中国农业科学 ›› 2014, Vol. 47 ›› Issue (1): 111-121.doi: 10.3864/j.issn.0578-1752.2014.01.012

• 园艺 • 上一篇    下一篇

中国50个甘蓝代表品种EST-SSR指纹图谱的构建

 王庆彪, 张扬勇, 庄木, 杨丽梅, 刘玉梅, 吕红豪, 方智远   

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081
  • 收稿日期:2013-02-06 出版日期:2014-01-01 发布日期:2013-09-26
  • 通讯作者: 庄木,Tel:010-82108756;E-mail:zhuangmu@caas.cn
  • 作者简介:王庆彪,E-mail:qbwang2008@163.com
  • 基金资助:

    公益性行业(农业)科研专项经费项目(200903008)、国家自然科学基金(31272180)、中央级公益性科研院所基本科研业务费专项(ICSCAAS2010-11)、国家“863”计划(2012AA100101)、农业部园艺作物遗传改良重点开放实验室项目

EST-SSR Fingerprinting of Fifty Cabbage Representative Varieties from China

中国农业科学院蔬菜花卉研究所,北京100081   

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2013-02-06 Online:2014-01-01 Published:2013-09-26

摘要: 【目的】研究甘蓝DNA指纹鉴定方法,并构建50份中国甘蓝代表品种DNA指纹数据库,为甘蓝品种特异性、真实性、纯度鉴定提供参考依据。【方法】首先,利用6%变性聚丙烯酰胺凝胶电泳技术和来源不同的甘蓝材料对已开发的甘蓝EST-SSR引物进行筛选,获得多态性引物;然后将可用于甘蓝DNA指纹鉴定的核心引物正义链5′末端分别标记TAMRA、HEX、 ROX、6-FAM四种荧光,利用DNA分析仪检测不同等位变异的扩增片段大小,并确定每个等位变异的标准品种。利用核心引物扩增结合标准品种的片段大小,构建中国50个甘蓝代表品种的SSR指纹数据库。通过人工构建模拟群体对‘中甘21’品种真实性进行鉴定,进而对该指纹鉴定技术进行验证。【结果】利用6份来自中国不同生态条件、植物学性状差异较大的甘蓝品种对978对EST-SSR引物进行初步筛选,获得带型清晰、具有多态性的引物128对。进一步根据引物扩增带型清晰、多态性信息指数较高、不同等位变异的带型易于区分、在染色体上分布均匀原则,筛选出20对核心引物。该套引物可以检测甘蓝染色体20个位点的58个等位变异,平均每条染色体上被检测位点2.22个,平均每个位点包含2.9个等位变异,其中引物BoE607、BoE723等位变异数最多为5个,PIC值处于0.34—0.76之间。通过DNA分析仪检测显示等位变异扩增片段长度范围143—296 bp。利用20对核心引物构建50份中国甘蓝代表品种DNA指纹数据库,并阐述了甘蓝SSR-DNA指纹鉴定的应用和技术流程。本研究还对来自甘蓝主产区的31份‘中甘21’品种进行真实性鉴定,结果显示指纹鉴定与田间鉴结果完全一致。【结论】筛选获得20对核心引物,用于构建中国50个甘蓝代表品种DNA指纹数据库,通过构建人工模拟群体对‘中甘21’品种的真实性进行鉴定,准确率达100%。

Abstract: 【Objective】In this study, the method of cabbage DNA fingerprint was drawn, and fifty cabbage representative varieties from China were fingerprinted with EST-SSR primers to provide reference for variety distinctness, authenticity, and purity identification. 【Method】First, EST-SSR primers were screened by using the technologies of 6% denaturing polyacrylamide gel electrophoresis and six cabbage varieties that come from different ecogeography. The length of amplified fragments was detected on DNA Analyzer platform using four fluorescent-labels (TAMRA, HEX, ROX and 6-FAM) in 5′end of forward primer, and then defined the reference variety for every alleles. Total twenty core primers were used to establish fifty cabbage varieties SSR fingerprinting, and for ‘Zhonggan 21’ variety identification. 【Result】 Six cabbage varieties of different resources were used to screen 978 EST-SSR primers, out of 128 polymorphic primers were obtained according to the PCR bands stability, high polymorphism information content (PIC), easy discrimination of different alleles and even distribution of molecular markers on each chromosome, and 20 pairs of primers were selected to detect a total of 58 alleles at 20 loci, with 2.22 loci per chromosome and 2.9 alleles per locus on average. The PIC values varied among the primers ranging from 0.34 to 0.76. The length of amplified fragment varied in the range of 143-296 bp. The maximum number of alleles for each primer pairs of BoE607 and BoE723 was five. Fingerprinting database of 50 cabbage representative varieties from China was established with 20 pairs of core primers. The authenticity of ‘Zhonggan 21’ was identified by artificial simulated population and these results were identical with that made from field investigation.【Conclusion】Twenty pairs of core primers were selected and used to establish DNA fingerprint database of 50 cabbage representative varieties from China, and the authenticity of ‘Zhonggan 21’ was identified by artificial simulated population and the accuracy was 100%.

Key words: cabbage , SSR marker , fingerprinting , varieties identification