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中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (13): 20-24.doi: 10.11924/j.issn.1000-6850.2013-2814

所属专题： 园艺

• 林学 园艺 园林 • 上一篇    下一篇

杨树RAPD标记技术体系的建立与优化

张玉平   

  • 收稿日期:2013-10-29 修回日期:2014-01-02 出版日期:2014-05-05 发布日期:2014-05-05

Establishment and Optimization of RAPD Technique in Poplar

  • Received:2013-10-29 Revised:2014-01-02 Online:2014-05-05 Published:2014-05-05

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摘要： 本研究主要对杨树RAPD体系进行优化，以银白杨为试材，利用L16(45)正交试验设计和2个单因素试验，研究各主要参数的适宜浓度。结果表明，杨树RAPD优化后的反应体系：20μL反应体系中，含Mg2+ 1.5 mmol/L、Taq酶2.0 U、引物0.2μmol/L、dNTPs 0.2 mmol/L、模板DNA 50 ng。扩增程序为：94℃预变性3 min、94℃变性40 s、38℃退火60 s、72℃延伸90 s，共38个循环；最后在72℃延伸7 min后4℃保温。通过1.5%的琼脂糖凝胶电泳检测其扩增产物，证明该体系稳定可靠，可以用于杨树的遗传学分析。

关键词: 诊断方法, 诊断方法

Abstract: The aim of this study was to optimize the RAPD reaction system in poplar. The L16(45) orthogonal tests design and two single factors tests were used to study the appropriate concentrations of the main factors with Populus alba. The results showed that an optimized RAPD reaction system was established: the total 20μL contained Mg2+ 1.5 mmol/L, Taq polymerase 2.0 U, primer 0.2μmol/L, dNTPs 0.2 mmol/L, DNA template 50 ng. Amplified procedure was: 94℃for 3 min, 38 cycles at 94℃for 40 s, 38℃for 60 s, 72℃for 90 s, followed by 72℃for 7 min and then ramped to 4℃to hold the reaction. Amplified products were detected by electrophoresis in 1.5% agarose gel. It was evident that the reaction system established in this study was stable and reliable for RAPD analysis, and could be used on genetic analysis of poplar.