Fine mapping and genetic dissection of PmCWI16926, a broad-spectrum powdery mildew resistance gene from cultivated emmer wheat

Fine mapping and genetic dissection of PmCWI16926, a broad-spectrum powdery mildew resistance gene from cultivated emmer wheat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fine mapping and genetic dissection of PmCWI16926, a broad-spectrum powdery mildew resistance gene from cultivated emmer wheat Ningning Yu, Xiaozhe Xu, Dongming Li, Fengtao Wang, Yuli Jin, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7647818/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici ( Bgt ), severely threatens global wheat production. Cultivated emmer wheat, a direct progenitor of common wheat, harbors rich genetic diversity and represents a valuable source of novel resistance genes. In the present study, the cultivated emmer accession CWI16926-4Y exhibited high-level and broad-spectrum resistance at both seedling and adult plant stages. Genetic analysis revealed that the powdery mildew resistance in CWI16926-4Y is controlled by a single dominant gene, designated PmCWI16926 . Employing bulked segregant RNA analysis combined with high-density mapping, PmCWI16926 was delimited to a 590 kb physical interval (21.70-22.29 Mb) on chromosome 2BS of the durum wheat cv. Svevo genome. Comparative analysis confirmed its distinction from nine previously reported loci on 2BS. Within the interval, two NLR-type genes were identified, with TRITD2Bv1G010140 emerging as the most promising candidate based on pathogen-induced expression and a unique haplotype defined by three nonsynonymous SNPs absent in other resistant and susceptible genotypes. Moreover, four co-segregated markers and a gene-specific marker KASP689-1 were validated, enabling marker-assisted transfer of PmCWI16926 into elite cultivars in breeding. This study expands the repertoire of deployable Pm genes from emmer wheat and provides new genetic tools for durable and precise resistance breeding. Biological sciences/Genetics/Agricultural genetics Biological sciences/Plant sciences/Plant breeding Cultivated emmer wheat Powdery mildew PmCWI16926 Fine mapping Marker-assisted selection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Common wheat ( Triticum aestivum L., 2n = 6x = 42, AABBDD) serves as a globally significant staple crop, providing a significant proportion of human caloric intake and essential nutrients 1 . However, its productivity is continually threatened by a range of biotic and abiotic stresses. Among these, powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici ( Bgt ), ranks as one of the most destructive foliar diseases worldwide 2 . In China, the disease has affected more than six million hectares annually over the past decade (NATESC). The continual emergence of virulent pathogen races, compounded by climate variability, has intensified the erosion of resistance, making the development of durable genetic protection a high priority 3 . Genetic resistance is widely recognized as the most economical, effective and environmentally sustainable approach to managing wheat powdery mildew 4 . To date, more than 100 formally or temporarily designated powdery mildew ( Pm ) resistance genes/alleles have been identified in common wheat and related species, most of which encode coiled-coil nucleotide-binding leucine-rich repeat (CC-NBS-LRR) proteins mediating race-specific immune recognition 5 . Among of them, only a few Pm genes have been widely used in developing disease-resistant wheat varieties 6 . Yet several extensively deployed genes, such as Pm1 , Pm2 , Pm3 , Pm8 , and Pm17 , have been overcome by rapidly evolving Bgt populations 7 – 10 . In addition, domestication and polyploidization have markedly narrowed the genetic base of modern cultivars 11 – 13 , limiting the scope for discovering novel alleles within elite wheat. Notably, recent advances have uncovered non-classical immune modules from wheat relatives—distinct from the canonical CNL architecture—that confer effective powdery mildew resistance 14 – 17 . The discovery of such unconventional resistance frameworks not only expands the molecular diversity available for defense but also provides alternative mechanistic routes for designing durable, broad-spectrum resistance in breeding programs. Ancestral tetraploid wheats, which form part of the secondary gene pool, represent a valuable source of untapped genetic variation. Cultivated emmer ( T. dicoccum , 2n = 4x = 28, AABB) and its wild progenitor T. dicoccoides originated in the Fertile Crescent and adapted to diverse environments under long-term exposure to complex biotic and abiotic pressures 18 , 19 . Compared with more distantly related Triticeae donors, genes from tetraploid progenitors are more easily introgressed into hexaploid wheat and generally incur fewer drawbacks from alien translocations, such as linkage drag or poor genetic compensation 20 – 22 . Several Pm loci, including Pm16 , Pm26 , Pm30 , Pm36 , Pm41 , Pm42 , Pm64 , Pm69 , and Pm71 , trace back to tetraploid ancestors, many conferring resistance profiles distinct from those found in cultivated wheat 5 , 23 . The development of high-quality reference genomes for wheat and its relatives, together with advances in high-throughput sequencing technologies, has transformed the pace and precision of gene discovery, fine mapping, and cloning 24 – 29 . Bulk segregant RNA-Seq integrated the advantage of bulked segregant analysis (BSA) and high-throughput transcriptomic profiling, enabling rapid delimitation of causal intervals and efficient identification of candidate genes 30 . We have previously used BSR-Seq to identify and characterize multiple Pm loci, including PmLS5082 and PmXQ-0508 from wheat breeding line LS5082 and XQ-0508, respectively 31 , 32 , demonstrating its versatility for exploiting diverse resistance sources. Crucially, coupling gene discovery with the development of tightly linked, breeder-friendly markers provide an essential bridge between molecular research and breeding deployment. In this study, we investigated the cultivated emmer accession CWI16926-4Y, which has consistently exhibited high-level and broad-spectrum resistance to powdery mildew under multi-year field conditions. By integrating BSR-Seq with classical genetic analysis, we aimed to (i) finely map the powdery mildew resistance gene(s) in CWI16926-4Y, (ii) dissect its genetic basis and inheritance pattern, (iii) identify and characterize potential candidate gene(s) underlying the resistance, and (iv) develop molecular markers for marker assisted selection (MAS) in wheat breeding. By sourcing resistance from an ancestral tetraploid donor with minimal linkage drag, this work expands the repertoire of deployable Pm loci and provides practical tools for precise gene pyramiding and stable field performance. Results Inheritance of powdery mildew resistance in CWI16926-4Y In field evaluations at the adult plant stage, CWI16926-4Y showed complete resistance (infection type, IT = 0) to a mixture of inoculated Bgt isolates and naturally occurring isolates in northern China. At the seedling stage, CWI16926-4Y exhibited compete seedling resistance to all 15 tested Bgt isolates, with IT 0 (Fig. 1 a). To dissect the genetic basis of this resistance, Bgt isolate E09 was selected for controlled inoculation. All ten tested plants of the resistant parent CWI16926-4Y remained immune (IT = 0), whereas all ten plants of the susceptible parent Langdon (LDN) were highly susceptible (IT = 4) (Fig. 1 b). The F₁ progeny from the CWI16926-4Y × LDN cross were all resistant (IT = 0), indicating that the resistance to E09 is conferred by a dominant gene. Phenotypic assessment of 207 F₂ individuals segregated into 154 resistant and 53 