Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high-throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed-borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U.maydis were identified. Of the remaining SSRS, 44.5% accounted for di-nucleotide repeats followed by 29.8% and 2.7% tri- and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T.horrida, of which 20.2% were di-nucleotide, 50.4% tri- and 20.5% tetra-nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross-amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed-borne pathogens.