Abstract Background Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), poses a persistent threat to global wheat production, and many widely deployed Pm genes have been overcome by new virulent races. Wild emmer wheat (Triticum turgidum var. dicoccoides) harbors abundant but underutilized resistance alleles that can broaden the genetic diversity of cultivated wheat. This study aimed to genetically dissect the powdery-mildew resistance in the wild emmer accession [38]CWI45575. Results [39]CWI45575 exhibited strong resistance to 14 of 16 Bgt isolates at the seedling stage and complete field resistance under inoculation with a mixture of four Bgt isolates (A3, A10, E09, and E18) and naturally occurring field isolates in northern China. Genetic analysis revealed that the resistance is controlled by a single dominant gene, temporarily designated PmCWI45575. Bulked-segregant RNA-Seq (BSR-Seq) and linkage mapping delimited PmCWI45575 to a 5.3 Mb interval (716.6–721.9 Mb) on chromosome arm 4AL of the wild emmer wheat reference genome Zavitan (WEW_v2.0). This region corresponds to 718.5–724.4 Mb in the Chinese Spring reference genome (RefSeq v2.1) and does not overlap with any previously reported Pm loci on 4AL, except for QPm.tut-4A, which originates from a 7G-derived alien segment inserted into this chromosome. Transcriptome profiling identified 2,932 differentially expressed genes (DEGs) between resistant and susceptible bulks, including 96 DEGs within the mapped interval; GO and KEGG enrichment highlighted pathways related to mitogen-activated protein kinase (MAPK) signaling, hormone responses, redox regulation and phenylpropanoid biosynthesis, suggesting multilayered defense activation. A closely linked co-dominant marker, HebustP15, was developed and displayed clear polymorphism between [40]CWI45575 and four elite but susceptible cultivars, supporting its utility for marker-assisted selection (MAS). Conclusion We identified a novel powdery mildew resistance gene, PmCWI45575, from wild emmer wheat and physically mapped it to a 5.3 Mb interval on chromosome arm 4AL. Comparison of physical location with previously reported Pm genes on 4AL suggested that PmCWI45575 is likely a novel resistance gene. BSR-Seq-based transcriptomic analysis revealed enriched defense-related pathways, providing molecular insights into the resistance mechanism. The development and validation of closely linked molecular markers offer practical tools for marker-assisted selection in wheat resistance breeding. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-025-07222-1. Keywords: Wild emmer wheat, Powdery mildew, PmCWI45575, BSR-Seq, MAS Background Common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most important food crops worldwide, providing approximately 20% of the daily caloric intake for the global population and playing a central role in ensuring food security [[41]1]. However, wheat production faces increasing challenges from both biotic and abiotic stresses, among which powdery mildew, caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), remains one of the most devastating foliar diseases [[42]2, [43]3]. Under favorable conditions, severe epidemics can result in yield losses of 10–15%, and in extreme cases, up to 50–60% [[44]4]. In China, powdery mildew has affected more than 6 million hectares annually over the past decade, posing a persistent threat to both yield stability and grain quality ([45]https://www.natesc.org.cn/). The rapid evolution of virulent Bgt isolates, further exacerbated by global climate change, has accelerated the breakdown of resistance and intensified the need for more durable solutions [[46]5, [47]6]. Genetic resistance is widely regarded as the most effective and environmentally sustainable strategy for managing powdery mildew. To date, over 140 Pm genes or alleles have been identified, most of which encode nucleotide-binding leucine-rich repeat (NLR) proteins involved in race-specific immune recognition [[48]5, [49]7]. However, many widely deployed genes, such as Pm1, Pm2, Pm3, and Pm8, have been rendered ineffective by new virulent Bgt races, exposing the limitations of single-gene-based resistance systems [[50]8–[51]10]. Moreover, the domestication and polyploidization of wheat have led to a substantial loss of genetic diversity compared to its wild ancestors, limiting the potential for intraspecific resistance enhancement [[52]11, [53]12]. Wild emmer (Triticum turgidum var. dicoccoides, 2n = 4x = 28, AABB), the progenitor of both cultivated tetraploid and hexaploid wheat, originated in the Fertile Crescent and is widely distributed across the Middle East. Through long-term evolutionary selection in diverse ecological environments, it has accumulated extensive genetic variation and developed broad resistance to multiple biotic and abiotic stresses, including powdery mildew, rusts, drought, salinity, and heat [[54]13, [55]14]. Unlike more distantly related wild species, wild emmer readily crosses with hexaploid wheat, producing partially fertile F₁ hybrids and facilitating efficient gene transfer via backcrossing. These features render it a strategic donor for the introgression of novel resistance genes into elite wheat backgrounds. To date, numerous Pm genes have been identified from wild emmer, including Pm16 [[56]15], Pm26 [[57]16], Pm30 [[58]17], Pm36 [[59]18], Pm41 [[60]19], Pm42 [[61]20], Pm64 [[62]14], Pm69 [[63]21], as well as several Ml- or temporarily designated loci such as MlIW72, MlIW172, MlIW39; MlNFS10, MlWE74 and MlZec1 [[64]5]. Many of these loci confer resistance profiles distinct from those found in cultivated wheat, providing valuable resources for expanding the resistance gene pool and enhancing the durability of resistance in breeding programs. Recent advances in sequencing technologies and the availability of high-quality genome assemblies for both cultivated and wild Triticum species have significantly accelerated the discovery and characterization of disease resistance genes [[65]22–[66]25]. Among the various gene mapping strategies, bulked segregant RNA sequencing (BSR-Seq) has emerged as a powerful tool that combines genetic mapping with transcriptomic profiling. This method enables the simultaneous identification of candidate loci and differentially expressed genes associated with the target trait, and has been successfully employed to map numerous disease resistance genes in wheat [[67]26–[68]29]. For practical breeding applications, the development of tightly linked molecular markers is critical. Marker-assisted selection (MAS) facilitates precise introgression, shortens breeding cycles, and improves selection efficiency [[69]30, [70]31]. In this study, the wild emmer wheat accession [71]CWI45575 exhibited high-level resistance to powdery mildew at both seedling and adult stages. To elucidate the genetic basis of this resistance, we constructed genetic mapping populations and employed BSR-Seq to localize the resistance gene to a defined chromosomal interval. Functional enrichment analyses using gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) databases were performed to explore the regulatory pathways potentially involved in resistance, based on transcriptome data. Furthermore, closely linked markers were developed and validated for application in MAS. Collectively, our findings enrich the repertoire of Pm genes derived from wild emmer wheat and offer promising resources for breeding cultivars with durable resistance. Results Phenotypic assessment and inheritance of powdery mildew resistance in [72]CWI45575 At the seedling stage, [73]CWI45575 exhibited resistance to 14 out of 16 tested Bgt isolates (87.5%), with infection types (ITs) scored as 0, indicating complete immunity. Only isolates F06 and F28 elicited a susceptible response (Table [74]1). In field evaluations at the adult plant stage, [75]CWI45575 remained highly resistant (IT = 0) to a mixture of four Bgt isolates (A3, A10, E09, and E18), together with naturally occurring Bgt isolates collected from wheat fields in northern China. Isolates F06 and F28, which induced susceptibility at the seedling stage, were not included in the field inoculum. In contrast, Langdon (LDN) showed high susceptibility with ITs ranging from 7 to 9 (Fig. [76]1a). The susceptible control cultivar Mingxian 169 consistently exhibited highly susceptible infection types under both seedling and adult plant conditions, confirming the effectiveness of the inoculation and validating the resistance evaluation. Table 1. Response of [77]CWI45575 and Langdon to 16 Blumeria graminis f. sp. tritici (Bgt) isolates Bgt isolate [78]CWI45575 Landon Bgt isolate [79]CWI45575 Landon A3 0 4 F08 0 4 A10 0 4 F09 0 4 E09 0 4 F10 0 4 E18 0 4 F11 0 4 F03 0 4 F23 0 4 F05 0 4 F24 0 4 F06 4 4 F25 0 4 F07 0 4 F28 4 4 [80]Open in a new tab Note: Responses were recorded as infection types at the seedling stage using a 0–4 scale [[81]32], in which 0–2 were considered resistant and 3–4 susceptible Fig. 1. [82]Fig. 1 [83]Open in a new tab Reaction patterns of [84]CWI45575, Langdon (LDN), and their F[2:3] progeny to Blumeria graminis f. sp. tritici (Bgt). a Disease responses of the resistant parent [85]CWI45575 and the susceptible parent LDN under field conditions inoculated with a mixture of Bgt isolates at the adult plant stage. b Seedling-stage reactions of [86]CWI45575, LDN, and their F[2:3] plants to the Bgt isolate E09 under controlled conditions To investigate the genetic basis of the observed resistance, Bgt isolate E09 was selected for controlled inoculation experiments. All ten tested plants of the resistant parent [87]CWI45575 displayed complete resistance (IT = 0), whereas all ten plants of the susceptible parent LDN were highly susceptible (IT = 4) (Fig. [88]1b). The F₁ progeny derived from the [89]CWI45575 × LDN cross were uniformly resistant (IT = 0), suggesting that the resistance in [90]CWI45575 against E09 is conferred by a dominant gene. Phenotypic assessment of 189 F₂ individuals segregated into 146 resistant and 43 susceptible plants, conforming to a 3:1 Mendelian ratio expected for a single dominant gene (χ^2 = 0.51, P = 0.48). Further validation using 174 F[2:3] families revealed 41 homozygous resistant, 94 segregating, and 39 homozygous susceptible lines, which also fit the expected 1:2:1 segregation ratio for a single dominant locus (χ^2 = 1.18, P = 0.56). These results indicate that powdery mildew resistance to isolate E09 in [91]CWI45575 is governed by a single dominant gene, temporarily designated as PmCWI45575. To assess whether PmCWI45575 also confers resistance to other Bgt isolates, five additional isolates (F05, F07, F08, F09, and F11) were randomly selected to inoculate the 174 F[2:3] families. The segregation patterns observed for each