dermo(caused by the parasite Perkinsus marinus). Not only different geographic populations but also different families before and after mortality were used in the analysis.1.Single nucleotide polymorphism analysis in the CvSerpinl geneIn an attempt to identify useful markers that could predict susceptibility to the disease and mortality of the American oyster(C. virginica),we resequenced all 24 exons from 3 different geographical populations (MA, DB and AL).Fourteen SNPs were identified, all in the coding exons.Of these, nine were non-synonymous SNPs,while the remaining five were synonymous.SNPs genotyping were used for screening by Real-time PCR with High Resolution Melting curve analysis in four wild populations ((Massachusetts (MA),New Jersey (NJ),Florida (FL) and Texas (TX))from north to south of the USA and three cultured families before and after mortality. Only one synonymous SNP CvSPI224(A-C)was well genotyped and polymorphic in all the populations and families detected. This SNP showed significant difference not only between north and south populations,which may due to the geographic isolation, but also significant difference between Texas and Florida population.This evolutionary selection between Texas and Florida may due to the different situation of infection by oyster disease such as MSX and Dermo. Also significant differentiation was detected in the main cultured families before and after mortality. This may suggest it has significant association between the SNP CvSPI224(A-C)CC genotype and the disease phenotype, which means this locus was related to the disease-resistance.These results were identified to the conclusion of La Peyre et al.(2009),which based on the CvSerpinl gene expression and plasma protease inhibitor activity in American oysters.2.Single nucleotide polymorphism analysis in the CvBD geneWe also resequenced all 24 exons of gene highly homologous to the big defensin gene of American oyster(CvBD) from three different geographical populations.Twenty-five SNPs were identified, twenty-four in the coding regions and one in the 5'Untranslated region. Of these, twenty-three were non-synonymous SNPs,two were synonymous.The surprisingly high NS/S ratio is one feature thought to be consistent with strong positive selection in evolutionary change of the gene.Two non-synonymous and polymorphic SNPs were used for screening by High Resolution Melting Curve analysis in four wild populations (MA,NJ, FL, TX) and three families before and after mortality. SNP CvBD378 (T-G)just showed significant difference between the north and south populations. But SNP CvBD90 (T-G)was interesting due to its significant differences between FL and other populations,which may suggest it has evolutionary selection between FL population and others.Screening on families before and after mortality with these SNPs was also addressed. No difference between families before and after mortality in both two SNPs could be concluded, which may not support the association between the genetic polymorphism of CvBDand the oyster mortality caused by MSX and Dermo in these families.However, the deviation from the mendelian inheritance ration in XG3 family and the deviation from Hardy-Weinberg equilibrium in both the two SNPs.These results indicated that the copy numbers variant (CNV) occuring in the CvBD gene may affect the assessment of SNPs adjacent to. More SNPs need to be developed and details of CNVs locations still need to be studied to convince their relationships to the innate defense mechanisms and susceptibility to disease.In the present study, we isolated and characterized a panel of microsatellite markers with three different methods,and also studied their polymorphism,laying a foundation for the mapping and MAS in Japanese flounder. SNPs of two candidate gene for disease resistance were isolated and screened.The-results suggested that,the SNP CvSPI224(A-C)CC genotype of CvSerpinl gene was significantly associated with the resistance of American oyster MSX, Dermo and other factors involved in the mortality, and could be potential marker applied in future selection of American oyster with enhanced disease resistance.These results would hopefully provide reference for MAS in mollusks.