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小麦矮腥黑穗病菌(Tilletia controversa Kühn,简称TCK)是小麦上的一种重要检疫性真菌。本研究利用内部简单重复序列(Inter-simple sequence repeat,ISSR)技术研究TCK及其近缘种的DNA多态性,开发了一种可靠而简单的方法用于TCK的分子鉴定。用ISSR引物P4从TCK中扩增出一条1 113 bp的特异性条带,据此设计了一对特异性引物TCKF/TCKR,在12个TCK菌株中均能扩增得到一条882 bp的特异性条带,而其他近缘种包括小麦网腥黑穗病菌(T.caries)和小麦光腥黑穗病菌(T.foetida)及相关黑粉菌的14个菌株均无扩增条带。用该特异性引物检测TCK的下限为25μL反应体系中可检测到1 ng DNA模板。本研究开发的种特异性引物,可将TCK与其形态上相似的近缘种尤其是小麦网腥黑穗病菌准确区分开,本研究基于ISSR标记建立的小麦矮腥黑穗病菌的分子鉴定方法为腥黑粉菌的检疫提供了一种便捷的方法,是对现有分子鉴定方法的一个补充。
Tilletia controversa Kühn (TCK) is an important quarantine fungus on wheat. In this study, we investigated the DNA polymorphism of TCK and its related species using the Inter-simple sequence repeat (ISSR) technique and developed a reliable and simple method for the molecular identification of TCK. A specific 1 113 bp band was amplified from TCK by ISSR primer P4. Based on this, a pair of specific primers TCKF / TCKR was designed and a 882 bp specific fragment was amplified from all 12 TCK strains However, no other bands were found in other 14 species including T.caries and T. foetida and related strains of Yersinia pestis. With this specific primer to detect TCK lower limit of 25μL reaction system can detect 1 ng DNA template. Species-specific primers developed in this study can accurately distinguish TCK from its closely related species, especially Triticum aestivum, in this study. The molecular identification method of Triticum aestivum based on ISSR marker was Quarantine of the Stenotrophomonas maltophilia provides a convenient way to complement existing molecular methods of identification.