基于ISSR标记开发的一种用于小麦矮腥黑穗病菌的分子鉴定方法

 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25 μL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。

Molecular Identification of Tilletia controversa KÜhn by Inter-simple Sequence Repeat Marker

Tilletia controversa Kühn (TCK), causing wheat dwarf bunt, is an internationally quarantine fungal pathogen of economic importance. The aim of the study was to develop a reliable and simple method suitable for identification of TCK. Inter-simple sequence repeat (ISSR) analysis was performed to reveal the DNA polymorphism of TCK and its related species. A pair of species-specific primers of T. controversa, TCKF/TCKR, was designed according to the sequence of a unique fragment of 1 113 bp obtained using ISSR primer P4. An amplicon of 882 bp was detected by PCR with the primer set in 12 isolates of TCK but absent in 14 isolates of other related pathogenic fungi including T. caries and T. foetida. The sensitivity limit of the primers TCKF/TCKR was 1 ng of template DNA in a 25 μL PCR reaction mixture. The designed species-specific primers could accurately differentiate TCK from its morphologically similar and phylogenetically closely related species, especially T. caries. The molecular identification of TCK using ISSR marker established in the study provides a simple detection system for the quarantine of TCK and is a supplement to the current molecular identification methods.