An efficient DNA Extraction Method from Ruditapes philippinarum larvae and Its Application in Genetic Analysis

Abstract

Ruditapes philippinarum—a marine bivalve mollusc known for its adaptability across a range of temperatures, salinities, and habitats—thrives primarily in tidal flats and shallow marine areas. It is an ideal candidate for high-density cultivation in tidal flats, making it one of the four major cultivated shellfish in China. Genetic exchanges are prevalent among populations in adjacent marine areas, and artificial translocation and cultivation of this species in China have affected the genetic resources of local wild populations, notably altering the genetic structure and ecological balance of these groups. Thus, there is an urgent need for an effective genetic identification method to assess their genetic diversity and germplasm resource status. Efficient extraction of larval DNA is essential for genetic research on shellfish; however, methods for extracting and identifying trace amounts of DNA have not been widely reported. This study addresses these limitations by establishing an efficient method suitable for individual larvae of the marine shellfish, R. philippinarum, and other bivalve mollusks. This method can extract high-quality genomic DNA from larvae as small as a few hundred micrometers, with the aim of providing an accurate and efficient DNA extraction method for species identification and genetic analysis of marine shellfish larvae.Here, D-shaped and umbo larvae were selected for DNA extraction. A rapid and efficient method for extracting genomic DNA from a single larva was developed and successfully applied for genetic identification and haplotype analysis. This method involved the use of physical and chemical means to disrupt the cell structure, and release and collect DNA through thermal denaturation and rapid cooling. A single larva was transferred to a PCR tube containing 10 μL of PCR buffer, followed by heating at 100 ℃ for 5 min and quick transfer to an ice bath. After adding protease K (0.5 μL) for digestion at 55 ℃ for 60 min, the tube was heated again at 100 ℃ for 10 min and then centrifuged at 4 ℃ to collect the DNA. The larval DNA quality was evaluated using a NanoDrop UV-Vis Spectrophotometer, agarose gel electrophoresis, and DNA sequencing.The results demonstrated that PCR products amplified by 16SrRNA and COX1 showed a single clear band of target products, corroborated by their precise sequences. This suggests that the extracted larval DNA can be effectively used for genetic identification and haplotype analysis using these markers. Haplotype analysis revealed 14 COX1 haplotypes with a diversity of 0.937, and 13 haplotypes detected by 16SrRNA with a diversity of 0.705. These findings indicate that the PCR buffer DNA extraction method can yield high-quality genomic DNA from larvae several hundred micrometers in size. This method offers an accurate, efficient, and reliable approach for species identification and genetic analysis of shellfish larvae, with broad potential applications in genetic research and molecular breeding.

Keywords

• genomic dna
• d-shaped larvae
• genetic analysis
• cox1
• 16srrna
• ruditapes philippinarum