susceptible plants, fitting the expected 3:1 ratio for a single dominant locus (χ² = 0.04, P = 0.84). Consistent results were obtained in 198 F 2:3 families, which segregated into 48 homozygous resistant, 103 segregating, and 47 homozygous susceptible lines, consistent with the expected 1:2:1 ratio (χ² = 0.33, P = 0.84). These results demonstrate that powdery mildew resistance to isolate E09 in CWI16926-4Y is governed by a single dominant gene, which we tentatively designated PmCWI16926 . To further evaluate its spectrum, 198 F 2:3 families were inoculated with ten additional Bgt isolates. Segregation patterns for each isolate mirrored those observed with E09, suggesting that PmCWI16926 confers broad-spectrum resistance across diverse Bgt isolates. SNP calling and screening of candidate intervals BSR-Seq analysis generated 20.67 and 22.15 Gb clean data from the R and S bulks, respectively, with Q30 percentages above 94.5% and 93.6%. Following sequence alignment to the reference genome, a total of 55,524 high-quality SNPs were identified between R and S bulks, distributed across all the 14 cultivated emmer wheat chromosomes (AA and BB genomes) (Fig. 2 ). Of these, 5798 (accounting for 10.4%) SNPs were located on the chromosome arm 2B. According to the △SNP index value, one estimated candidate region was detected, located at the end of chromosome arm 2BS (13.0-25.3 Mb). Using ED analysis, an evident allelic difference between the R and S bulks on the similar interval was also detected (Fig. 2 ), re-validating the candidate interval of PmCWI16926. Molecular mapping of PmCWI16926 To map PmCWI16926 , 26 reported markers and 87 newly developed markers within the candidate interval were tested for polymorphism. Thirteen newly developed simple sequence repeats (SSRs) and insertions/deletions (InDels) markers in candidate interval were linked to PmCWI16926 , after screening for polymorphism between the parents and two bulks and subsequent genotyping on 202 F 2:3 families (Fig. 3 , Table S1 ). Using these polymorphic markers, a high-density linkage map was constructed, placing PmCWI16926 between markers YTU2BS-045 and YTU2BS-058 at genetic distances of 1.3 and 0.2 cM, respectively. Furthermore, genotyping of 2000 F 2:3 families refined the interval to 590 kb (Chr2B_21700330-Chr2B_22293417) in the durum cv. Svevo genome, flanked by the markers YTU2BS-045 and YTU 2BS-062 (Figs. 3 and 4 , Table S1 ). Comparison of PmCWI16926 with known Pm genes on chromosome 2BS To elucidate the relationship between the Pm gene(s) in CWI16926-4Y and previously reported Pm genes located in similar genomic intervals, CWI16926-4Y and wheat genotypes carrying Pm26 (TTD140), pm42 ( B05429 ) , Pm68 (TRI1796), MlIW170 (IW170), pmWE99 (WE99) and PmXQ-0508 (XQ-0508) were inoculated with 15 distinct Bgt isolates to characterize their reaction pattern. As shown in Fig. 1 , CWI16926-4Y exhibited resistance to all 15 Bgt isolates. By contrast, TTD140, B05429, IW170, WE99 and XQ-0508 showed susceptibility to one or more Bgt isolates, indicating narrower resistance spectra. Notably, although both CWI16926-4Y and TRI1796 ( Pm68 ) showed broad resistance, CWI16926-4Y exhibited differential responses to at least six Bgt isolates. Micro-collinearity analysis of the target interval Micro-collinearity analysis was conducted between durum wheat cv. Svevo and several common wheat accessions using the flanking markers YTU2BS-045 and YTU2BS-062 of PmCWI16926 . The homologous intervals in reference genomes T. turgidum (Svevo v1), T. dicoccoides (WEWSeq v1), T. aestivum (10 + Genome Mace), T. aestivum (10 + Genome Jagger), T. aestivum (Fielder), and T. aestivum (IWGSC RefSeq v1.1), were 0.18 Mb, 0.18 Mb, 0.18 Mb, 0.17 Mb, 0.17 Mb and 0.18 Mb, and displayed high collinearity across different Triticum genomes (Fig. 5 ). Analysis and identification of candidate gene Within the fine-mapped interval of PmCWI16926 , three high-confidence genes were annotated, among which TRITD2Bv1G010130 and TRITD2Bv1G010140 were predicted to be involved in disease resistance or stress response pathways (Table 1 ). To explore their potential role, we monitored the infection process with Bgt E09. As shown in Fig. 6 a, conidial germination and penetration occurred at 0.5–12 hour post-inoculation (hpi), haustorium formation at 24 hpi, secondary penetration at 48 hpi, and microcolony development at 72 hpi. Clear differences in fungal growth were observed between resistant CWI16926-4Y and susceptible LDN after 24 hpi. Expression profiling revealed that both TRITD2BV1G010130 and TRITD2BV1G010140 were up-regulated on Bgt E09 infected CWI16926-4Y (Fig. 6 b), suggesting that the two genes may be induced by Bgt invasion. TRITD2Bv1G010130 was strongly induced in the susceptible genotype LDN, with transcript levels peaking close to 9-fold at 48–72 hpi, whereas expression in CWI16926-4Y remained consistently low (< 5). These results suggest that TRITD2Bv1G010130 is responsive to pathogen challenge; however, the stronger activation in the susceptible genotype raises the possibility that this gene may function as a susceptibility factor or be exploited by the pathogen to facilitate infection, rather than contributing to resistance. By contrast, TRITD2Bv1G010140 exhibited rapid and strong induction in CWI16926-4Y, reaching nearly 13-fold at 12 hpi, while remaining at basal levels (< 3) in LDN. Infection versus control comparisons further confirmed its pathogen-inducible nature, as transcripts were significantly upregulated in CWI16926-4Y at multiple time points (2, 4, 12, 24, and 72 hpi). This profile is consistent with the behavior of known resistance genes that are rapidly activated to trigger defense responses. Table 1 Annotation of high-confidence genes in the candidate interval of PmCWI16926 . No Gene Physical genomic location Functional annotation 1 TRITD2BV1G010120 Chr2B: 21815911..21822104 Exocyst complex component,putative 2 TRITD2BV1G010130 Chr2B: 21983992..21987186 Disease resistance protein/NB-ARC domain-containing protein 3 TRITD2BV1G010140 Chr2B: 21994145..21997849 Disease resistance protein (NBS-LRR class) family To verify sequence variation in the two candidate genes, full-length sequences of TRITD2Bv1G010130 and TRITD2Bv1G010140 were amplified from CWI16926-4Y, four susceptible accessions (LDN, Huixianhong (HXH), Kenong 199 (KN199), Tainong 18 (TN18)), and five resistant donors carrying reported 2BS loci ( Pm26 , pm42 , Pm68 , MlIW170 , and PmXQ - 0508 ). TRITD2Bv1G010130 showed identical sequences between CWI16926-4Y and the susceptible parent LDN, indicating that this gene is unlikely to underlie the resistance. In contrast, analysis of TRITD2Bv1G010140 revealed two isoforms in the T. durum cv. Svevo reference genome: TRITD2Bv1G010140 .1, consisting of four exons (1799 bp, 994 bp, 90 bp, 30 bp), and TRITD2Bv1G010140 .2, a single-exon transcript of 3357 bp (Fig. 7 a). Both isoforms encode NLR-type proteins with an NB-ARC domain and an R13L1/DRL21-like LRR repeat region. Sequence similarity between the two isoforms was 89.2%, with major differences attributed to InDel polymorphisms. Among them, TRITD2Bv1G010140.2 was successfully amplified and sequenced in this study (Fig. 7 b). Comparative analysis identified four unique SNPs in CWI16926-4Y at positions 1643, 1999, 2182, and 2614 bp, resulting in amino acid substitutions R→H, S→S, S→G, and D→N. These variants were absent in all susceptible accessions and resistant donors carrying other 2BS Pm loci, indicating that TRITD2Bv1G010140 in CWI16926-4Y represents a distinct haplotype potentially underlying the resistance. Furthermore, we developed a specific Kompetitive Allele-Specific PCR (KASP) marker KASP689-1 for TRITD2Bv1G010140 and found that it co-segregated with PmCWI16926 after