Developing simple DNA extraction and PCR-RFLP forMALE STERILITY 4(MS4) gene inCryptomeria japonicaD. Don: Toward an environmentally friendly protocol

Abstract

This study presents the development of a simple DNA extraction method and a novel PCR-RFLP protocol for genotyping theMALE STERILITY 4(MS4) gene inCryptomeria japonicaas a proof of concept. Traditional CTAB-based DNA extraction methods, while effective, involve hazardous chemicals and require high-speed centrifugation, which are impractical in many field settings. Our approach utilizes a household dish detergent-based buffer, sodium chloride, and polyvinylpyrrolidone K-30 to extract DNA fromC. japonicaneedle leaf tissues—without the need for liquid nitrogen. The simplicity of this method makes it more accessible and environmentally friendly. The extracted DNA was successfully used in PCR-RFLP analysis, targeting a single nucleotide polymorphism in theMS4gene, demonstrating its efficacy for genotyping. The PCR-RFLP markers reliably discriminated between individual genotypes, confirming the practical application of our simple extraction method, even for conifers containing inhibitory substances. This technique is particularly advantageous for use in arboretums and field stations, where the use of hazardous chemicals, specialized equipment, and a draft chamber is limited. Our study contributes to genetic resource management by providing an easy, reliable, and safer method for DNA extraction and genetic analysis.