尖镰孢EST-SSR信息分析及分子标记建立

doi:10.3864/j.issn.0578-1752.2014.20.006

摘要/Abstract

摘要： 【目的】探讨尖镰孢（Fusarium oxysporum）表达序列标签（expressed sequence tag，EST）序列中简单重复序列（simple sequence repeat，SSR）位点的分布特点，开发尖镰孢EST-SSR引物，为镰孢菌遗传多样性分析提供技术支持。【方法】从NCBI公共数据库下载尖镰孢EST 9 304条，利用SSRIT软件查找SSR位点，Primer Premier 5.0软件设计EST-SSR引物，用非变性聚丙烯酰胺凝胶（PAGE）电泳分离SSR-PCR扩增产物，并选用能够扩增出与预期产物大小一致且具有多态性的引物，对23株尖镰孢菌株进行SSR遗传多样性分析。【结果】在尖镰孢EST序列中，共搜索到92个SSR位点，分布于90条EST序列中，出现频率为1.0%。其中二核苷酸重复类型是最主要的SSR类型，占总SSR的比例为59.78%。其次为三核苷酸与五核苷酸重复类型，占总SSR的比例分别为17.39%、11.96%。出现频率最高的基序是（AC/GT）（0.51%）。设计合成的30对引物中有20对（66.7%）能够有效扩增，其中14对引物（46.7%）能够扩增出与预期产物大小一致且具有多态性的产物。尖镰孢的遗传多样性分析结果表明，利用筛选出的14对引物扩增出62条条带，其中多态性条带59条，多态性位点比例为95.2%，平均每对引物可扩增4.2条。供试菌株间的遗传相似系数为0.487—0.933，平均为0.686。供试菌株间存在明显的遗传分化。聚类分析结果表明，当遗传相似系数为0.781时，除14号菌株外，所有菌株均根据寄主来源划分为不同的SSR类群。【结论】基于尖镰孢EST序列开发SSR标记是一种简便有效的方法，研究开发的14对引物可用于尖镰孢的遗传多样性分析。

关键词: 尖镰孢, 表达序列标签, 简单重复序列, 遗传多样性

Abstract: 【Objective】 The objective of this study is to investigate the distribution of Fusarium oxysporum SSRs (simple sequence repeat) derived from ESTs (expressed sequence tag) and to develop EST-SSR markers. These researches could provide technical supports for the genetic diversity analysis of Fusarium. 【Method】 All the 9 304 EST sequences of F. oxysporum were obtained from NCBI. SSRIT (simple sequence repeat identification tool) was used to search SSRs in these sequences and Primer Premier 5.0 was used to design primers. Fourteen pairs of primers were used to analyze the genetic diversity of 23 F. oxysporum strains from different hosts and areas, and the PCR products of these primers were detected by PAGE.【Result】A total of 92 SSRs were identified from 90 F. oxysporum EST sequences, with the frequency of 1.0%. The dinucleotide repeats (59.78%) were most abundant followed by tri-(17.39%) and penta-nucleotide repeats (11.96%). AC/GT (0.51%) occurred more often than any other repeat motif. Among the 30 synthesized primers, 20 (66.7%) amplified distinct bands, and 14 (46.7%) amplified expected polymorphic products. The genetic diversity analysis based on the SSR data showed that a total of 62 bands were amplified by 14 selected SSR primers, 59 (95.2%) of which were polymorphic, with an average of 4.2 bands per primer. The genetic similarity ranged from 0.487 to 0.933, with an average of 0.686. The genetic variation of the strains was obvious. The cluster analysis based on the SSR data showed that except No.14 strain, F. oxysporum strains were distinctly clustered into different groups according to their hosts at 0.781.【Conclusion】Exploitation of SSR markers from F. oxysporum ESTs is convenient and practicable. Fourteen EST-SSR markers identified in this study would be useful for genetic variation study of F. oxysporum.

Key words: Fusarium oxysporum, expressed sequence tag (EST), simple sequence repeat (SSR), genetic diversity

李新凤，张作刚，王建明，高俊明，田宏先. 尖镰孢EST-SSR信息分析及分子标记建立[J]. 中国农业科学, 2014, 47(20): 3982-3991.

LI Xin-feng, ZHANG Zuo-gang, WANG Jian-ming, GAO Jun-ming, TIAN Hong-xian. EST-SSR Information Analysis and Markers Development in Fusarium oxysporum[J]. Scientia Agricultura Sinica, 2014, 47(20): 3982-3991.

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