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

Abstract

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.

Identification and development of novel salt-responsive candidate gene based SSRs (cg-SSRs) and MIR gene based SSRs (mir-SSRs) in bread wheat (Triticum aestivum)

“…Salt stress-responsive candidate gene-based SSRs (cg-SSR) and MIR gene based SSRs (miR-SSRs) were mined from the identified wheat salt stress responsive genes using the BatchPrimer3 tool 47 , 48 . Out of 171 genes (including 10 MIR genes) screened, 115 genes yielded a total of 264 SSR loci (69%) (Fig.…”

“…The BatchPrimer3 tool was used for mining SSRs. The primers were designed using the BatchPrimer3 tool from the flanking sequences of the identified microsatellite repeats 47 , 48 . We included di-, tri-, tetra-, peta- and hexa- nucleotide repeats; repetition of motifs less than three times were excluded.…”

Phylogeographic Diversity Analysis of Bipolaris sorokiniana (Sacc.) Shoemaker Causing Spot Blotch Disease in Wheat and Barley

“…For extraction of fungal genomic DNA, 40 mg of dried mycelium of each isolate of B. sorokiniana was processed using the CTAB method as described with minor modification [ 29 ]. A UV/VIS spectrophotometer (Simadzu, Kyoto, Japan) was used for DNA quantification, and the DNA was stored at −20 °C for further use.…”

Four In Silico Designed and Validated qPCR Assays to Detect and Discriminate Tilletia indica and T. walkeri, Individually or as a Complex

“…Different approaches have recently been taken to attempt a better differentiation method for T. indica and its close relatives. For instance, Sharma et al [ 59 ] screened for simple sequence repeats, or microsatellites, for diagnostics and genetic diversity studies of smut and bunt fungi and they included cross-transferable markers for T. indica . Promisingly, the project appears to be the first one to develop microsatellites for identification and validation of T. indica .…”