欢迎访问《中国农学通报》，
中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (31): 214-218.doi: 10.11924/j.issn.1000-6850.2014-1577

所属专题： 园艺

• 林学 园艺 园林 • 上一篇    下一篇

利用SSR快速鉴定甜菜品种纯度和真实性的研究

吴则东,王茂芊,王华忠,马龙彪   

  1. 黑龙江大学农作物研究院,黑龙江大学/中国农业科学院甜菜研究所,中国农业科学院甜菜研究所/黑龙江大学,黑龙江省普通高校甜菜遗传育种重点实验室/黑龙江大学
  • 收稿日期:2014-06-04 修回日期:2014-06-04 接受日期:2014-09-05 出版日期:2014-11-20 发布日期:2014-11-20
  • 通讯作者: 吴则东
  • 基金资助:
    黑龙江省普通高校甜菜遗传育种重点实验室；甜菜现代产业技术体系建设项目“甜菜丰产抗病种质创新及新品种选育”(CARS-210104- 01)。

Research of the Rapid Identification System for Purity and Authenticity of Sugarbeet Variety by Using SSR Markers

  • Received:2014-06-04 Revised:2014-06-04 Accepted:2014-09-05 Online:2014-11-20 Published:2014-11-20

摘要： 为了建立一套快速鉴定甜菜品种纯度和真实性的实验体系，对DNA的提取、检测、PCR反应体系、程序、电泳以及显影方法等多个方面进行了优化, 最终确立了一套简单、快速、节约、污染小的甜菜品种纯度和真实性的鉴定体系。该方法使用96孔PCR板，利用碱裂解法提取干种子（或者叶片干粉）DNA；使用无毒的Gelred替代致癌性的EB，利用λ DNA检测提取样品DNA的浓度； PCR反应的体系为5μL，使用多重PCR替代单一PCR；PCR反应程序为94℃变性15s，退火15s，72℃延伸30秒，30个循环,最后72℃延伸5min； PCR产物的分离使用8%非变性聚丙烯酰胺凝胶电泳，最后使用快速银染法对电泳后的聚丙烯酰胺凝胶进行显影。利用优化后的程序，一个成熟的实验室操作人员只需一天就可以完成192份样品的检测。

关键词: 普通野生稻, 普通野生稻, 遗传多样性, 分子标记

Abstract: The study aims to establish an experimental system of rapid identification of beet variety purity and authenticity, to optimized extraction, detection of DNA, PCR reaction system, program, electrophoresis and the method of developing multiple aspects, and finally to set up a set of simple, rapid, economical and less polluted identification system for sugarbeet variety purity and authenticity. With 96- hole PCR plates, alkali decomposition method was used to extract DNA from dried seed (or leaf powder). Using nontoxic Gelred substitute carcinogenic EB, DNA concentration was extracted with λDNA. PCR reaction system was 5 μL, using multiple PCR to replace a single PCR; PCR reaction procedure was 94℃, degeneration 15 s, annealing 15 s, extension 72℃ for 30 s, 30 cycles and extended at 72℃ for 5 min finally. Separation of the PCR products was conducted using 8% of Native-PAGE, and rapid argentation was used to develop polyacrylamide gel after electrophoresis. Using the optimized program, a skillful laboratory operator can complete 192 samples of detection within one day.