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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (6): 50-53.

• 林学 园艺 园林 • 上一篇    下一篇

梨属植物ISSR技术体系的建立与优化

张玉星 马艳芝 赵国芳   

  • 收稿日期:2010-05-13 修回日期:2010-07-10 出版日期:2011-03-20 发布日期:2011-03-20
  • 基金资助:

    梨属种质资源分子遗传连锁图谱的构建

Establishment and Optimization of ISSR Technique in Pyrus L.

  • Received:2010-05-13 Revised:2010-07-10 Online:2011-03-20 Published:2011-03-20

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摘要：

本研究以雪花梨为材料，对ISSR反应体系中的模板用量、引物浓度、dNTPs浓度、TaqDNA聚合酶用量等一些重要参数进行探索和优化，初步建立了稳定的具有广泛适用性的梨属植物ISSR技术的反应体系及扩增程序，即20 μL体系中含1×buffer (内含1.5 mmol? L-1 MgCl2)、模板DNA 40～60 ng、引物浓度为0.2 μmol?L-1、 dNTP 200 μmol?L-1、Taq DNA聚合酶 1U。扩增程序为：94℃ 5 min、退火60s、72℃延伸120 s，然后连续39个循环为94℃ 60s、退火温度(52℃左右)60s 、72℃延伸120s，完成最后一个循环后，在72℃继续延伸10 min，然后在4℃保温，最后通过1.4%的琼脂糖凝胶电泳检测其扩增产物。

关键词: 渍害指标, 渍害指标, 特征, 江淮地区, 冬小麦

Abstract:

In this study, the genome DNA was extracted from Xuehua pear and was used for inter-simple sequence repeat(ISSR)analysis. By evaluating amplification concentrations and conditions,the optimum reaction system of ISSR-PCR for Pyrus L. were established in a total volume of 20μL, containing template DNA 40 ng, 1.0 U Taq-DNA polymerase, 0.2μmol?L-1ISSR primer, 200μmol?L-1, dNTP, Buffer (Tris-HCl (pH8.3) 10m mol?L-1、KCl 50 mmol?L-1、 MgCl21.5 mmol?L-1). Amplified procedure was programmed for 5 minutes at 94℃, at annealing temperature for 60s,extension 120s at 72℃; 39 cycles: 60 seconds at 94℃, at annealing temperature(52℃) for 60s, extension 120s at 72℃and then a final extension 72℃ for 10 min after the last cycle and then keep at 4℃.Amplified products were detected by electrophoresis in 1.4% agarose gel (containing 1μg?mL-1 EB).

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