MutExomeSeq Accelerates the Cloning of PmNCA6 Conferring Powdery Mildew Resistance from Triticum boeoticum

Powdery mildew poses a major threat to global wheat production, highlighting the urgent need to identify resistance genes. In this study, we report the cloning of PmNCA6 , a powdery mildew resistance gene originating from Triticum boeoticum . Using bulked segregant exome capture sequencing (BSE-Seq) and genetic mapping, we mapped PmNCA6 to a 17-Mb recombination-suppressed interval (680.1–697.1 Mb) on chromosome 7AL. By applying a mutant exome sequencing (MutExomeSeq) approach, we analyzed six ethyl methanesulfonate (EMS)-induced susceptible mutants and identified non-synonymous mutations in a nucleotide-binding leucine-rich repeat ( NLR ) gene, NLR1 . Functional validation through barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) and transgenic complementation confirmed that two alternatively spliced NLR1 transcripts ( NLR1_V1 and NLR1_V2 ) confer resistance to powdery mildew. Phylogenetic analysis revealed that PmNCA6 is orthologous to the stem rust resistance gene Sr22a . Domain-swapping experiments between PmNCA6 and Sr22a demonstrated that the leucine-rich repeat (LRR) domain of PmNCA6 is critical for powdery mildew specificity. Field trials of near-isogenic and recombinant inbred lines (RILs) indicated that PmNCA6 -mediated resistance does not compromise yield performance. Screening of 553 Chinese wheat cultivars confirmed the absence of PmNCA6 , emphasizing its potential for diversifying resistance sources in breeding programs. This study establishes MutExomeSeq as a robust tool for cloning genes in recombination-suppressed intervals and highlights the potential of engineering synthetic NLRs with tailored LRR domains to combat evolving pathogens.