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中国农学通报

中国农学通报 ›› 2010, Vol. 26 ›› Issue (21): 147-151.

所属专题： 园艺

• 农学 农业基础科学 • 上一篇    下一篇

基于ISSR和AFLP标记开发甜菜 SSR 引物的研究

牛泽如 杨文柱 庞 磊 田自华 邵金旺 史树德   

  • 收稿日期:2010-04-12 修回日期:2010-06-01 出版日期:2010-11-05 发布日期:2010-11-05
  • 基金资助:

    内蒙古自治区高等学校科学研究项目(NJzy08045)；内蒙古农业大学博士科研启动基金

A Method for Developing Sugar Beet (Beta vulgaris L.) Microsatellite Markers

  • Received:2010-04-12 Revised:2010-06-01 Online:2010-11-05 Published:2010-11-05

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摘要：

本研究以ISSR-PCR和AFLP标记原理为基础，介绍一种新的分离甜菜基因组微卫星引物的方法。首先对甜菜基因组DNA进行酶切并连接已知序列的接头，构建基因组DNA酶切文库，同时用一个或两个ISSR引物，扩增文库中两端含微卫星序列片段并进行克隆测序，根据测序结果设计微卫星序列间的IP1引物和IP1与微卫星序列间IP2 引物；再根据侧翼序列克隆原理，采用巢式PCR进行基因组步移，扩增IP2引物下游序列，根据巢式PCR产物测序结果，设计微卫星序列另一侧的引物IP3 ，IP2和IP3即为SSR标记引物，对获得的SSR引物进行PCR验证，结果表明SSR引物产率为16%，本研究获得的SSR引物具有较高的多态性，对于后续的遗传多样性检测和遗传连锁图构建具有重要意义。

关键词: 烤烟 烘烤 密集烤房 散叶堆积式, 烤烟 烘烤 密集烤房 散叶堆积式

Abstract:

based on principle and characteristics of the molecular marker techniques of amplified AFLP and ISSR, we have developed a valid method for isolating microsatellites from sugar beet (Beta vulgaris L.) genomic DNA. Fragments flanked by one or two microsatellies were amplified using a microsatellie primer, (AG)10 or (AT)10 etc.. A primer IPl of 27bp designed from the sequenced region at one end of the microsatellie, and for nested PCR another primer IP2 of 21bp based on the sequence between IP1 and the microsatellie were prepared. Then primer IP3 of other side of the microsatellite DNA was designed by nested PCR. The success rate was 21%, which is significantly higher than that of traditional methods. The SSR primers which were got from this study showing higher polymorphic, it is great significance for the following testing of the genetic diversity and genetic linkage map construction.