Multiple sequence alignment and phylogenetic analysis of wheat pathogens using conserved genes for identification and development of diagnostic markers

Molecular markers are fast, reliable and robust for early detection of pathogens. They need unique stable regions specific to pathogens for developing good diagnostics. The multiple sequence alignment and phylogenetic studies of conserved loci such as Internal Transcribed Spacer (ITS), Translation elongation factor 1α (TEF-1α), β-tubulin gene and glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of wheat pathogens identified intergeneric (5–30%) and intrageneric (2–24%) variability with multiple sequence polymorphism (5–250 bp) for selection of pathogen-specific regions. ITS region precisely showed specificity for Tilletia indica and storage pathogens (Aspergillus spp., Penicillium spp. and Fusarium spp.), while TEF-1α gene distinctly differentiated 5 Fusarium species with high potential (10%) and found highly suitable for developing qPCR or LAMP assays for detection of Fusaruim head blight disease. β-tubulin gene showed, enormous variabilities among species of Puccinia (15%) and Aspergillus (23%) for developing their diagnostics. GAPDH gene with the highest intergenic sequence variability (> 30%) found suitable for developing markers at generic level. Further, it has differentiated different species of Aspergilli and Bipolaris. The variability among different pathogens enabled the selection and development of appropriate diagnostic marker such as, PCR, qPCR, RPA, LAMP assay, macro- or micro-array. As a proof of concept, a PCR-based diagnostics (MN754026) for Tilletia indica was developed, which amplified a band of 148 bp only in Tilletia indica and detected as little as 10 pg of DNA. Developed maker has tremendous potential for timely management of this quarantined pathogen.