isolate were consistent with those obtained using E09, suggesting that PmCWI45575 confers broad-spectrum resistance to these Bgt isolates. SNP calling and screening of the candidate intervals Following BSR-Seq, the resistant bulk generated 23.05 Gb clean data with a Q30 score of 93.8%, while the susceptible bulk produced 24.41 Gb clean data with Q30 score of 94.4%, indicating high sequencing quality. After sequence alignment to the wild emmer wheat reference genome Zavitan (WEW_v2.0) [[92]23], 154,339,698 reads from the resistant bulk and 163,569,572 reads from the susceptible bulk were successfully mapped, providing high-quality dataset for further analysis. A total of 1,637 polymorphic SNPs were detected between the two bulks. Among these, 664 SNPs were located on chromosome 4A. Notably, 280 SNPs were clustered within a 39 Mb genomic interval spanning from 704.5 Mb to 743.5 Mb on chromosome 4 A (Fig. [93]2a). Independent validation using the ED algorithm identified a highly similar candidate region, further supporting the reliability of the mapped interval associated with powdery mildew resistance (Fig. [94]2b). Fig. 2. [95]Fig. 2 [96]Open in a new tab Candidate interval analysis of PmCWI45575 revealed by BSR-Seq. a Genome-wide distribution of single nucleotide polymorphisms (SNPs) on the 14 chromosomes of wild emmer wheat (2n = 4x = 28, AABB), based on comparisons between the resistant and susceptible bulks of [97]CWI45575 × Langdon. b Euclidean distance (ED) values across all chromosomes, reflecting the association strength between genomic regions and powdery mildew resistance. ED values were calculated using a 10 Mb sliding window with parameters refFreq = 0.3 and minDepth = 25. Peaks in the ED plot indicate candidate intervals that may harbor the PmCWI45575 locus Mapping of PmCWI45575 To refine the chromosomal location of PmCWI45575, we developed 25 gel-based markers and 5 KASP markers based on InDel (≥ 4 bp) and SNP variants identified between the resistant and susceptible bulks. Marker design was performed using the PrimerServer module in WheatOmics 1.0 ([98]http://202.194.139.32/). Among these, nine gel-based markers (HebustP1, HebustP2, HebustP4, HebustP6, HebustP8, HebustP11, HebustP15, HebustP17, and HebustP19) and one KASP marker (HebustK721) showed clear polymorphisms between the resistant and susceptible parents as well as the corresponding bulks (Fig. [99]3; Table [100]2). In parallel, we evaluated eight previously reported markers located on chromosome arm 4AL for polymorphism between the parental lines and bulks. Of these, only three markers, including XWsdau73134.3, Xicsx520, and [101]BE446815 [[102]33, [103]34] were polymorphic and thus included in the genotyping of the F[2:3] population derived from the [104]CWI45575 × LDN cross. Genetic linkage analysis revealed that these 12 markers were closely associated with PmCWI45575. A linkage map was constructed using the genotypic and phenotypic data, positioning PmCWI45575 between HebustP2 and HebustK721 at genetic distances of 2.4 cM and 1.4 cM, respectively. HebustP6 co-segregated with the resistance gene (Fig. [105]4a). Based on alignment to the WEW_v2.0 reference genome, this interval corresponds to a physical region spanning 716.6–721.9 Mb on chromosome arm 4AL. Fig. 3. [106]Fig. 3 [107]Open in a new tab PCR amplification patterns of molecular markers HebustP6, HebustP11, and HebustP15, and genotyping results of the KASP marker HebustK721 in selected F[2:3] families. M, Puc19/Msp I. P[R], [108]CWI45575. P[S,] Langdon. R, homozygous resistant F[2:3] families. H, heterozygous resistant F[2:3] families. S, homozygous susceptible F[2:3] families. Red arrows indicate the polymorphic DNA bands linked to PmCWI45575. In the KASP genotyping plot, NTC indicates no template control Table 2. Polymorphic markers linked to the powdery mildew resistance gene PmCWI45575 developed in this study Marker Prime sequence (5′–3′) Physical location (bp) (WEW_v2.0) Product size (bp) HebustP1 F:GTCGTCTCCTCCTGCTC chr4A_711938080 124 R:TACAACTGAGGCACCAA HebustP2 F:AGGGTCCAGGTTTTTCGTGT chr4A_716631369 401 R:TGCAAGCTCCAGAACACTG HebustP4 F:GCTTGCTTGCAGTTCCTCCT chr4A_718833021 423 R:GCACCGCAGATAAGCTAACACC HebustP6 F:ACCAATTGATGAGGGTAGCTCT chr4A_721438312 444 R:GCTCTTGCCAAGTACATGCC HebustP8 F:GCTCCTACTCCATCGCTTCCC chr4A_737951480 268 R:CCTGCGATAAACACCGATGAACA HebustP11 F:TCTCACCTCTGAATCTGCGC chr4A_724440528 430 R:AGAACACAGGAATTCGGTCAGT HebustP15 F:CTGCACTCCGTCCCTTGTTT chr4A_731741352 358 R:GCCATCAAGCGTCGTGTAC HebustP17 F:CAATCCCTGATCGATCCGG chr4A_732605863 221 R:AAAGGTCGACTGCTGCTC HebustP19 F:TGCAGTTACTGCCATCTTCCCA chr4A_735204001 264 R:TTGGGGTCGTGTTAATTGCTAG HebustK721 F(FAM):gaaggtgaccaagttcatgct GAGGATCGGCGAGAAGGGC chr4A_721975197 - F(HEX):gaaggtcggagtcaacggatt GAGGATCGGCGAGAAGGGG R:CCAACCAACCATGAAATGTATCAAT [109]Open in a new tab Fig. 4. [110]Fig. 4 [111]Open in a new tab Genetic and physical mapping of PmCWI45575 on chromosome arm 4AL. a Genetic linkage map of PmCWI45575, constructed using polymorphic markers in the F[2:3] population derived from the cross between [112]CWI45575 and Langdon. The physical positions of the molecular markers were corresponding to the wild emmer wheat reference genome Zavitan (WEW_v2.0) (b) Comparative physical positions of PmCWI45575 and previously reported Pm genes on chromosome arm 4AL. The x-axis represents the physical position (in