PmD479 is an Unutilized Gene for Powdery Mildew Resistance in Common Wheat

Abstract

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), threatens global wheat production. Wild emmer wheat (Triticum turgidum ssp. dicoccoides, DIC) is a progenitor of common wheat (T. aestivum L.) and harbours abundant powdery mildew (Pm) resistance genes (Wulff and Liu 2025). Here, we mapped and cloned PmD479, an unutilized Pm gene on chromosome 2BL in the DIC accession ‘DIC479’ (Marais et al. 2005), and introduced PmD479 into common wheat. Given its broad-spectrum efficacy and current rarity in common wheat germplasm, PmD479 is now a valuable resource for diversifying powdery mildew resistance in wheat.

DIC479 seedlings were resistant to 11 Chinese Bgt isolates (Table S1). In response to Bgt isolate E09, DIC479 produced reactive oxygen species (ROS) and exhibited localized necrosis (Figure 1A). DIC479 and Langdon (LDN, a Pm-susceptible durum wheat, T. turgidum ssp. durum) were used to develop an F₂ population, which showed a 3:1 segregation (157 resistant vs. 47 susceptible; χ² = 0.418, p > 0.05). Among 83 randomly selected F_2:3 families, 23 showed homozygous resistance, 39 segregated for Pm resistance and 21 showed homozygous susceptibility, fitting a 1:2:1 ratio (χ² = 0.397, p > 0.05) (Table S2). Thus, PmD479 acts as a single dominant gene for Pm resistance.

Nine Pm-susceptible F₂ plants were genotyped by the 90 K iSelect SNP array identified nine PmD479-linked SNPs on the long arm of the homoeologous group 2 chromosome in wheat (Table S3). Sixteen molecular markers (Table S4) were developed by integrating the linked SNPs and the genomic collinearity among rice, barley, Aegilops tauschii and common wheat. Using 204 F₂ plants, PmD479 was mapped to a 2.35 cM interval between Xsdau169 and Xsdau149 on distal 2BL (Figure 1A).

To fine map PmD479, we sequenced DIC479 with PacBio HiFi, generated 8923 contigs with an N50 of 6.05 Mb, and developed 13 additional markers (Table S4). Using 124 homozygous recombinants from 3045 F_2:3 plants, PmD479 was precisely mapped between Xsdau186B and Xsdau193 (< 0.1 cM; Figure 1A; Tables S4 and S5). Both Xsdau186B and Xsdau193 reside on the ptg000752l contig (3.17 Mb), delimit a 90 kb region in DIC479, and correspond to 811.78–811.93 Mb (2BL) in the Chinese Spring reference genome v2.1 (Zhu et al. 2021) (Figure 1A). NLR148, a homologue of TraesCS2B03G1573100, is the only complete gene in contig ptg000752l (Figure 1A; Table S6). NLR148 was constitutively expressed at 0 to 24 h post infestation (hpi) and then significantly upregulated at 48 to 96 hpi (Figure 1A). NLR148 encodes for a typical resistance protein with 978 amino acids and three domains: a Rx N-terminal domain (Rx_N), a NB-ARC domain (NB-ARC) and leucine-rich repeats (LRR) (Figure 1A; Figure S1).

Using the Pm-susceptible wheat ‘Fielder’ as the recipient, we transformed and expressed NLR148 via three vectors: PC1218 (cDNA with maize ubiquitin promoter), PC1219 (genomic DNA with maize ubiquitin promoter) and PC1220 (native expression) (Figure S2). Transgenic plants, positive for either PC1218, PC1219 or PC1220, were highly resistant (infection types, ITs = 0) to E09, whereas non-transgenic controls were highly susceptible (ITs = 4) (Figure 1B). For knockouts, we used CRISPR/Cas9 (PC1205) on a Pm-resistant F₅ line that was derived from a DIC479/LDN cross (Pop2-167-6). The knockouts from PC1205 resulted in either 1 or 15 bp deletions or a 1 bp insertion, and resulted in susceptibility to E09 (Figure 1B; Figure S3). Thus, NLR148 is PmD479 (GenBank: PX828566).

PmD479 was further tested with 106 Bgt isolates from different areas in China. The Pop2-167-6 line was resistant to 93 isolates and immune or highly resistant to 78 of them (Figure 1C and Table S7). Thus, PmD479 is effective against 88% of Chinese Bgt isolates, demonstrating broad-spectrum resistance.

We surveyed the distribution and haplotype of PmD479 in 147 DIC accessions from the Fertile Crescent region. Homologous genes were PCR-amplified from 75 accessions, revealing 28 haplotypes, with none identical to PmD479 (Figure 1D; Table S8). In contrast, PmD479 was absent in all 491 common wheat cultivars surveyed (Figure S4). Apparently, PmD479 has not been deployed previously in common wheat.

Here, PmD479 was then introduced into common wheat. A synthetic wheat, Syn-8, was obtained between Pop3000-2332-17 (an F₅ derivative of the DIC479/LDN cross) and Aegilops tauschii PI511383. The Pop3000-2332-17 was also crossed and backcrossed with common wheat cultivars and generated the hexaploid wheat 1454. Both Syn-8-3 and 1454 were euploid (42 chromosomes, confirmed by FISH; Figure 1E and Figure S5), positive for the PmD479-FM marker, and resistant to E09 (Figure 1E,F; Tables S9 and S10). Therefore, PmD479 introduces Pm resistance in common wheat and can be deployed in wheat breeding.

To date, 14 Pm genes have been mapped on chromosome 2BL in wheat. Twelve Pm genes, i.e., Pm52/Pm6 (Qiu et al. 2025), Pm33, Pm51, Pm63, Pm64, PmCG15-009, PmKN0816, PmLS5082, pmQ, pmYN99102 (Wang et al. 2023) and PmYD588 (Ma et al. 2021) (Table S11), differ from PmD479 genetically and physically. However, the DIC-derived resistance genes MlZec1, MlAB10 and PmD479 were localized to the 2BL deletion bin (0.89–1.00) (Mohler et al. 2005; Maxwell et al. 2010). Whether PmD479 is allelic to Mlzec1 and/or MlAB10 remains unclear.

In summary, we cloned an unutilized Pm resistance gene PmD479 from wild emmer wheat. Introduction of PmD479 into common wheat markedly enhanced Pm resistance. Thus, this study provides a valuable gene and germplasm for powdery mildew resistance in common wheat.

The Key R&D Program of Shandong Province, China (2024LZGC034, 2024CXPT072) and the Innovation Funding of the State Key Laboratory of Wheat Improvement (WIFY202519).

The authors declare no conflicts of interest.

The sequencing data produced in this study have been deposited in the NCBI BioProject database under accession number PRJNA1394530.