Dasypyrum villosum L. is one of the most valuable gene resources in wheat improvement, especially for disease resistance. The mining of favorable genes from D. villosum is frustrated by the lack of genome sequence. Here, by generating a doubled haploid line 91C43 using microspore culture, we obtained its 4.05 GB high-quality, chromosome-scale genome assembly. The assembly contains 85.31% repetitive sequences and 39,727 high-confidence genes. Two reciprocal translocation events were detected and the 7VS-4VL is a unique translocation in D. villosum. The prolamin seed storage protein-coding genes were duplicated, particularly, the genes encoding low-molecular-weight glutenin in Glu-V3 locus were significantly expanded. RNA-seq indicated that after Blumeria graminearum f.sp tritici (Bgt) inoculation, the up-regulated genes involving the pattern-triggered immunity and effector-triggered immunity defense pathways were more enriched in D. villosum than those in Triticum urartu. MNase hypersensitive sequencing (MH-seq) identified two Bgt-inducible MH sites (MHSs) in the promoter and 3’terminal regions of powdery mildew resistance (Pm) gene NLR1-V, respectively. They had two subpeaks each and were termed MHS1 (MHS1.1/1.2) and MHS2 (MHS2.1/2.2). Bgt inducible MHS2.2 was uniquely present in D. villosum and MHS1.1 was more inducible in D. villosum than in wheat. The identified MHSs may be critical for NLR1-V expression regulation and subsequent defense pathways. In summary, this study provides a valuable genome resource for functional genomics study and wheat-D. villosum introgression breeding. The identified regulatory mechanisms may provide a new strategy for enhancing Pm resistance by optimizing gene expression in wheat.
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