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Development of a diagnostic assay for differentiation between genetic groups in clades I, II, III and IV of Puccinia graminis f. sp. tritici.

Wheat stem rust has re-emerged as a serious disease caused by new variants of Puccinia graminis f. sp. tritici. Variants with significant virulences and broad geographical distribution (Africa, Central Asia, and Europe) include the Ug99 race group, race TTRTF, and TKTTF race group. Genetic analysis has placed isolates representing these critical new virulent races into 12 genetic groups that make up clades I to IV. Development of molecular diagnostic assay for these 12 genetic groups will be an important component of global surveillance efforts. A single-nucleotide polymorphism database was mined for candidate markers that would differentiate between these 12 genetic groups. Thirty-five candidate markers were screened and a core set of 17 markers were tested against a set of 94 isolates representing a broad range of genotypes and race phenotypes. This core set of markers were 100% accurate in identifying the 12 genetic groups for 52 isolates in clades I to IV and no false positives were observed with non-target isolates. The assay has built-in redundancy so that minor genetic changes, or errors in genotyping calling will not affect the accuracy of the results. This assay is also effective in identifying the genetic groups in clade V from Germany and Georgia, the three main subgroups in North American clade VI, and the clade VII consisting of race TTTTF found in North and South America. This assay provides a rapid diagnostic tool for both living and non-living samples for detecting these critical new race or race groups of P. graminis f. sp. tritici.