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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (36): 241-245.

所属专题： 畜牧兽医； 园艺

• 生物技术科学 • 上一篇    下一篇

羊耳蒜遗传多样性研究中AFLP反应体系的建立与初步应用

管俊杰 丁锐 李梦媛 段茹 陈旭辉 曲波 张立军   

  • 收稿日期:2012-03-29 修回日期:2012-05-18 出版日期:2012-12-25 发布日期:2012-12-25
  • 基金资助:

    纤毛虫表膜内表面与射出体、皮层颗粒、色素颗粒的形态学研究;高等学校博士学科点专项科研基金;辽宁省教育厅科学研究一般项目;沈阳农业大学青年教师科研基金

Establishment and Primary Application of AFLP Analysis System for Genetic Diversity Analysis of Liparis japonica

  • Received:2012-03-29 Revised:2012-05-18 Online:2012-12-25 Published:2012-12-25

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摘要：

为研究羊耳蒜种质资源遗传多样性，建立并初步应用羊耳蒜AFLP反应体系。以羊耳蒜幼嫩叶片为材料，采用常规SDS法提取基因组DNA，通过优化AFLP反应体系中的几个关键因素，建立适合羊耳蒜的AFLP银染反应体系，并利用筛选出的引物组合对羊耳蒜的遗传多样性进行初步研究。经EcoRⅠ和MseⅠ酶组合37℃酶切3 h后可将500 ng基因组DNA完全切开；酶切产物和接头经16℃连接过夜后，用带有1个选择性碱基的预扩引物和带有3个选择性碱基的选扩引物分别进行扩增，扩增产物经变性聚丙烯酰胺凝胶电泳分离，银染，能够得到清晰的扩增图谱。3对引物组合对8个羊耳蒜种群共185个体的扩增共得到221条条带，其中多态性条带195条，多态性条带百分率为88.24%。本研究结果表明建立的AFLP反应体系可用于羊耳蒜种质资源遗传多样性研究。

关键词: 防控方法, 防控方法

Abstract:

To establish and apply of AFLP analysis system for genetic diversity analysis of Liparis japonica. The genomic DNA in the fresh leaves of Liparis japonica was extracted using the traditional SDS method, and an AFLP silver-stained analysis system suitable for L. japonica was established by optimizing several main factors and then applied for genetic diversity analysis of L. japonica populations in northeast China. 500 ng genomic DNA was digested for 3 h with EcoRⅠand MseⅠrestriction enzymes, ligated to adaptors for 12 h at 16 ℃, pre-amplified using EcoRⅠ+1 and MseⅠ+1 primers and then amplified using EcoRⅠ+3 and MseⅠ+3 primers. The amplified products were resolved in denaturing ployacrylamide gels and stained with silver, and then the clear ploymorphic fingerprints were obtained. AFLP analysis of 185 individuals of L. japonica using three pairs of primers generated a total of 221 bands, of which 195 were polymorphic and the PPB value was 88.24%. The AFLP analysis system we established could be used in the research of genetic diversity of L. japonica species.