苦瓜抗白粉病SRAP标记筛选的PCR体系优化与验证  

Optimizing and verification of PCR system for SRAP marker screening related to powdery disease resistance in bitter melon (Momordica charantia)

作　　者：米军红[1,2] 尚小红[2,3] 周生茂[2,3] 车江旅[4] 黄如葵[2,3] 梁任繁[2,3] 文俊丽[2,3] 郭元元[2,3] 梁和[1] 

机构地区：[1]广西大学农学院,南宁530005 [2]广西作物遗传改良生物技术重点开放实验室,南宁530007 [3]广西农业科学院蔬菜研究所,南宁530007 [4]广西农业科学院,南宁530007

出　　处：《南方农业学报》2013年第6期898-902,共5页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31240060);广西科学研究与技术开发计划项目(桂科攻10100005-6);广西自然科学基金项目(2012GXNSFAA053061);广西农业科学院基本科研业务专项项目(桂农科2012YT05);广西农业科学院公益性维持费项目(桂农科2012GW04)

摘　　要：【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 present study was conducted to optimize the PCR system of SRAP molecular marker related to powdery disease resistance in bitter melon for providing reference on breeding new bitter melon cultivars resistant to powdery disease. Method On the basis of the known SRAP-PCR amplification procedure, the concentration combinations of DNA template, dNTPs, Mg2＋ , primer, Taq DNA polymerase were optimized and verified by orthogonal design L16 （45 ） with the genome DNA of powdery disease -resistant and -susceptible parents used as templates and four pair primers. ResultThe extrated bitter melon genomic DNA showed better purity and integrity, and had no pollution for protein and RNA, etc. The primer pair ME9/EM11 was used for PCR amplification and got obvious difference in 16 PCR systems of genomic DNA in parents MC18 and MC1-2. Among 16 PCR systems, the 3th system was the optimum system of SRAP-PCR for getting clear and more amplified bands, which contained DNA 5 ng/μL, dNTPs 0.05 mmol/L, Mg2＋ 2.5 mmol/L, primer 0.6 μmol/L, Taq DNA polymerase 0.088 U/μL, in total 20 μL. Four primer pairs were used to verify the optimized SRAP-PCR system for different bitter melon materials, It showed good repetition and no polymorphism. Conclusion The optimized SRAP-PCR system was suitable for screening SRAP marker related to powdery disease resistance in bitter melon.

关 键 词：苦瓜 SRAP-PCR 体系优化 筛选 验证 

分 类 号：S642.5[农业科学—蔬菜学]