genotyping the 2000 F 2:3 families of CWI16926-4Y×LDN (Fig. S1 ). By integrating sequence information, gene expression patterns after Bgt inoculation, and gene-specific marker analysis, TRITD2Bv1G010140 was identified as the key candidate gene for PmCWI16926. Analysis of potential value in MAS As a tetraploid wheat, cultivated emmer wheat CWI16926-4Y exhibits distinct agronomic characteristics compared with common wheat, including taller plant height, enhanced tillering capacity, and denser spikes featuring two-grained spikelets (Fig. 8 ). Although its yield potential is generally lower than that of common wheat, CWI16926-4Y demonstrates superior resistance to powdery mildew, underscoring its breeding value as a genetic donor for resistance improvement. To facilitate MAS for PmCWI16926 , four co-segregated markers tightly linked to this locus were validated across a diverse panel of 46 elite wheat cultivars/lines representing major wheat-growing regions in China. Among them, YTU2BS-011 and YTU2BS-072 consistently differentiated CWI16926-4Y from all susceptible genotypes. In addition, YTU2BS-045 and YTU2BS-072 were polymorphic in most accessions (95.65% and 78.26%, respectively) (Table S2 ). All markers produced clear polymorphic bands between CWI16926-4Y and majority of the tested genotypes, demonstrating their effectiveness as diagnostic tools for tracking PmCWI16926 in breeding programs. Discussion Exploiting emmer wheat for novel Pm gene discovery Wheat powdery mildew remains one of the most destructive foliar diseases of wheat, severely affecting both yield and quality 33 . The deployment of host resistance genes is regarded as the most effective, environmentally friendly, and sustainable approach for disease control, thereby reducing dependence on chemical fungicides 34 , 35 . Cultivated emmer wheat ( Triticum turgidum ssp. dicoccum ), one of the direct progenitors of common wheat, harbors abundant genetic diversity and serves as an invaluable reservoir of alleles for enhancing disease resistance and stress tolerance 18 . Its close genetic relationship with common wheat makes it particularly amenable to introgression, as desirable alleles from emmer wheat can be transferred into bread wheat backgrounds through relatively simple breeding schemes 24 . To date, several Pm genes have been reported from cultivated emmer wheat, including Pm4a and Pm50 on chromosome arm 2AL 36 , 37 , Pm49 / Ml5323 on chromosome arm 2BS 38 , and Pm71 on chromosome arm 6AS 23 . These findings underscore the potential of emmer wheat as a promising source for novel resistance loci. In this study, the cultivated emmer wheat accession CWI16926-4Y displayed high resistance to all 15 tested Bgt isolates at the seedling stage. Genetic analysis revealed that the resistance is controlled by a single dominant gene, designated PmCWI16926 , which was finely mapped to a 590 kb physical interval (21.70-22.29 Mb) on chromosome arm 2BS referred to the durum wheat cv. Svevo genome. Expression profiling further indicated that TRITD2Bv1G010140 is the most likely candidate resistance gene, highlighting both the potential of emmer wheat germplasm for broadening the genetic basis of powdery mildew resistance and the potential of PmCWI16926 for further wheat improvement. Comparison of PmCWI16926 with known powdery mildew resistance genes on 2BS The short arm of chromosome 2B harbors a well-documented cluster of powdery mildew resistance loci. To clarify whether PmCWI16926 corresponds to a previously reported gene or represents a novel locus, we compared its physical position (21.17–21.76 Mb in the WEW cv. Zavitan (v2.0) reference genome) with nine known loci mapped to 2BS. These include Pm68 (20.73–22.24 Mb) 30 , Pm49/Ml5323 (22.20–25.47 Mb) 38 , MlIW170 (26.41–27.25 Mb) 39 , PmL962 (7.03–23.09 Mb) 40 , pmWE99 (distal terminal–118.92 Mb) 41 , MlIW39 (21.95–22.24 Mb) 42 , and MlWE74 (25.48–26.28 Mb) 43 . Pm26 was originally derived from the wild emmer accession TTD140 and later identified to be the same gene as MlIW170 . Based on the uniform WEW Zavitan v2.0 reference genome positions, PmCWI16926 (21.17–21.76 Mb) can be clearly distinguished from eight loci, but overlaps partially with Pm68 (20.73–22.24 Mb). Through fine-scale genetic mapping, we delineated PmCWI16926 to a compact 0.59 Mb interval (21.70-22.29 Mb) in the durum wheat cv. Svevo reference genome, while Pm68 occupied a substantially larger 1.78 Mb genomic segment (21.59–23.37 Mb) on 2BS chromosome. Importantly, comparative phenotyping with 15 distinct Bgt isolates revealed distinct resistance spectra between PmCWI16926 and Pm68 (Fig. 1 ). Therefore, the genetic interval comparisons and phenotypic differences strongly indicate that PmCWI16926 is distinct from all nine previously reported loci on 2BS. This discovery expands the repertoire of resistance genes contributed by emmer wheat and highlights the evolutionary diversification of the 2BS resistance cluster. While several NLR-like candidates were identified within the fine-mapped interval, the precise molecular identity of PmCWI16926 remains to be determined. Nevertheless, its broad-spectrum and stable resistance makes it a valuable genetic resource for durable powdery mildew resistance breeding in wheat. Analysis and identification of pivotal candidate gene(s) Within the mapped interval of PmCWI16926 on chromosome arm 2BS, two candidate genes ( TRITD2Bv1G010130 and TRITD2Bv1G010140 ) were identified based on their annotation and expression profiles following Bgt inoculation. TRITD2Bv1G010130 showed transient induction after Bgt inoculation, but its stronger activation in the susceptible background suggests a possible role as a pathogen-responsive or susceptibility-related gene rather than a resistance determinant. In contrast, TRITD2Bv1G010140 was rapidly and strongly upregulated in CWI16926-4Y, peaking at nearly 13-fold at 12 hpi, whereas its expression remained basal in controls. This pathogen-inducible profile resembles known resistance genes that trigger defense upon infection. Sequence analysis further reinforced the candidacy of TRITD2Bv1G010140 . Four unique nucleotide polymorphisms were detected in CWI16926-4Y but not in any of the susceptible accessions (HXH, KN199, LDN, TN18) or in genotypes carrying previously reported 2BS resistance loci ( Pm26, pm42, Pm68, MlIW170 , and PmXQ-0508 ). These polymorphisms caused non-synonymous substitutions (R→H, S→S, S→G, D→N), generating a distinct haplotype that may underlie the resistance phenotype of CWI16926-4Y. Both TRITD2Bv1G010130 and TRITD2Bv1G010140 encode NLR-type proteins. Recent reports of cloned resistance genes such as Pm26 17 , Pm6SI 4 , and Yr87/Lr85 15 have highlighted the crucial role and unique mechanisms of non-canonical NLR proteins or NLR pairs in plant immunity. Collectively, these findings suggest that TRITD2Bv1G010140 is the most likely candidate for PmCWI16926 , with TRITD2Bv1G010130 potentially acting as a secondary or co-regulatory component. Functional validation, including transgenic overexpression or gene silencing, will be essential to confirm the causal role of TRITD2Bv1G010140 and/or TRITD2Bv1G010130 in conferring resistance. Development of molecular markers for wheat breeding The ultimate goal of identifying disease resistance genes is to enhance wheat production by providing genetic resources for disease management strategies 44 . To facilitate the practical use of PmCWI16926 in breeding, we developed four co-segregated markers and one gene-specific KASP marker, which can serve as robust tools for MAS breeding. Given that emmer wheat is one of the direct progenitors of common wheat and can easily hybridize to generate fertile offspring, PmCWI16926 holds considerable potential for transfer into common