An ISSR‐based Approach for the Molecular Detection and Diagnosis of Dwarf Bunt of Wheat, Caused by Tilletia controversa Kühn

Abstract Dwarf bunt of wheat, caused by Tilletia controversaKu¨hn, is an important international quarantine diseasein many countries. The objective of this investigationwas to develop a diagnostic molecular marker gener-ated from intersimple sequence repeat (ISSR) for rapididentiﬁcation of T. controversa. A total of 60 primerswere tested by ISSR to detect DNA polymorphismsbetween T. controversa and related species. The primerISSR818 generated a polymorphic pattern displaying a952- bp DNA fragment speciﬁc for T. controversa. Themarker was converted into a sequence characterizedampliﬁed region (SCAR), and speciﬁc primers(TCKSF2⁄TCKSR2) were designed for use in a PCRdetection assay. Its detection limit was 1 ng of DNA,which could be yielded by 1.1 lg of teliospores in a25- ll PCR. Conclusively, a method to distinguishT. controversa from similar pathogenic fungi has beensuccessfully developed based on the use of a SCARmarker. Introduction Dwarf bunt of wheat, caused by Tilletia controversaKu¨hn, is an important international quarantine dis-ease. The teliospore morphology and genetic structureof T. controversa, Tilletia caries and Tilletia foetida areso similar that they are not easy to distinguish.ITS regions of the ribosomal subunit have been usedas a target region for species-speciﬁc primers (OGor-man et al. 1994; Salaza et al. 2000; Errampalli et al.2001). However, in the case of common bunt, it wasnot possible to design species-speciﬁc primers in thesequenced ITS region, as there is no variation betweenthe ITS2 region of T. caries, T. foetida, T. controversa,and Tilletia bromi (Josefsen and Christiansen 2002). A similar conclusion was drawn by Mulholland andMcEwan (2000), who designed Tilletia-speciﬁc primersin the large subunit of the ribosomal RNA genes to beused in a PCR-based seed health assay. The identiﬁca-tion of smut fungi has been reported for Tilletia indica,T. controversa ,T. caries T. foetida and other relatedfungi (Frederick et al. 1998; Eibel et al. 2005; Lianget al. 2006). However, none of these investigationsreadily and clearly distinguished T. controversa fromsimilar pathogens.Therefore, screening random regions of the fungalgenome for the species may reveal species speciﬁcity(Liu et al. 2009). DNA molecular marker technologyprovides a powerful tool for identiﬁcation with theadvantages of time-saving, less labour-consumptionand more eﬃciency (Dongre and Parkhi 2005; Liuet al. 2007). Intersimple sequence repeat (ISSR) is anovel molecular marker technique and is a PCR-based method that is similar to random ampliﬁcationof polymorphic DNA (RAPD) technique except thatthe ISSR primer sequences are longer and aredesigned from microsatellite regions (Agarwal et al.2008). Therefore, the annealing temperatures used arehigher than those used for RAPD markers, whichcould lead to higher consistency of the PCR prod-ucts. Hantula et al. (1996) found that ISSRs are com-mon in Ustilago genomes by conducting a computersearch on all available Ustilago sequences. The use ofspeciﬁc sequence characterized ampliﬁed region(SCAR) markers is also a powerful tool to unambig-uously identify a particular strain (Weber et al. 2002). Thus, for further speciﬁc and reliable discriminationof T. controversa, development of SCAR markers isvery essential. In this study, ISSR technique has beensuccessfully applied to develop a SCAR marker fordiagnosis of dwarf bunt of wheat and detection ofT. controversa.