Allele-specific PCR genotyping of the HSP70 gene polymorphism discriminating the green and red color variants sea cucumber (Apostichopus japonicus)

Abstract

Color variation is a well-known feature of sea cucumbers (Apostichopus japonicus), which are classified into three groups based on their colors of red, green and black. It is also one of the most important traits related to how they taste, and it thereby affects their market price. Attempts were made to identify single-nucleotide polymorphisms (SNPs) and to analyze differences associated with SNP genotypes between green and red color variants using HSP70 as the target gene. The HSP70 gene, which is found universally in organisms from bacteria to humans, is one of the most evolutionarily conserved genes and the most widely studied biomarker of stress response. DNA fragments of 1074 bp covering a partial sequence of the sea cucumber HSP70 gene, were amplified from both red and green variants, and subsequently analyzed for the presence of SNPs. Twenty-seven polymorphic sites in total, including heterozygous sites, were observed. Of these, six sites were found to be significantly different SNP genotypes between green and red variants. Furthermore, PCR with an internal primer designed to include an allele-specific SNP at the 3' end (site 443) showed differentiation between the two variants, 100% and 4.2% amplification in green and red variants, respectively. The validated SNPs may serve as informative genetic markers that can be used to distinguish variants at the early developmental stage, prior to color differentiation.