wheat to improve resistance. Moreover, further exploration of the line CWI16926-4Y may uncover additional favorable genes that could be harnessed for wheat genetic improvement. Importantly, the development of tightly linked molecular markers not only accelerates the deployment of PmCWI16926 in breeding but also provides a foundation for pyramiding this gene with other resistance loci to achieve durable and broad-spectrum resistance. Materials and methods Plant materials The cultivated emmer wheat accession CWI16926-4Y (CIMMYT, accession CWI16926-4Y, kindly provided by Dr. Hongxing Xu, Henan University), exhibited stable powdery mildew resistance throughout all growth stages, while durum wheat accession LDN was highly susceptible. CWI16926-4Y was crossed with LDN to generate F 1 , F 2 and F 2:3 generations for determining the inheritance of the resistance to powdery mildew in CWI16926-4Y and mapping the Pm gene(s) in CWI16926-4Y. The wheat cultivar TN18 served as a susceptible control during powdery mildew phenotyping. For comparative analyses, wheat accessions harboring Pm26 (TTD140), pm42 ( B05329 ) , Pm68 (TRI1796), MlIW170 (IW170), pmWE99 (WE99) and PmXQ-0508 (XQ-0508) were included to evaluate their responses to Bgt isolates. In addition, 46 susceptible wheat cultivars/breeding lines from different wheat producing regions in China were used to analyze the candidate gene and to evaluate the availability of the closely linked markers for MAS. Powdery mildew resistance phenotyping To evaluate adult-stage resistance to powdery mildew, CWI16926-4Y was inoculated with a mixture of Bgt isolates, together with naturally occurring Bgt isolates collected from wheat fields in northern China during the 2022–2024 cropping seasons. Field experiments were arranged in a randomized complete block design with three replications. Each plot consisted of three rows, 2 m in length, with 25 cm between rows. The highly susceptible cultivar TN18 was planted as spreader rows surrounding each plot to ensure uniform disease pressure. Disease reactions were assessed after heading using a 0–9 infection scale, in which scores of 0–4 denoted resistance and 5–9 denoted susceptibility 9 . Assessments were performed twice at one-week intervals to confirm consistency of the evaluations. Fifteen Bgt isolates with distinct virulence spectra were collected from different wheat-growing provinces in China and used for resistance evaluation under controlled conditions. All Bgt isolates were maintained and propagated on the susceptible TN18 seedlings. For inheritance analysis, the F 1 , F 2 and F 2:3 progenies of the cross CWI16926-4Y×LDN, together with their parents, were inoculated with Bgt isolate E09 for assessment of response to powdery mildew. In addition, 15 other Bgt isolates were employed to assess the seedling reaction patterns of CWI16926-4Y, LDN, and wheat genotypes with the reported Pm genes on chromosome arm 2BS, using TN18 as susceptible control. For each isolate, five seeds per genotype were sown in separate trays to prevent cross-contamination. Seedlings were grown under controlled conditions (24°C/14 h light and 18°C/10 h dark; relative humidity > 60%). At the two-leaf stage, plants were inoculated with freshly increased conidiospores on the TN18 seedlings, and then placed in the dark environment with relative humidity above 60% at 18℃ for 24 h, and then moved back to the greenhouse. After about 14 days post-inoculation (dpi), the phenotypes were divided into two categories, resistant (R, IT 0–2) and susceptible (S, IT 3–4). Assessments were performed in three independent replications. Genetic analysis The Bgt isolate E09 was employed to inoculate 10 F 1 plants, 207 F 2 individuals, and their corresponding F 2:3 families (20 seeds for each family), together with the parents CWI16926-4Y and LDN, at the seedling stage for genetic analysis. A goodness-of-fit analysis of these phenotype data was performed to determine the deviation of the observed phenotypic data and the theoretical segregation ratios by chi-squared (χ 2 ) test on SPSS 16.0 (SPSS Inc., Chicago, IL, USA). BSR-Seq and data analysis At 10 dpi with the Bgt isolate E09, equal amounts of leaf tissue from 30 homozygous resistant and 30 homozygous susceptible F 2:3 plants of CWI16926-4Y×LDN were pooled to construct R and S bulks for RNA isolation. The two contrasting bulks were subjected to BSR-Seq (Chengdu Tcuni Technology, Chengdu, China). Raw reads were trimmed to remove adapters and low-quality sequences using Trimmomatic v0.38 45 , and high-quality reads were aligned to the durum wheat cv. Svevo reference genome via STAR v2.5.1b 46 on the WheatOmics platform ( https://202.194.139.32 ). PCR duplicates were removed, and split reads spanning introns were retained for variant calling with GATK v4.2.3.0 47 . Variants were filtered using BCFtools v1.9 48 with thresholds of QUAL > 30 and DP ≥ 5. To identify candidate genomic intervals, single nucleotide polymorphisms (SNPs) and small InDels were analyzed using the varBScore algorithm 49 . Euclidean distance (ED) values of SNPs were further filtered with a quantile-based cutoff, retaining the top 1% to minimize noise from low-density regions 50 . Markers with varBScore > 2.0 × 10⁹ were considered significantly associated with the target locus. Additional association statistics, including the ΔSNP index, G′, and ED⁴, were calculated using a 10 Mb sliding window with 1 Mb increments. All analyses were performed using the WheatGmap online platform ( https://www.wheatmap.org ) 51 . Molecular marker development and gene mapping After delimiting the candidate interval(s), SSRs, InDels, and SNPs between R and S bulks within the target region(s) were hunted for designing polymorphic markers. SSR and InDel primers were designed using the software WEKits (v1.0.2) ( https://github.com/GP-sir/wekits/releases ) and Primer 5. To develop KASP markers, 200 bp sequences flanking each target SNP were extracted using Polymarker ( https://www.polymarker.info/ ). Newly developed and previously reported markers from homologous regions were screened for polymorphisms between the resistant and susceptible parents as well as the two contrasting bulks. Polymorphic markers were subsequently used to genotype the F 2:3 families derived from CWI16926-4Y×LDN for gene mapping. Markers co-segregating with PmCWI16926 were further applied to fine mapping with a larger population comprising 2000 F 2:3 families. All the technical procedures, including reaction mixture, DNA amplification, and polymorphic marker visualization, were performed according to Wu et al . (2022) with minor modifications. The markers used for mapping are listed in Table S1 . Linkage relationship between the markers and the Pm gene(s) in CWI16926-4Y was determined by Mapmaker 3.0b with a logarithm of odds (LOD) score of 3.0 as the threshold. Micro-collinearity analysis of the candidate interval Micro-collinearity analysis among different genomes was performed using the Triticeae-Gene Tribe database ( http://wheat.cau.edu.cn/TGT ). The nearest flanking markers of PmCWI16926 were used to extract homologous genomic regions from wild emmer v1.0, durum wheat cv. Svevo, hexaploid wheat cv. Chinese Spring v1.0, Fielder, Mace, and Jagger on WheatOmics 1.0 ( https://202.194.139.32 ). Sequences with the highest identity to the target genes were retained for comprehensive collinearity analysis. Gene annotations on the database EnsemblPlants ( http://plants.ensembl.org/index.html ) and UniProt ( https://www.uniprot.org ) were used to explore potential disease resistance genes with homologous regions. Candidate gene screening and quantitative real-time PCR (qRT-PCR) analysis After fine mapping, high-confidence genes within the region were annotated