megabases, Mb) along chromosome arm 4AL of the Chinese Spring reference genome (RefSeq v2.1), and the y-axis lists the names of Pm genes mapped to this chromosome arm. Each horizontal box indicates the mapped interval spanned by the corresponding gene Comparison of PmCWI45575 with the known Pm genes on chromosome arm 4AL To date, ten Pm genes have been mapped to chromosome arm 4AL, including Pm61 [[113]34], MlIW30 [[114]35], QPm.tut-4A [[115]36], MlNSF10 [[116]33], PmPBDH [[117]37], PmHHXM [[118]38], PmXNM [[119]39], PmHSM [[120]40], PmHHM [[121]41], and PmLF540 [[122]42]. To evaluate the novelty of PmCWI45575, its physical position (718.5–724.4 Mb) corresponding to the Chinese Spring reference genome (RefSeq v2.1) [[123]43], was compared with the mapping positions of these genes. The mapped interval of PmCWI45575 does not overlap with any of the known loci on 4AL, except for QPm.tut-4A, which originates from a 7G-derived alien segment inserted into 4AL (Fig. [124]4b). This positional distinction suggests that PmCWI45575 is likely a novel Pm gene on chromosome arm 4AL. Discovery and analysis of DEGs To elucidate the molecular mechanisms underlying powdery mildew resistance in [125]CWI45575, we conducted transcriptomic comparisons between resistant and susceptible bulks using BSR-Seq. This analysis identified 2,932 differentially expressed genes (DEGs), comprising 1,136 upregulated and 1,796 downregulated genes in the resistant bulk relative to the susceptible one (Fig. [126]5; Table [127]S1). Within the refined candidate interval on chromosome arm 4AL (716.6–721.9 Mb) of WEW_v2.0 genome, 96 DEGs were detected, representing strong candidates potentially involved in the resistance response of [128]CWI45575. Notably, ten of these genes were functionally annotated as being directly associated with disease resistance pathways, suggesting they may serve as priority candidates for further functional characterization and gene cloning. Fig. 5. [129]Fig. 5 [130]Open in a new tab Volcano plot of differentially expressed genes (DEGs) between the resistant and susceptible bulks of [131]CWI45575 × Langdon. Each point represents a single gene, with the x-axis showing the log₂ fold change (logFC) and the y-axis representing the –log₁₀ of the false discovery rate (FDR). Upregulated genes in the resistant bulk are shown in blue, while downregulated genes are shown in yellow. DEGs were identified from transcriptomic comparisons based on BSR-Seq and mapped to the 14 chromosomes of wild emmer wheat (2n = 4x = 28, AABB) GO enrichment analysis revealed that the DEGs were primarily associated with stress and defense responses (Fig. [132]6; Table S2). In the biological process (BP) category, significantly enriched terms included response to oxygen-containing compounds, response to acid chemical, response to abiotic stimulus, response to chitin, and response to hormone, suggesting activation of multiple defense pathways involving oxidative stress, pathogen recognition, and hormone-mediated signaling (e.g., jasmonic acid and ethylene pathways). In the molecular function (MF) category, enrichment was observed for oxidoreductase activity, transmembrane transporter activity, and tetrapyrrole binding, implicating roles in redox regulation, metabolite transport, and chlorophyll-related responses during pathogen attack. The cellular component (CC) analysis showed enrichment of integral component of plasma membrane and transmembrane transport, indicating that membrane-associated processes are crucial for early defense signaling and molecular trafficking. Fig. 6. [133]Fig. 6 [134]Open in a new tab Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in wild emmer wheat. GO analysis was performed for DEGs mapped to the 14 wild emmer chromosomes (2n = 4x = 28, AABB) between resistant and susceptible bulks. Enriched GO terms were classified into three categories: biological process, cellular component, and molecular function To gain further insight into the biological pathways involved, KEGG enrichment analysis was performed (Fig. [135]7; Table S3). The DEGs were significantly enriched in multiple defense-related pathways, including photosynthesis-antenna proteins and biosynthesis of secondary metabolites, reflecting a reprogramming of photosynthetic machinery and activation of specialized metabolites during infection. Enrichment in the MAPK signaling pathway–plant and plant hormone signal transduction confirmed the involvement of conserved signaling cascades in response to Bgt. Additionally, pathways such as phenylpropanoid biosynthesis, brassinosteroid biosynthesis, glutathione metabolism, and cutin, suberine, and wax biosynthesis were notably represented, highlighting their roles in reinforcing cell wall structures, modulating redox homeostasis, and maintaining barrier integrity. Enrichment in nitrogen metabolism and ABC transporter pathways further indicated potential involvement of nutrient mobilization and transmembrane transport in mediating resistance. Fig. 7. [136]Fig. 7 [137]Open in a new tab Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes (DEGs) in wild emmer wheat. KEGG analysis identified significantly enriched pathways among the DEGs distributed across the 14 wild emmer chromosomes (2n = 4x = 28, AABB). Pathways related to disease resistance and stress response were predominantly enriched Evaluation of molecular markers available for MAS To