A new insight into the SNP genotyping using high-resolution melting method after the correlation analysis of the SNPs with WSSV-resistant traits

Procambarus clarkii is an important freshwater cultured crayfish in China. With the gradual development of its aquaculture industry, research on white spot disease, which is harmful to healthy culture of P. clarkii, increases gradually. The prophenoloxidase (proPO) system is an important part of crayfish's innate immunity and plays a role in virus resistance. In this study, based on the early discovery of three SNP sites in the intron of proPO gene, the linkage disequilibrium and haplotype were analyzed for the SNPs, and it was found that there was a strong linkage disequilibrium relationship among them. Through the analysis on association between the haplotypes and genotype of each SNP site with the WSSV-resistant traits, the detection of the SNP_7081 genotype was considered as the most convenient and efficient way for WSSV-resistant group selection. Furtherly, the high-resolution melting curve (HRM), which is a rapid and economic genotyping method, was chosen to establish for SNP_7081 site genotyping. The 68 bp target fragment with 27.94% GC content was amplified and melting curve analysis were performed. However, the appearance of false negatives which led to unable automatically grouped although the melting curves of genotypes CC, C>T and T>C were obviously different, and could be treated as standard to manually genotype the samples with an accuracy rate of 97.61%. The low GC content which correlated with the Tm value, was confirmed as the reason for the false negatives by the assay about the recombinant plasmid PMD18-T-SNP_7081 constructed with 45.24% GC content. Eventually, the adaptor primers were used to increase the GC content of the target fragment, and a modified HRM method for genotyping SNP_7081 site that could group automatically was established, which could provide a new insight for the HRM method to genotype SNPs.