The Identification And Genetic Interaction Analysis Of The Vibrio-resistance Related Markers/genes In The Clam Meretrix Meretrix

The clam Meretrix meretrix belongs to Lamellibranchia, Mollusca, and has broad market prospects. However, the development of M. meretrix aquaculture is limited by disease problems, especially Vibrio infection. Given that M. meretrix live in the open sea, it is difficult to reduce disease outbreaks by isolating the pathogens. Therefore, it is important to cultivate resistant strains in genetic breeding of M. meretrix. Recently, the development of molecular marker-assisted breeding(MAS) provides a new approach for resistance breeding of M. meretrix. Molecular markers are not affected by gene expression and environment, and can be detected in the early biological development. So the combination of MAS and traditional breeding methods will improve the efficiency of resistance breeding of M. meretrix.Development of reliable molecular markers associated with resistance is premise for MAS in resistance breeding. The vibrio-resistance related molecular markers in M. meretrix were developed and validated in the present study. Firstly, the stock materials of M. meretrix with different vibrio-resistance were constructed through vibrio infection experiments(i.e. 09-R, 09-C to Vibrio harveyi; 11-R, 11-S, 14-R and 14-S to V. parahaemolyticus). Secondly, the contigs involved in immune were excavated from M. meretrix transcriptome by bioinformatics methods, which contained 48 SSRs and 323 SNPs. These molecular markers would be applied as candidate markers for subsequent marker-trait association analysis.For the development of SSRs associated with vibrio-resistance, 8% nondenaturing polyacrylamide gels electrophoresis was used for genotyping, and 27 SSRs were verified to be polymorphic in 2011 Shandong M. meretrix population. Chi-square test was used for marker-trait association analysis, and MM959, MM4765 MM8364 were identified to be associated with vibrio-resistance. Multi-dimensional scaling(MDS) analysis showed that clusters generated by genetic similarity revealed by these 3 SSRs were consistent with the subgroups distinguished by survival time in 11-S. The putative functions also suggested that the three SSR-involved genes might play important roles in immunity of M. meretrix.For the development of SNPs associated with vibrio-resistance, Multiplex SNaPshot genotyping method was used and the genotypes of 40 SNPs were obtained in 2011 Shandong M. meretrix population. Chi-square test was applied for marker-trait association analysis, and 8 SNPs were found to be associated with vibrio-resistance trait, among which 4 SNPs were further validated in 09-R and 09-C population. Sequence alignments and annotations indicated that the contigs containing the associated SNPs had high similarity to the immune related genes.The interactions between molecular markers were also analyzed by multifactor dimensionality reduction(MDR), and the results showed that there were different levels of interactions between markers. The general linear model(GLM) indicated the interactions between 4 pairs of SSRs and 3 pairs of SNPs had significant influence on vibrio-resistance trait in M. meretrix. The binary logistic regression model contained both SSRs and SNPs could distinguish 11-R and 11-S groups.The full-length cDNA of MmeHairy was obtained and phylogenetic tree revealed the MmeHairy belongs to the Hairy protein subfamily. Real-time quantitative PCR showed that MmeHairy had the highest expression in hepatopancreas, and the expression of MmeHairy up-regulated in hepatopancreas after V. parahaemolyticus challenge. Quantitative analysis also showed that the MmeHairy mRNA expression levels had significant differences among clams with different genotypes at SNP1066 and 1067(P < 0.05), which indicated that these two SNPs may affect the expression of MmeHairy.The 1936 bp of Mme Id2 gene sequence was cloned which contained one 227 bp of intron, and 32 SNPs of MmeId2 were detected by direct sequencing. Marker-trait association analysis was applied and 5 V. harveyi resistance related SNPs and 3 V. parahaemolyticus resistance related SNPs were indentified, among which SNP6 and SNP15 were coincident. In addition, linkage disequilibrium and haplotype analysis identified a haplotype associated with vibrio-resistance. Both mRNA and protein of MmeId2 were up-regulated after V. parahaemolyticus infection also indicated MmeId2 was involved in vibiro immune of M. meretrix.