| 正交设计优化玉米SSR—PCR反应体系的研究 |
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| 引用本文： | 王士磊,李玉鹏,高树仁.正交设计优化玉米SSR—PCR反应体系的研究[J].广西农业生物科学,2009(1):119-122. |
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| 作者姓名： | 王士磊 李玉鹏 高树仁 |
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| 作者单位： | 黑龙江八一农垦大学农学院;黑龙江农垦双峰农场; |
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| 基金项目： | 农业部寒地作物生理生态重点开放实验室课题;;黑龙江省2008年研究生创新科研资金项目(YJSCX2008-064HLJ);;黑龙江省教育厅科研项目(11521196)资助 |
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| 摘 要： | 为建立适宜玉米SSR—PCR的反应体系和扩增程序，利用正交设计L16（4^5）表，对反应体系的模板DNA、dNTPs、Primers、Taq DNA聚合酶的浓度进行4因素4水平优化筛选和扩增程序的优化，确立了最优反应体系和扩增程序。即在10μL体系中，模板DNA20ng，1×PCR buffer，dNTPs62．5pmol／μL，Primers0．25pmol／μL，Taq DNA polymerase0．5U。反应程序为：94℃预变性5min；94℃变性45S，60℃退火45S，72℃延伸1min，32个循环；72℃延伸5min，4℃保存。使用300对SSR引物对重组近交系的两亲本扩增，筛选出条带清晰，有差异的引物94对，用于基因连锁图谱构建和QTL定位。
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| 关 键 词： | 玉米 正交设计 SSR—PCR |
Study on Optimization of SSR-PCR Reaction System on Maize by Orthogonal Design |
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| Wang Shilei Li Yupeng Gao Shuren College of Agriculture,Heilongjiang August First L, Reclamation University,Daqing, Heilongjiang Reclamation Shuangfeng Farm,Mishan.Study on Optimization of SSR-PCR Reaction System on Maize by Orthogonal Design[J].Journal of Guangxi Agricultural and Biological Science,2009(1):119-122. |
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| Authors: | Wang Shilei Li Yupeng Gao Shuren College of Agriculture Heilongjiang August First L Reclamation University Daqing Heilongjiang Reclamation Shuangfeng Farm Mishan |
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| Institution: | Wang Shilei 1 Li Yupeng 2 Gao Shuren 1 1 College of Agriculture,Heilongjiang August First L, Reclamation University,Daqing,163319,2 Heilongjiang Reclamation Shuangfeng Farm,Mishan,158308 |
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| Abstract: | In order to establish the optimum SSR-PCR reaction amplification system and procedures of maize, the method oforthogonal design was used with 4 levels of 4 factors. Which are the template DNA, dNTPs, primers, Taq DNA polymerase concentration. Eventually established the optimal reaction amplification systems and procedures. In a total volume of 10 μL SSR-PCR system, it contains template DNA 20 rig, 1×PCR buffer, dNTPs 62.5 pmol/μL, primers 0.25 pmol/μL and Taq DNA polymerase 0.5 U. PCR reaction amplification procedures is： pre-denaturation for 5 min at 94℃, followed by 32 cycles of denaturation for 45 s at 94℃, anneal for 45 s at 60℃, extension for 1 min at 72℃, the amplification was completed after extension for 10 min at 72℃, then stored at 4℃. RIL＇s parents were amplification with 300 SSR primer pairs, and 94 primer pairs were selected for construction gene linkage map and QTL mapping based on their discrepant and clear banding patterns. |
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| Keywords: | Maize （Zea mays L ） Orthogonal design SSR-PCR |
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