facilitate the practical application of PmCWI45575 in MAS, two representative markers, HebustP6 (a dominant, co-segregating marker) and HebustP15 (a co-dominant, closely linked marker), were tested across nine powdery mildew-susceptible wheat cultivars. HebustP6 failed to generate distinguishable amplification patterns between [138]CWI45575 and the susceptible cultivars, limiting its utility in diverse backgrounds. In contrast, HebustP15 successfully produced polymorphic amplicons between [139]CWI45575 and four susceptible cultivars, Shi 4185, Kenong 199, Shixin 828 and Shixin 5071 (Table [140]3). These results suggest that HebustP15 is more suitable for use in MAS programs targeting PmCWI45575 introgression in selected elite wheat backgrounds. Table 3. Polymorphism analysis of molecular markers HebustP6 and HebustP15 between [141]CWI45575 and nine representative wheat cultivars/breeding lines Marker Shimai 22 Shi 4185 Kenong 199 Shiluan 02–1 Heng 4399 Shixin 733 Shixin 633 Shixin 828 Shixi 5071 HebustP6 - - - - - - - - - HebustP15 -  +   +  - - - -  +   +  [142]Open in a new tab Note: “ + ” represents polymorphic, whereas “-” represents nonpolymorphic Discussion Powdery mildew remains one of the most serious foliar diseases affecting global wheat production, with the continuous emergence of virulent Bgt populations posing a persistent challenge to disease management [[143]8]. In recent decades, substantial progress has been achieved in the cloning and functional characterization of Pm genes, with more than 20 resistance loci, including Pm1a, Pm2, Pm3, Pm4, Pm5e, Pm8, Pm12, Pm13, Pm17, Pm21, Pm24, Pm26, Pm36, Pm38, Pm41, Pm46, Pm57, Pm60, Pm69, Pm6SI, and WTK4, successfully isolated [[144]13, [145]44, [146]45]. These advances have significantly deepened the understanding of the molecular mechanisms underpinning wheat resistance to Bgt. Most of these cloned Pm genes encode intracellular NLR proteins that mediate race-specific effector-triggered immunity (ETI). However, their specificity often limits their durability, as field resistance can be quickly overcome by evolving pathogen populations [[147]46]. Wild relatives of wheat, which harbor a wealth of untapped genetic diversity, represent a promising source for discovering new resistance genes [[148]47–[149]49]. To achieve more durable resistance, identifying novel Pm genes from structurally diverse classes and underexploited germplasm, particularly wild wheat relatives, is a strategic priority in breeding programs [[150]50, [151]51]. In this study, we identified and genetically mapped a novel dominant Pm gene derived from the wild emmer wheat accession [152]CWI45575. Phenotypic evaluation revealed that [153]CWI45575 exhibited strong resistance to 14 of 16 tested Bgt isolates at the seedling stage and displayed complete immunity under natural field conditions with mixed natural and artificial inoculation. Using BSR-Seq and molecular marker analysis, we delimited the candidate interval of PmCWI45575 to a 5.3 Mb region (716.6–721.9 Mb) on 4AL based on the WEW_v2.0 genome. To assess its novelty, we compared the physical position of PmCWI45575 with ten previously reported Pm loci mapped to chromosome arm 4AL based on their positions in the Chinese Spring reference genome (RefSeq v2.1), including those from wheat breeding lines (PmPBDH), landraces (Pm61, PmXNM, PmHHXM, PmHHM, and PmHSM), and wild emmer accessions (MlIW30, MlNFS10, and PmLF540). QPm.tut-4A, derived from T. militinae, is the only known locus partially overlapping with PmCWI45575 in physical position. However, QPm.tut-4A is a quantitatively inherited QTL introduced from a 7G chromosomal segment of T. militinae, which exhibits suppressed recombination and low homology with wheat 4AL chromatin. In contrast, PmCWI45575 is a dominant resistance gene located within native wheat chromatin derived from T. dicoccoides, suggesting a distinct evolutionary origin and genetic behavior. Therefore, despite partial positional overlap, PmCWI45575 is unlikely to be allelic to QPm.tut-4A and most likely represents a novel resistance locus. Nevertheless, due to differences in reference genomes and donor germplasm backgrounds, further gene cloning and functional validation will be required to conclusively determine whether PmCWI45575 and the other Pm genes on 4AL are distinct or allelic. Overall, these findings underscore the value of wild wheat relatives as a reservoir of novel resistance genes and provide a promising resource for broadening the genetic base of powdery mildew resistance in wheat breeding programs. Recent advances have revealed novel classes of resistance genes beyond the canonical NLR family, broadening the understanding of immune mechanisms in wheat [[154]48, [155]52]. For instance, Pm13, derived from Aegilops longissima, encodes a mixed lineage kinase domain-like (MLKL) protein that contains an N-terminal-domain of MLKL (MLKL_NTD) domain in its N-terminus and a C-terminal serine/threonine kinase (STK) domain, underscoring the important roles of kinase fusion proteins (KFPs) in wheat immunity [[156]53]. Pm36, derived from wild emmer wheat, encodes a tandem kinase with a transmembrane domain (WTK7-TM), implicating a signal-transduction-based mechanism in pathogen recognition and defense activation [[157]13]. In another example, Pm26, also originating from wild emmer wheat, is governed by a genetically linked atypical