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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (16): 202-207.doi: 10.11924/j.issn.1000-6850.2012-0154

所属专题： 生物技术

• 林学 园艺 园林 • 上一篇    下一篇

太行菊DNA提取和ISSR标记的筛选与优化

何敏杰 程月琴 王红卫 郭长宁 叶永忠 孟丽   

  • 收稿日期:2012-01-16 修回日期:2012-03-13 出版日期:2012-06-05 发布日期:2012-06-05
  • 基金资助:

    太行花种群历史动态及其亚种分化研究

DNA Extract, Screening and Optimization of ISSR Markers for Opisthopappus taihangensis

  • Received:2012-01-16 Revised:2012-03-13 Online:2012-06-05 Published:2012-06-05

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摘要：

为了探索太行菊DNA的适宜提取方法，获得适用于太行菊的ISSR标记，以太行菊为试验材料，采用常规CTAB法、改良CTAB法和SDS法提取太行菊总DNA，利用提取的太行菊DNA对ISSR引物进行了筛选和优化。结果表明，改良CTAB法提取的DNA产率高且稳定，无明显降解，杂质少。以改良CTAB法提取的DNA为模板，应用ISSR引物对总DNA进行扩增，进而从测试的50条ISSR引物中选出了扩增效率高，条带丰富的15条引物。通过设置温度梯度对每条引物的反应条件进行了优化。最后，使用引物UBC848对部分群体样品进行检测，得到了效果稳定、重复性好的扩增结果。上述结果表明，通过温度梯度筛选出的ISSR引物适用于太行菊的群体遗传学研究。

关键词: 分离鉴定, 分离鉴定

Abstract:

To find a DNA extract method suitable to Opisthopappus taihangensis and obtain a few ISSR markers for O. taihangensis, the total DNA was isolated from the O. taihangensis by the common CTAB method, improved CTAB method and SDS method, then O. taihangensis DNA as template, ISSR primers were screened and the PCR conditions were optimized. The results showed the improved CTAB method got a high production rate of the extracts, and that was pure with little degradation. These productions were good templates for ISSR molecular markers. Then, out of 50 ISSR primers, 15 ISSR primers were screened which could generate clear, stable bands. By setting gradient temperatures for each primer, reaction conditions were optimized. Eventually, population samples were detected by primer UBC848, clear and stable bands were gotten. These results showed the selected ISSR primers were suitable for the study on population genetics of O. taihangensis.