Identification and validation of SNP markers with relevance to downy mildew resistance in pearl millet [Pennisetum glaucum (L.) R. Br.]

Pearl millet (Pennisetum glaucum (L.) R. Br.) is a nutritionally rich and climate-resilient staple crop cultivated widely across the semi-arid tropics of India. Despite its adaptability, productivity is significantly hampered by downy mildew, a destructive disease caused by the obligate biotrophic pathogen Sclerospora graminicola, leading to yield losses of 20–40%. To elucidate molecular mechanisms underlying resistance, transcriptome sequencing was performed on resistant (P310-17) and susceptible (7042-S) genotypes under both inoculated and control conditions. Assembly using Trinity yielded 26,690 high-quality transcripts. SNP and indel discovery revealed 6,110 SNPs and 149 indels in the resistant genotype, and 6,718 SNPs and 1,626 indels in the susceptible genotype. Transition-to-transversion ratios were 1.83 and 1.88, respectively, with frequent substitutions including G/A and C/T transitions and C/G transversions. Functional annotation of SNP-containing transcripts showed strong homology with Zea mays and Setaria italica, highlighting conserved genomic regions. Gene Ontology analysis indicated enrichment in nucleotide binding, transport, and plastid-related functions. KEGG pathway classification revealed predominant roles in transferase (43%), hydrolase (28%), and oxidoreductase (15%) activities. From these, 20 SNP markers associated with putative disease resistance genes—such as transcription factors and pathogenesis-related proteins—were shortlisted. Validation across twelve diverse genotypes identified seven markers producing clear amplicons. Notably, three markers—ASP2 (beta-glucosidase 31-like), SNP14 (polyamine oxidase-like isoform x2), and SNP16 (SOBIR1-like receptor kinase)—were confirmed via sequencing to contain expected SNPs. These validated EST-derived SNP markers represent a valuable genomic resource for pearl millet, offering promising tools for marker-assisted selection (MAS) and the development of transcript-based genetic maps aimed at improving resistance to downy mildew. The findings contribute significantly to breeding strategies focused on enhancing disease resilience in this vital crop.