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【目的】优化苦瓜抗白粉病SRAP标记的PCR体系,为获得苦瓜抗白粉病的SRAP分子标记、加快苦瓜抗白粉病新品种的选育提供参考。【方法】采用正交设计L16(45),在已有SRAP-PCR扩增程序基础上,以苦瓜抗、感白粉病的父母本及其杂交后代基因组DNA为模板,用4对SRAP引物对模板DNA、dNTPs、Mg2+、引物、TaqDNA聚合酶等5个因素浓度进行优化组合和验证。【结果】提取的苦瓜基因组DNA纯度较好,无蛋白质、RNA等物质污染,其纯度和完整性较好。利用引物对ME9-EM11进行PCR扩增,16个不同因素组合处理在苦瓜父本MC18和母本MC1-2的基因组DNA间的扩增结果有明显差异,其中以处理3的SRAP-PCR体系(20.0μL:DNA5.0ng/μL、dNTP0.05mmol/L、Mg2+2.5mmol/L、引物0.6μmol/L、TaqDNA聚合酶0.088U/μL)获得较清晰、较多的扩增条带。用4对引物对验证优化的SRAP-PCR体系,其重复性好,不同苦瓜材料间无明显的多态性。【结论】优化的SRAP-PCR体系适用于苦瓜抗白粉病SRAP分子标记的筛选。
【Objective】 The objective of this study was to optimize the SRAP-PCR system for powdery mildew resistance to powdery mildew and to provide a reference for obtaining the SRAP molecular marker for powdery mildew resistance to powdery mildew and accelerating the selection of new bitter powdery mildew resistance cultivars. 【Method】 Using orthogonal design L16 (45), based on the existing SRAP-PCR amplification program, the genomic DNA of bitter melon and powdery mildew parents and their hybrid progenies were used as template, and four pairs of SRAP primer pairs DNA, dNTPs, Mg2 +, primers, Taq DNA polymerase concentration of five factors for optimal combination and verification. 【Result】 The purity of the extracted bitter gourd genomic DNA was good, without any contamination of protein, RNA and other substances, and its purity and integrity were better. PCR amplification of ME9-EM11 with primers showed significant differences in the amplification results between the genomic DNAs of the bitter melon parent MC18 and the female parent MC1-2 by a combination of 16 different factors. The SRAP-PCR system of treatment 3 20.0μL: DNA5.0ng / μL, dNTP0.05mmol / L, Mg2 + 2.5mmol / L, primer 0.6μmol / L, TaqDNA polymerase 0.088U / μL). The SRAP-PCR system verified by 4 pairs of primers has good repeatability and no obvious polymorphism among different bitter gourd materials. 【Conclusion】 The optimized SRAP-PCR system is suitable for the screening of bitter gourd powdery mildew resistant SRAP molecular markers.