NLR pair TdCNL1/TdCNL5, demonstrating structural diversity in immune gene architecture [[158]45]. These discoveries emphasize that wild wheat relatives harbor a wealth of non-NLR-based resistance mechanisms, reinforcing the importance of exploring genetically diverse germplasm for novel gene discovery. In this study, transcriptome profiling further supported the uniqueness of PmCWI45575. Among 2,932 DEGs identified between resistant and susceptible bulks, 96 were located within the candidate interval, with 10 functionally annotated as being directly involved in disease resistance, including genes encoding NBS-LRR and RLK proteins. GO enrichment analysis revealed that these DEGs were significantly associated with responses to chitin, plant hormones (notably jasmonic acid and ethylene), and oxidative stress, indicating that PmCWI45575 may orchestrate a complex, multilayered defense network. Functional enrichment of oxidoreductase activity and transmembrane transporter activity further suggested roles in redox regulation and metabolite trafficking, which are critical components of immune responses. Supporting this, KEGG pathway analysis indicated significant enrichment in MAPK signaling, secondary metabolite biosynthesis, and plant hormone signal transduction pathways, all of which are well-characterized in plant immune signaling. Notably, transcripts associated with photosynthesis-related processes, including antenna proteins, were also enriched, suggesting a potential reprogramming of energy allocation and plastid-derived signaling during pathogen challenge. This is consistent with prior studies reporting photosynthetic modulation as a strategy to maintain metabolic balance under biotic stress [[159]54]. Additionally, the upregulation of phenylpropanoid biosynthesis and glutathione metabolism pathways, which are implicated in lignin deposition and reactive oxygen species (ROS) detoxification, points to their involvement in reinforcing cell wall structures and mitigating oxidative damage during infection [[160]55, [161]56]. These findings collectively suggest that PmCWI45575 may constitute a distinct resistance gene with multilayered defense functions, differing from known Pm loci in both genetic and regulatory features. From a breeding perspective, PmCWI45575 represents a promising resource for broadening the genetic base of powdery mildew resistance in wheat. The overreliance on a narrow set of resistance genes such as Pm8 and Pm17 in commercial cultivars has led to frequent breakdowns under pathogen pressure [[162]57, [163]58]. The introduction of PmCWI45575, especially given its origin from wild emmer, offers an opportunity to enrich the diversity of resistance genes and mitigate the vulnerability associated with resistance gene monoculture. A critical step toward the effective utilization of PmCWI45575 is the development of reliable molecular markers for MAS. In this study, the dominant marker HebustP6 exhibited limited applicability across diverse genetic backgrounds. In contrast, the co-dominant marker HebustP15 displayed polymorphism between [164]CWI45575 and four elite but susceptible wheat cultivars, making it a valuable tool for MAS. The observed difference in marker performance highlights the genetic background dependence of molecular markers. The lack of polymorphism in HebustP6 among the tested cultivars may be attributed to sequence conservation in the targeted region, which limits its utility beyond the mapping population. Although HebustP15 exhibited polymorphism in four out of nine tested wheat cultivars, its applicability across broader germplasm may still be limited due to potential sequence conservation in the target region. Therefore, when deploying resistance genes in breeding programs, it is important to evaluate marker effectiveness across diverse germplasm, and to develop alternative or complementary markers when necessary. To fully realize the potential of PmCWI45575, future work should focus on pyramiding it with complementary resistance loci to enhance durability, and on gene cloning to elucidate its molecular mechanism. Given the ongoing evolution of Bgt populations and the frequent breakdown of single-gene resistance, pyramiding PmCWI45575 with other functionally distinct Pm genes offers a promising strategy to achieve broad-spectrum and long-lasting resistance. The unique origin of PmCWI45575 from wild emmer wheat suggests that it may operate through a mechanism different from those widely deployed in commercial cultivars, making it an ideal candidate for gene stacking in resistance breeding programs. Integration of PmCWI45575 into elite backgrounds via marker-assisted selection will provide breeders with new options for developing durable resistant varieties. Conclusion PmCWI45575 was mapped to a previously uncharacterized resistance locus on chromosome arm 4AL of wild emmer wheat. This locus is physically distinct from all known Pm genes on 4AL, except for partial overlap with QPm.tut-4A, an alien 7G-derived segment inserted into the chromosome, suggesting that PmCWI45575 likely represents a novel resistance gene. BSR-Seq-based transcriptomic analysis provides a solid foundation for future gene cloning and functional characterization. Moreover, the closely linked molecular markers developed in this study have been experimentally validated for marker-assisted selection, offering