| 割手密AFLP分子标记技术体系建立与优化 |
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| 引用本文： | 刘许辉,段维兴,刘新龙,杨海霞,高轶静,罗霆,宋焕忠,张革民.割手密AFLP分子标记技术体系建立与优化[J].广西农业科学,2013(4):552-557. |
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| 作者姓名： | 刘许辉 段维兴 刘新龙 杨海霞 高轶静 罗霆 宋焕忠 张革民 |
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| 作者单位： | [1]广西大学农学院,南宁530005 [2]广西农业科学院甘蔗研究所,南宁530007 [3]云南省农业科学院甘蔗研究所,云南开远661600 |
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| 基金项目： | 国家自然科学基金项目（30860150）;现代农业产业技术体系资助项目（CARS-20-1-3）;广西自然科学基金项目（2011GXNSFF018002,2012GXNSFBA053036）;广西农科院基本科研业务专项项目（桂农科2011YT01,桂农科201IYT04,桂农科2012YZ16,桂农科2012YM14） |
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| 摘 要： | 【目的】建立和优化割手密AFLP分子标记技术体系，为割手密遗传多样性分析、遗传图谱构建提供技术支持。【方法】以广西割手密GXS87—16、GXS85．30、GXS79—9、GXS96、GXS112、GXS212为材料，利用改良SDS法提取DNA，并用EcoR I和Mse I酶切，连接接头后，采用正交试验设计对影响预扩增反应和选择性扩增反应的主要成分如Mg^2＋、模板DNA、引物、dNTP、Taq聚合酶浓度进行优化。【结果】样品DNA用限制性内切酶＆0RI和MseI各3u于PCR仪过夜可完全酶切。经正交设计优化，较佳的预扩增体系包含0．4μL dNTPs（20mmol／mL），1．6μLMg^2＋（25mmol／mL），2．0μL EcoRI—P（5pmol／mL），2．0IxLMseI-P（5pmol／mL），1UTaq酶（1U／μL），DNA模板稀释10倍；选择性扩增体系包含0．4μL dNTPS（20mmol／mL），0．8μLMg^2＋（25mmol／mL），1．0μL EcoR I—AAG（6pmol／mL），1．0μL Mse I-CAG（6pmol／mL），3U Taq酶（1U／μL），DNA模板稀释20倍。以对优化反应体系扩增获得的PcR产物用5％变性聚丙烯酰胺凝胶电泳和银染后，可获得清晰的多态性指纹图谱。【结论】建立的割手密AFLP分子标记技术体系具有扩增条带清晰、多态性丰富的特点，可为构建割手密高密度遗传图谱提供技术支持。
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| 关 键 词： | 割手密 AFLP 改良SDS法 正交设计 优化 |
Establishment and optimization of AFLP molecular marker technology system for S. spontanuem L. |
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| LIU Xu-hui,DUAN Wei-xin,LIU Xin-long,YANG Hai-xia,GAO Yi-jing,LUO Ting,SONG Huan-zhong,ZHANG Ge-min.Establishment and optimization of AFLP molecular marker technology system for S. spontanuem L.[J].Guangxi Agricultural Sciences,2013(4):552-557. |
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| Authors: | LIU Xu-hui DUAN Wei-xin LIU Xin-long YANG Hai-xia GAO Yi-jing LUO Ting SONG Huan-zhong ZHANG Ge-min |
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| Institution: | . (1Agricuhural College, Guangxi University, Nanning 530005, China; 2 Sugarcane Research Insmute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China; 3Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences, Kaiyuan, Yunnan 661600, China) |
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| Abstract: | Objective]The AFLP molecular marker technology system for Saccharum spontaneum L. was established and optimized to provide support for the analysis of genetic diversity and genetic map construction. Method]The DNA of Saccharum spontaneum L. materials viz., GXS85-30, GXS87-16, GXS79-9 , GXS96, GXS112 and GXS212 collected from Guangxi were extracted by modified SDS method, and completely digested by enzyme EeoR I and Mse I .The digested fragments were legated by the EcoR I and Mse I adaptors. Then, on the basis of the orthogonal experimental design, the influencing components on preamplification and selective amplification reactions, namely Mg^2＋, DNA template, primer, dNTP, and Taq DNA polymerase, were optimized. Result]The DNA were completely digested by 3.0 U enzyme EcoR 1 and Mse I . By orthogonal design optimization, favorable preamplification system included 0.4 μL of dNTPs （20 mmol/ mL）, 1.6 μL of Mg^2＋（25 mmnl/mL）, 2.0 μL of EcoR I -P （5 pmol/mL）, 2.0μL of Mse -P（5 pmol/mL）, 1 U Taq polymerase （1 U/μL）, and 10 times diluted DNA template. Selective amplification system included 0.4 μL of dNTPs （20 mmol/ mL） ,0.8 μL of Mg^2＋（25 mmo//mL）, 1.0 μL ofEcoR I -AAG （6 pmol/mL）, 1.0 μL of Mse 1 -CAG（6 pmo/mL）, 3 U of Taq polymerase （1 U/μL） and 20 times diluted DNA template. The clear polymorphic fingerprint maps were obtained after PCR product from optimized reaction system was undergone 5% denatured polyacrylamide gel electrophoresis and silver stain. Conclusion ]The high efficient and stable AFLP technical system could provide strong technical support for constructing molecular genetic linkage map of Saeeharum spontaneum L. |
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| Keywords: | Saccharum spontaneum L AFLP modified SDS method orthogonal design optimization |
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