using the durum cv. Svevo reference genome on the WheatOmics platform ( http://202.194.139.32/ ). To examine their expression, qRT-PCR was conducted on leaf samples of CWI16926-4Y and LDN collected at 0, 0.5, 2, 4, 12, 24, 48 and 72 hpi with Bgt isolate E09. Similarly, expression patterns of the candidate genes between infected and non-infected controls were further analyzed. Total RNA was extracted from each sample using the TRIzol reagent (Invitrogen, Shanghai, China), and qRT-PCR assays were performed on the Bio-Rad CFX Connect Real-Time PCR detection system (Bio-Rad, Hercules, USA). Relative quantitation of the transcript levels was calculated using the 2 −∆∆CT method 52 , with the TaActin gene as normalization control. The real-time PCR reactions were performed in triplicate for each sample. Microscopic observation of fungal structures To monitor fungal invasion, seedlings of CWI16926-4Y and LDN were inoculated with Bgt isolate E09. Leaf segments (2 cm) were collected at 0, 4, 8, 12, 24, 48, and 72 hpi, fixed in Carnoy’s fixative (ethanol: glacial acetic acid, 3:1, v/v) for 24 h, and then stained with 0.6% (w/v) Coomassie brilliant blue solution for 5 min. Excess dye was removed by rinsing with distilled water, and samples were observed using an Axioscope 5 microscope (ZEISS, Oberkochen, Germany). Assessment of linked markers for MAS To assess their suitability of markers for MAS, closely linked markers were screened in CWI16926-4Y and 46 elite wheat cultivars/lines collected from major wheat-growing regions across China. Markers showing clear polymorphisms between CWI16926-4Y and the tested genotypes were considered effective for MAS and applicable for tracking the target Pm gene in diverse breeding backgrounds. Statistics and reproducibility The specific number (n) of replicates employed in each experiment was provided in the corresponding figure legends. In this study, student′s t -test was used for statistical analysis of intersample differences by Graphpad Prism5 software. A goodness-of-fit analysis of phenotype data of F 2 and F 2:3 generations was performed by chi-squared (χ 2 ) test were used for statistical analysis on SPSS 16.0 (SPSS Inc., Chicago, IL, USA). All quantitative data was expressed as the mean ± standard error (SE). The sample size of the qRT-PCR experiments in this article was set to three biological replicates. Statistically significant differences are denoted in graphs with * standing for P -value, where * refers to P < 0.05, ** to P < 0.01, *** to P < 0.001, and **** to P < 0.0001. Declarations Data availability All source data supporting the findings of this study are available within the paper and its supplementary information files. The genomic data for DNA sequence and gene analyzed in this study was obtained from the WheatOmics platform (https://202.194.139.32). All formatted data required to reproduce the results are available from the corresponding authors by request. Declaration of competing interest The authors declare that they have no conflict of interest. Author Contributions XZX: formal analysis, data curation, investigation, methodology; DML: formal analysis, investigation, methodology, visualization; FTW: formal analysis, investigation, validation; YLJ: data curation, formal analysis, investigation; KW: validation, methodology; NNS: validation, methodology; LZL: validation, methodology; JTL: validation, methodology; YTD: validation, methodology; TZ: validation, methodology; CL: funding acquisition, conceptualization, supervision; GHH: writing-original draft, funding acquisition, conceptualization, methodology, supervision; NNY: writing-review and editing, funding acquisition, conceptualization, supervision. 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MMAPPR: Mutation mapping analysis pipeline for pooled RNA seq. Genome Res . 23 , 687–697 (2013). Zhang, L. C. et al. WheatGmap: a comprehensive platform for wheat gene mapping and genomic studies. Mol Plant . 14 , 187–190 (2021). Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C (T) method. Nat. Protoc . 3 , 1101–1108 (2008). Additional Declarations There is NO Competing Interest. Supplementary Files TableS1.docx Table S1 TableS2.docx Table S2 RS1102.pdf Reporting Summary SupplementaryFigure.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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08:40:54","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123210,"visible":true,"origin":"","legend":"","description":"","filename":"COMMSBIO2591350structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/159d9e3e25dc2198be3138ef.xml"},{"id":93568270,"identity":"57bc4a80-347e-428e-a632-edcc25e98039","added_by":"auto","created_at":"2025-10-15 08:48:54","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138556,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/0c52e0b7265b019ae891a279.html"},{"id":93567939,"identity":"33ef069e-b3c7-4df4-9ada-0b34e478fef6","added_by":"auto","created_at":"2025-10-15 08:40:53","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":156547,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype identification against \u003cem\u003eBlumeria\u003c/em\u003e \u003cem\u003egraminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003eBgt\u003c/em\u003e) isolates. (a) Comparative phenotyping of CWI16926-4Y and wheat genotypes carrying known powdery mildew resistance genes on 2BS with 15 distinct \u003cem\u003eBgt\u003c/em\u003e isolates. (b) Responses of CWI16926-4Y, Langdon (LDN), and part of F\u003csub\u003e2:3\u003c/sub\u003e plants at ~14 d post inoculation with \u003cem\u003eBgt\u003c/em\u003e isolate E09. R, resistance phenotype; S, susceptible phenotype.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/c3bf624f2a52a96145070bc0.jpeg"},{"id":93568261,"identity":"d3caf3e3-671e-42f7-80ee-319b385b4290","added_by":"auto","created_at":"2025-10-15 08:48:53","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":105389,"visible":true,"origin":"","legend":"\u003cp\u003eBSR-Seq analysis of polymorphic SNPs between resistant (R) and susceptible (S) pools across 14 wheat chromosomes based on varBScore and Euclidean distance (ED) algorithm.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/b16afa5ef5e52ec6fede7326.jpeg"},{"id":93567953,"identity":"fb321fd9-1e90-43e4-9d05-85b946dca47b","added_by":"auto","created_at":"2025-10-15 08:40:54","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":159041,"visible":true,"origin":"","legend":"\u003cp\u003eLinkage map of \u003cem\u003ePmCWI16926\u003c/em\u003e using F\u003csub\u003e2:3\u003c/sub\u003e families from cross CWI16926-4Y×LDN. (a) Genetic mapping of \u003cem\u003ePmCWI16926\u003c/em\u003e on chromosome 2BS. (b) Initial mapping of \u003cem\u003ePmCWI16926\u003c/em\u003e. (c) The physical map of markers located on chromosome arm 2BS. (d) Gene annotation in the fine mapping interval.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/c70c2338c0d6cb4d9a327154.jpeg"},{"id":93567949,"identity":"8943dea1-d54c-4d0d-813a-95e12d98ecd4","added_by":"auto","created_at":"2025-10-15 08:40:54","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":122365,"visible":true,"origin":"","legend":"\u003cp\u003eBanding patterns of \u003cem\u003ePmCWI16926\u003c/em\u003e-linked markers \u003cem\u003eYTU2BS-008\u003c/em\u003e, \u003cem\u003eYTU2BS-015\u003c/em\u003e, \u003cem\u003eYTU2BS-045\u003c/em\u003e, and \u003cem\u003eYTU2BS-062 \u003c/em\u003efor\u003cem\u003e \u003c/em\u003eCWI16926-4Y, LDN and randomly selected F\u003csub\u003e2:3\u003c/sub\u003e families. Lane M, pUC18/\u003cem\u003eMsp\u003c/em\u003eⅠ; lane 1, CWI16926-4Y; lane 2, LDN; lanes 3-7, resistant F\u003csub\u003e2:3\u003c/sub\u003e families; lanes 8-12, susceptible F\u003csub\u003e2:3\u003c/sub\u003e families; lanes 13-17, heterozygous F\u003csub\u003e2:3\u003c/sub\u003e families. The polymorphic bands specific for CWI16926-4Y are indicated by red arrows.