practical tools for the efficient introgression of PmCWI45575 into wheat breeding programs. Materials and methods Plant materials The wild emmer wheat accession [165]CWI45575, provided by the International Maize and Wheat Improvement Center (CIMMYT), was used to evaluate powdery mildew resistance. The susceptible durum wheat accession LDN, also provided by CIMMYT, was crossed with [166]CWI45575 to generate F[1], F[2], and F[2:3] populations for genetic analysis and BSR-Seq analysis. The common wheat cultivar Mingxian 169, which is susceptible to all the tested Bgt isolates [[167]37], was used as a susceptible control in all phenotyping experiments. Additionally, Mingxian 169 was used as a spreader line to ensure uniform disease pressure in inoculation assays. Phenotypic assessment and genetic analysis Sixteen single-pustule-derived Bgt isolates were utilized to evaluate the resistant spectrum of [168]CWI45575 (Tabe [169]1). Independent inoculations were conducted at the one-leaf stage. For each Bgt isolate, five seeds from [170]CWI45575 and LDN were sown in individual square pots (7 × 7 × 5 cm) and spatially isolated to prevent cross-infection. Seedlings were grown in a greenhouse under controlled conditions: relative humidity > 60%, a photoperiod of 14 h light/10 h darkness, and day/night temperatures of 24 °C/18 °C, until reaching the two-leaf stage. Fresh conidiospores were collected from heavily sporulating Mingxian 169 plants and used to inoculate the test seedlings. After inoculation, the seedlings were incubated in the dark at 18 °C and 60% humidity for 24 h, and then transferred back to the greenhouse. ITs were scored 10–14 days post-inoculation using a 0–4 scale [[171]32], where 0–2 were considered resistant, and 3–4 susceptible. Phenotyping was conducted in three independent replications. To evaluate adult-stage resistance to powdery mildew, [172]CWI45575 was inoculated with a mixture of four Bgt isolates (A3, A10, E09, and E18), along with naturally occurring Bgt isolates collected from wheat fields in northern China during the 2022–2024 cropping seasons. Field trials were conducted in a randomized complete block design with three replications. Each plot consisted of three rows, 2 m in length and spaced 25 cm apart. The susceptible cultivar Mingxian 169 was planted around each plot to promote uniform disease pressure. Disease reactions were evaluated after the full heading stage [[173]59] using a 0–9 scale, where 0–4 indicated resistance and 5–9 indicated susceptibility [[174]60]. Assessments were performed twice, with a one-week interval. To evaluate powdery mildew resistance and dissect its genetic basis, [175]CWI45575, LDN, their 10 F[1] hybrids, 189 F[2] individuals, and 174 F[2:3] families were evaluated using the Bgt isolate E09, a prevalent Bgt isolate in North China and thus commonly used in genetic studies of powdery mildew resistance [[176]61]. For each F[2:3] family, approximately 30 seeds were sown in three adjacent cells within a seedling tray, while the susceptible hexaploid cultivar Mingxian 169 was randomly distributed across all trays. All seedlings were grown under controlled greenhouse conditions as described above. When the seedlings reached the one-leaf stage, fresh conidiospores collected from heavily sporulating Mingxian 169 plants were evenly dusted onto the trays using the sweeping method. Disease responses were recorded ten days post-inoculation, when sporulation on the control Mingxian 169 leaves was fully developed. Each evaluation was repeated three times to ensure phenotypic consistency. For the F[2:3] families, those in which all plants exhibited resistant infection types (ITs 0–2) were classified as homozygous resistant, whereas those with all plants showing susceptible ITs (3–4) were classified as homozygous susceptible. Families segregating for both resistant and susceptible plants were considered heterozygous. These classifications were based on the segregation pattern of infection types within each family. Segregation ratios in the F[2] and F[2:3] generations were subjected to chi-squared (χ^2) tests to determine the goodness-of-fit to expected Mendelian inheritance patterns, thereby assessing whether the resistance was controlled by a single dominant gene. If the segregation ratio against isolate E09 was consistent with that of monogenetic inheritance, five other isolates avirulent on [177]CWI45575 will be further used to inoculate the F[2:3] generations. BSR-Seq analysis For BSR-seq, leaf tissues were collected from 30 homozygous resistant and 30 homozygous susceptible F[2:3] individuals, each selected from an independent family derived from the [178]CWI45575 × LDN cross, based on consistent extreme phenotypes in response to Bgt isolate E09 under greenhouse conditions. Total RNA was extracted separately from each bulk using RNAiso Plus reagent (Takara Bio, Shiga, Japan), and equal quantities of RNA from each sample were pooled to construct resistant and susceptible bulks. cDNA libraries were prepared using the Illumina TruSeq RNA Library Prep Kit (Illumina Inc., San Diego, CA) following the manufacturer’s protocol. Sequencing was conducted on the Illumina HiSeq 4000 platform at Tcuni Bioscience (Chengdu, China). Raw sequencing data were preprocessed to remove adapter sequences and low-quality reads. Clean reads were aligned to WEW_v2.0 reference genome using STAR aligner [[179]62]. Duplicate reads and reads spanning splice junctions were filtered using SAMtools v1.9 [[180]63]. SNP calling was performed using GATK v4.2.3.0 [[181]64], and the SNP index for each bulk was calculated following the MutMap method [[182]65]. The ΔSNP index was then computed as the difference between the SNP index of the resistant bulk and that of the susceptible bulk (ΔSNP index = SNP index of resistant bulk—SNP index of susceptible bulk), following the approach described in [[183]66]. Euclidean distance (ED) algorithms were applied to refine the candidate interval associated with powdery mildew resistance. Candidate intervals with higher confidence (99%) and SNPs with larger than the threshold ΔSNP index value (set as 0.5) were defined as candidate loci associated with resistance. Genetic mapping of the powdery mildew resistance gene in [184]CWI45575 Following the identification of a candidate genomic region by BSR-Seq, both previously reported markers within the mapped interval and newly designed markers based on the BSR-Seq data were employed to further validate and refine the resistance locus. For marker development, Insertions/deletions (InDels) and SNPs within the candidate region were identified and used to design gel-based markers for gel electrophoresis and high-throughput kompetitive allele-specific PCR (KASP) markers. Marker design was conducted using the PrimerServer tool available in WheatOmics 1.0 ([185]http://202.194.139.32/). All the markers were first tested for polymorphism between the resistant and susceptible parents and bulks. Polymorphic markers were subsequently genotyped across the F[2:3] mapping population derived from the [186]CWI45575 × LDN cross. PCR amplification and product separation followed the protocol described by [[187]67] with minor modifications. In detail, PCR amplification was performed in a 10 μL reaction volume containing 1 μL of template DNA (50–150 ng/μL), 4 μL of 2 × Taq Master Mix (Vazyme Biotech, Nanjing, China), 4 μL of ddH₂O, and 1 μL of primer mix (10 μM each). The thermal cycling program was as follows: an initial denaturation at 94 °C for 5 min; followed by 36 cycles of denaturation at 94 °C for 30 s, annealing at 55–60 °C for 40 s (depending on primer), and extension at 72 °C for 40 s; and a final extension at 72 °C for 10 min. PCR products were resolved on 8% non-denaturing polyacrylamide gels (29:1 acrylamide:bisacrylamide) followed by silver staining. KASP genotyping was performed using a Bio-Rad CFX96 real-time PCR system in a total reaction volume of 5 μL, consisting of 1.5 μL genomic DNA (approximately 100 ng), 2.8 μL KASP Master Mix, 0.1 μL primer mix, and 0.6 μL ddH₂O. The thermal cycling profile included an initial denaturation at 94 °C for 15 min, followed by 10 touchdown cycles (94 °C for 20 s; 65 °C to 55 °C, decreasing by 0.6 °C per cycle, for 60 s), and 36 standard amplification cycles (94 °C for 20 s, 55 °C for 60 s). Fluorescence signals were detected at 37 °C using Bio-Rad CFX Manager 3.1 software. Linkage analysis between genotypic and phenotypic data was conducted using Mapmaker 3.0b [[188]68], with a logarithm of odds (LOD) threshold of 3.0 to determine significant associations. DEGs analysis associated with the powdery mildew resistance in [189]CWI45575 To investigate the molecular basis of powdery mildew resistance in the wild emmer accession [190]CWI45575, differential gene expression (DEG) analysis was conducted between the resistant and susceptible bulks derived from the [191]CWI45575 × LDN F[2:3] population used for BSR-Seq. Following quality filtering, clean reads were aligned to WEW_v2.0 reference genome. Gene expression levels were normalized and quantified as fragments per kilobase of transcript per million mapped reads (FPKM). DEGs were identified using EBSeq v3.5, with significance thresholds set at a fold change (FC) ≥ 2 and a false discovery rate (FDR) < 0.01, applying the Benjamini–Hochberg method to correct for multiple hypothesis testing. Genes meeting these criteria were considered to be significantly differentially expressed between the two phenotypic bulks. Subsequently, functional annotation of DEGs was performed using GO and KEGG databases, implemented through the R package clusterProfiler [[192]69]. Enrichment analysis enabled the classification of DEGs into key biological processes, molecular functions, and cellular components, and also revealed significantly overrepresented metabolic and signaling pathways potentially associated with disease resistance. These results provided a valuable resource for prioritizing candidate resistance genes for further functional validation. Evaluation of the markers for MAS breeding To evaluate the applicability of the developed markers for MAS, closely linked markers were used to genotype the resistant donor [193]CWI45575 and nine powdery mildew-susceptible wheat cultivars or breeding lines (Table [194]3). These materials represent a diverse set of elite, yet powdery mildew-susceptible, genetic backgrounds that are widely used in wheat improvement programs across Hebei province, China. Markers displaying clear polymorphisms between [195]CWI45575 and the susceptible genotypes were deemed effective and potentially deployable for MAS-based introgression of PmCWI45575 in diverse breeding populations. Supplementary Information [196]Supplementary Material 1.^ (8MB, xlsx) Acknowledgements