Developing simple DNA extraction and PCR-RFLP for MALE STERILITY 4 ( MS4 ) gene in Cryptomeria japonica D. Don: Toward an environmentally friendly protocol

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Abstract
This study presents the development of a simple DNA extraction method and a novel PCR-RFLP protocol for genotyping the MALE STERILITY 4 ( MS4 ) gene in Cryptomeria japonica as 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 from C. japonica needle 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 the MS4 gene, 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.
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