Identification of candidate gene for the wheat powdery mildew resistance gene Pm40 by bulked segregant analysis coupled with whole-genome sequencing (BSA-seq).

An NLR gene has been identified as the candidate for the powdery mildew resistance gene Pm40 in the 236.9-241.0 Mb interval on wheat chromosome 7B. Wheat powdery mildew severely threatens wheat production, while Pm40 is a durable and effective resistance gene against the disease. To accelerate the practical application and theoretical understanding of Pm40, the resistant parent PI672537 carrying Pm40, the susceptible parent L958, and their F2 and F2:3 populations were employed for genetic analysis. A total of 191.4, 191.9, 336.2, and 316.5 Gb of whole-genome sequencing data were obtained from PI672537, L958, and the resistant and susceptible bulked pools, respectively. In total, 3,879,037 single nucleotide polymorphism (SNP) markers were identified and used for mapping. The results of bulked segregant analysis combined with whole-genome sequencing (BSA-seq) revealed that Pm40 was narrowed down to a chromosomal region spanning 233.3-254.8 Mb on wheat chromosome 7B. Subsequent marker development and genetic analysis localized the Pm40 locus to a region of 236.9-241.0 Mb. Within this 4.1 Mb chromosomal region, 21 annotated genes were identified, among which only TraesCS7B03G0444100 and TraesCS7B03G0444300 were potentially associated with disease resistance. TraesCS7B03G0444100 encodes a RING-type zinc finger protein, whereas TraesCS7B03G0444300 encodes a nucleotide-binding leucine-rich repeat (NLR) protein. Sequence analysis revealed three nonsynonymous SNPs in the coding region of TraesCS7B03G0444300, resulting in amino acid substitutions. Furthermore, a kompetitive allele-specific polymerase chain reaction (KASP) found that one SNP marker (kasp-2) co-segregated with powdery mildew resistance. Collectively, these results suggested that TraesCS7B03G0444300 could be the candidate gene underlying Pm40. This study provided a valuable foundation for marker-assisted breeding and future functional characterization of Pm40-mediated resistance in wheat.