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/761f1e5fabe59dbd6501fd88.jpeg"},{"id":93568266,"identity":"b6039a31-1035-43fc-96c5-661efe284a8c","added_by":"auto","created_at":"2025-10-15 08:48:54","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":132828,"visible":true,"origin":"","legend":"\u003cp\u003eMicro-collinearity analysis of the candidate interval of \u003cem\u003ePmCWI16926 \u003c/em\u003ebetween wild emmer, durum and hexaploidy wheat. Orthologous genes are linked by lines.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/325e08be6215d3772c34c1c1.jpeg"},{"id":93568265,"identity":"f7ac1be0-8686-404c-a3a8-695f51cd06be","added_by":"auto","created_at":"2025-10-15 08:48:54","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":187724,"visible":true,"origin":"","legend":"\u003cp\u003eInfection process monitoring and gene expression analysis after \u003cem\u003eBgt\u003c/em\u003e isolate E09 invasion. (a) Infection process of \u003cem\u003eBgt \u003c/em\u003eisolates E09 on leaves of CWI16926-4Y and LDN. Leaf samples were taken at different hours post-inoculation (hpi) for Coomassie brilliant blue staining. Scale bar = 200 μm. (b) Expression patterns of candidate genes in CWI16926-4Y/LDN, and infected/uninfected CWI16926-4Y at different hpi.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/5855941702dee213d2371ffb.jpeg"},{"id":93567954,"identity":"a6bb5282-351c-46a3-b0a1-2ad255d84b99","added_by":"auto","created_at":"2025-10-15 08:40:54","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":142910,"visible":true,"origin":"","legend":"\u003cp\u003eStructure and variation of the candidate gene \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e. (a) Gene and protein structures. (b) Genetic divergence in nucleotide and amino acid sequences between CWI16926-4Y and other genotypes.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/dfe75721d478d88d0f24f6ab.jpeg"},{"id":93568267,"identity":"fbd989de-93ee-4663-af41-a81c4606b7f8","added_by":"auto","created_at":"2025-10-15 08:48:54","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":237346,"visible":true,"origin":"","legend":"\u003cp\u003eAgronomic traits of CWI16926-4Y and three wheat cultivars Huixianhong (HXH), Tainong 18 (TN18), and Kenong 199 (KN199).\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/7d4878ba90a4bd6d4b573b49.jpeg"},{"id":93571661,"identity":"04b3b9b5-917e-4786-8d27-1687091eae3a","added_by":"auto","created_at":"2025-10-15 09:04:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2466681,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/dd85af30-cca4-47c0-9907-192a3d193e69.pdf"},{"id":93568260,"identity":"5cf05d95-abdc-49d8-a785-5346ea2c7506","added_by":"auto","created_at":"2025-10-15 08:48:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20393,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/281cc965456df25c2d8d9493.docx"},{"id":93567946,"identity":"efc7a7d8-c718-49f9-8592-5bfd89bced20","added_by":"auto","created_at":"2025-10-15 08:40:53","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21574,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2\u003c/p\u003e","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/ec952596df70ecde70405f76.docx"},{"id":93567952,"identity":"005c0bbc-44ed-45e0-976c-ba2fdd4d9162","added_by":"auto","created_at":"2025-10-15 08:40:54","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1663627,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"RS1102.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/16a1ae09e4ea547887c5d7ca.pdf"},{"id":93568263,"identity":"5fb79510-04b6-406f-9bfc-a1379fe307b5","added_by":"auto","created_at":"2025-10-15 08:48:53","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":113865,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7647818/v1/1fa4a4d432eec94396423f64.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Fine mapping and genetic dissection of PmCWI16926, a broad-spectrum powdery mildew resistance gene from cultivated emmer wheat","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCommon wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L., 2n\u0026thinsp;=\u0026thinsp;6x\u0026thinsp;=\u0026thinsp;42, AABBDD) serves as a globally significant staple crop, providing a significant proportion of human caloric intake and essential nutrients\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, its productivity is continually threatened by a range of biotic and abiotic stresses. Among these, powdery mildew, caused by the biotrophic fungus \u003cem\u003eBlumeria graminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003eBgt\u003c/em\u003e), ranks as one of the most destructive foliar diseases worldwide\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In China, the disease has affected more than six million hectares annually over the past decade (NATESC). The continual emergence of virulent pathogen races, compounded by climate variability, has intensified the erosion of resistance, making the development of durable genetic protection a high priority\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGenetic resistance is widely recognized as the most economical, effective and environmentally sustainable approach to managing wheat powdery mildew\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. To date, more than 100 formally or temporarily designated powdery mildew (\u003cem\u003ePm\u003c/em\u003e) resistance genes/alleles have been identified in common wheat and related species, most of which encode coiled-coil nucleotide-binding leucine-rich repeat (CC-NBS-LRR) proteins mediating race-specific immune recognition\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Among of them, only a few \u003cem\u003ePm\u003c/em\u003e genes have been widely used in developing disease-resistant wheat varieties\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Yet several extensively deployed genes, such as \u003cem\u003ePm1\u003c/em\u003e, \u003cem\u003ePm2\u003c/em\u003e, \u003cem\u003ePm3\u003c/em\u003e, \u003cem\u003ePm8\u003c/em\u003e, and \u003cem\u003ePm17\u003c/em\u003e, have been overcome by rapidly evolving \u003cem\u003eBgt\u003c/em\u003e populations\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In addition, domestication and polyploidization have markedly narrowed the genetic base of modern cultivars\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, limiting the scope for discovering novel alleles within elite wheat. Notably, recent advances have uncovered non-classical immune modules from wheat relatives\u0026mdash;distinct from the canonical CNL architecture\u0026mdash;that confer effective powdery mildew resistance\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The discovery of such unconventional resistance frameworks not only expands the molecular diversity available for defense but also provides alternative mechanistic routes for designing durable, broad-spectrum resistance in breeding programs.\u003c/p\u003e\u003cp\u003eAncestral tetraploid wheats, which form part of the secondary gene pool, represent a valuable source of untapped genetic variation. Cultivated emmer (\u003cem\u003eT. dicoccum\u003c/em\u003e, 2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;28, AABB) and its wild progenitor \u003cem\u003eT. dicoccoides\u003c/em\u003e originated in the Fertile Crescent and adapted to diverse environments under long-term exposure to complex biotic and abiotic pressures\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Compared with more distantly related \u003cem\u003eTriticeae\u003c/em\u003e donors, genes from tetraploid progenitors are more easily introgressed into hexaploid wheat and generally incur fewer drawbacks from alien translocations, such as linkage drag or poor genetic compensation\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Several \u003cem\u003ePm\u003c/em\u003e loci, including \u003cem\u003ePm16\u003c/em\u003e, \u003cem\u003ePm26\u003c/em\u003e, \u003cem\u003ePm30\u003c/em\u003e, \u003cem\u003ePm36\u003c/em\u003e, \u003cem\u003ePm41\u003c/em\u003e, \u003cem\u003ePm42\u003c/em\u003e, \u003cem\u003ePm64\u003c/em\u003e, \u003cem\u003ePm69\u003c/em\u003e, and \u003cem\u003ePm71\u003c/em\u003e, trace back to tetraploid ancestors, many conferring resistance profiles distinct from those found in cultivated wheat\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe development of high-quality reference genomes for wheat and its relatives, together with advances in high-throughput sequencing technologies, has transformed the pace and precision of gene discovery, fine mapping, and cloning\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Bulk segregant RNA-Seq integrated the advantage of bulked segregant analysis (BSA) and high-throughput transcriptomic profiling, enabling rapid delimitation of causal intervals and efficient identification of candidate genes\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We have previously used BSR-Seq to identify and characterize multiple \u003cem\u003ePm\u003c/em\u003e loci, including \u003cem\u003ePmLS5082\u003c/em\u003e and \u003cem\u003ePmXQ-0508\u003c/em\u003e from wheat breeding line LS5082 and XQ-0508, respectively\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, demonstrating its versatility for exploiting diverse resistance sources. Crucially, coupling gene discovery with the development of tightly linked, breeder-friendly markers provide an essential bridge between molecular research and breeding deployment.\u003c/p\u003e\u003cp\u003eIn this study, we investigated the cultivated emmer accession CWI16926-4Y, which has consistently exhibited high-level and broad-spectrum resistance to powdery mildew under multi-year field conditions. By integrating BSR-Seq with classical genetic analysis, we aimed to (i) finely map the powdery mildew resistance gene(s) in CWI16926-4Y, (ii) dissect its genetic basis and inheritance pattern, (iii) identify and characterize potential candidate gene(s) underlying the resistance, and (iv) develop molecular markers for marker assisted selection (MAS) in wheat breeding. By sourcing resistance from an ancestral tetraploid donor with minimal linkage drag, this work expands the repertoire of deployable \u003cem\u003ePm\u003c/em\u003e loci and provides practical tools for precise gene pyramiding and stable field performance.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eInheritance of powdery mildew resistance in CWI16926-4Y\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eIn field evaluations at the adult plant stage, CWI16926-4Y showed complete resistance (infection type, IT\u0026thinsp;=\u0026thinsp;0) to a mixture of inoculated \u003cem\u003eBgt\u003c/em\u003e isolates and naturally occurring isolates in northern China. At the seedling stage, CWI16926-4Y exhibited compete seedling resistance to all 15 tested \u003cem\u003eBgt\u003c/em\u003e isolates, with IT 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To dissect the genetic basis of this resistance, \u003cem\u003eBgt\u003c/em\u003e isolate E09 was selected for controlled inoculation. All ten tested plants of the resistant parent CWI16926-4Y remained immune (IT\u0026thinsp;=\u0026thinsp;0), whereas all ten plants of the susceptible parent Langdon (LDN) were highly susceptible (IT\u0026thinsp;=\u0026thinsp;4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The F₁ progeny from the CWI16926-4Y \u0026times; LDN cross were all resistant (IT\u0026thinsp;=\u0026thinsp;0), indicating that the resistance to E09 is conferred by a dominant gene. Phenotypic assessment of 207 F₂ individuals segregated into 154 resistant and 53 susceptible plants, fitting the expected 3:1 ratio for a single dominant locus (χ\u0026sup2; = 0.04, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.84). Consistent results were obtained in 198 F\u003csub\u003e2:3\u003c/sub\u003e families, which segregated into 48 homozygous resistant, 103 segregating, and 47 homozygous susceptible lines, consistent with the expected 1:2:1 ratio (χ\u0026sup2; = 0.33, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.84). These results demonstrate that powdery mildew resistance to isolate E09 in CWI16926-4Y is governed by a single dominant gene, which we tentatively designated \u003cem\u003ePmCWI16926\u003c/em\u003e. To further evaluate its spectrum, 198 F\u003csub\u003e2:3\u003c/sub\u003e families were inoculated with ten additional \u003cem\u003eBgt\u003c/em\u003e isolates. Segregation patterns for each isolate mirrored those observed with E09, suggesting that \u003cem\u003ePmCWI16926\u003c/em\u003e confers broad-spectrum resistance across diverse \u003cem\u003eBgt\u003c/em\u003e isolates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSNP calling and screening of candidate intervals\u003c/h3\u003e\n\u003cp\u003eBSR-Seq analysis generated 20.67 and 22.15 Gb clean data from the R and S bulks, respectively, with Q30 percentages above 94.5% and 93.6%. Following sequence alignment to the reference genome, a total of 55,524 high-quality SNPs were identified between R and S bulks, distributed across all the 14 cultivated emmer wheat chromosomes (AA and BB genomes) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Of these, 5798 (accounting for 10.4%) SNPs were located on the chromosome arm 2B. According to the △SNP index value, one estimated candidate region was detected, located at the end of chromosome arm 2BS (13.0-25.3 Mb). Using ED analysis, an evident allelic difference between the R and S bulks on the similar interval was also detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), re-validating the candidate interval of \u003cem\u003ePmCWI16926.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular mapping of\u003c/b\u003e \u003cb\u003ePmCWI16926\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo map \u003cem\u003ePmCWI16926\u003c/em\u003e, 26 reported markers and 87 newly developed markers within the candidate interval were tested for polymorphism. Thirteen newly developed simple sequence repeats (SSRs) and insertions/deletions (InDels) markers in candidate interval were linked to \u003cem\u003ePmCWI16926\u003c/em\u003e, after screening for polymorphism between the parents and two bulks and subsequent genotyping on 202 F\u003csub\u003e2:3\u003c/sub\u003e families (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Using these polymorphic markers, a high-density linkage map was constructed, placing \u003cem\u003ePmCWI16926\u003c/em\u003e between markers \u003cem\u003eYTU2BS-045\u003c/em\u003e and \u003cem\u003eYTU2BS-058\u003c/em\u003e at genetic distances of 1.3 and 0.2 cM, respectively. Furthermore, genotyping of 2000 F\u003csub\u003e2:3\u003c/sub\u003e families refined the interval to 590 kb (Chr2B_21700330-Chr2B_22293417) in the durum cv. Svevo genome, flanked by the markers \u003cem\u003eYTU2BS-045\u003c/em\u003e and YTU\u003cem\u003e2BS-062\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparison of\u003c/b\u003e \u003cb\u003ePmCWI16926\u003c/b\u003e \u003cb\u003ewith known\u003c/b\u003e \u003cb\u003ePm\u003c/b\u003e \u003cb\u003egenes on chromosome 2BS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the relationship between the \u003cem\u003ePm\u003c/em\u003e gene(s) in CWI16926-4Y and previously reported \u003cem\u003ePm\u003c/em\u003e genes located in similar genomic intervals, CWI16926-4Y and wheat genotypes carrying \u003cem\u003ePm26\u003c/em\u003e (TTD140), \u003cem\u003epm42\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eB05429\u003cb\u003e)\u003c/b\u003e, \u003cem\u003ePm68\u003c/em\u003e (TRI1796), \u003cem\u003eMlIW170\u003c/em\u003e (IW170), \u003cem\u003epmWE99\u003c/em\u003e (WE99) and \u003cem\u003ePmXQ-0508\u003c/em\u003e (XQ-0508) were inoculated with 15 distinct \u003cem\u003eBgt\u003c/em\u003e isolates to characterize their reaction pattern. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, CWI16926-4Y exhibited resistance to all 15 \u003cem\u003eBgt\u003c/em\u003e isolates. By contrast, TTD140, B05429, IW170, WE99 and XQ-0508 showed susceptibility to one or more \u003cem\u003eBgt\u003c/em\u003e isolates, indicating narrower resistance spectra. Notably, although both CWI16926-4Y and TRI1796 (\u003cem\u003ePm68\u003c/em\u003e) showed broad resistance, CWI16926-4Y exhibited differential responses to at least six \u003cem\u003eBgt\u003c/em\u003e isolates.\u003c/p\u003e\n\u003ch3\u003eMicro-collinearity analysis of the target interval\u003c/h3\u003e\n\u003cp\u003eMicro-collinearity analysis was conducted between durum wheat cv. Svevo and several common wheat accessions using the flanking markers \u003cem\u003eYTU2BS-045\u003c/em\u003e and \u003cem\u003eYTU2BS-062\u003c/em\u003e of \u003cem\u003ePmCWI16926\u003c/em\u003e. The homologous intervals in reference genomes \u003cem\u003eT. turgidum\u003c/em\u003e (Svevo v1), \u003cem\u003eT. dicoccoides\u003c/em\u003e (WEWSeq v1), \u003cem\u003eT. aestivum\u003c/em\u003e (10\u0026thinsp;+\u0026thinsp;Genome Mace), \u003cem\u003eT. aestivum\u003c/em\u003e (10\u0026thinsp;+\u0026thinsp;Genome Jagger), \u003cem\u003eT. aestivum\u003c/em\u003e (Fielder), and \u003cem\u003eT. aestivum\u003c/em\u003e (IWGSC RefSeq v1.1), were 0.18 Mb, 0.18 Mb, 0.18 Mb, 0.17 Mb, 0.17 Mb and 0.18 Mb, and displayed high collinearity across different \u003cem\u003eTriticum\u003c/em\u003e genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eAnalysis and identification of candidate gene\u003c/h3\u003e\n\u003cp\u003eWithin the fine-mapped interval of \u003cem\u003ePmCWI16926\u003c/em\u003e, three high-confidence genes were annotated, among which \u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e and \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e were predicted to be involved in disease resistance or stress response pathways (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To explore their potential role, we monitored the infection process with \u003cem\u003eBgt\u003c/em\u003e E09. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, conidial germination and penetration occurred at 0.5\u0026ndash;12 hour post-inoculation (hpi), haustorium formation at 24 hpi, secondary penetration at 48 hpi, and microcolony development at 72 hpi. Clear differences in fungal growth were observed between resistant CWI16926-4Y and susceptible LDN after 24 hpi. Expression profiling revealed that both \u003cem\u003eTRITD2BV1G010130\u003c/em\u003e and \u003cem\u003eTRITD2BV1G010140\u003c/em\u003e were up-regulated on \u003cem\u003eBgt\u003c/em\u003e E09 infected CWI16926-4Y (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), suggesting that the two genes may be induced by \u003cem\u003eBgt\u003c/em\u003e invasion. \u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e was strongly induced in the susceptible genotype LDN, with transcript levels peaking close to 9-fold at 48\u0026ndash;72 hpi, whereas expression in CWI16926-4Y remained consistently low (\u0026lt;\u0026thinsp;5). These results suggest that \u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e is responsive to pathogen challenge; however, the stronger activation in the susceptible genotype raises the possibility that this gene may function as a susceptibility factor or be exploited by the pathogen to facilitate infection, rather than contributing to resistance. By contrast, \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e exhibited rapid and strong induction in CWI16926-4Y, reaching nearly 13-fold at 12 hpi, while remaining at basal levels (\u0026lt;\u0026thinsp;3) in LDN. Infection versus control comparisons further confirmed its pathogen-inducible nature, as transcripts were significantly upregulated in CWI16926-4Y at multiple time points (2, 4, 12, 24, and 72 hpi). This profile is consistent with the behavior of known resistance genes that are rapidly activated to trigger defense responses.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAnnotation of high-confidence genes in the candidate interval of \u003cem\u003ePmCWI16926\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhysical genomic location\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFunctional annotation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTRITD2BV1G010120\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChr2B: 21815911..21822104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExocyst complex component,putative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTRITD2BV1G010130\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChr2B: 21983992..21987186\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDisease resistance protein/NB-ARC domain-containing protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eTRITD2BV1G010140\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChr2B: 21994145..21997849\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDisease resistance protein (NBS-LRR class) family\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo verify sequence variation in the two candidate genes, full-length sequences of \u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e and \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e were amplified from CWI16926-4Y, four susceptible accessions (LDN, Huixianhong (HXH), Kenong 199 (KN199), Tainong 18 (TN18)), and five resistant donors carrying reported 2BS loci (\u003cem\u003ePm26\u003c/em\u003e, \u003cem\u003epm42\u003c/em\u003e, \u003cem\u003ePm68\u003c/em\u003e, \u003cem\u003eMlIW170\u003c/em\u003e, and \u003cem\u003ePmXQ\u003c/em\u003e-\u003cem\u003e0508\u003c/em\u003e). \u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e showed identical sequences between CWI16926-4Y and the susceptible parent LDN, indicating that this gene is unlikely to underlie the resistance. In contrast, analysis of \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e revealed two isoforms in the \u003cem\u003eT. durum\u003c/em\u003e cv. Svevo reference genome: \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e.1, consisting of four exons (1799 bp, 994 bp, 90 bp, 30 bp), and \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e.2, a single-exon transcript of 3357 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Both isoforms encode NLR-type proteins with an NB-ARC domain and an R13L1/DRL21-like LRR repeat region. Sequence similarity between the two isoforms was 89.2%, with major differences attributed to InDel polymorphisms. Among them, \u003cem\u003eTRITD2Bv1G010140.2\u003c/em\u003e was successfully amplified and sequenced in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Comparative analysis identified four unique SNPs in CWI16926-4Y at positions 1643, 1999, 2182, and 2614 bp, resulting in amino acid substitutions R\u0026rarr;H, S\u0026rarr;S, S\u0026rarr;G, and D\u0026rarr;N. These variants were absent in all susceptible accessions and resistant donors carrying other 2BS \u003cem\u003ePm\u003c/em\u003e loci, indicating that \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e in CWI16926-4Y represents a distinct haplotype potentially underlying the resistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, we developed a specific Kompetitive Allele-Specific PCR (KASP) marker \u003cem\u003eKASP689-1\u003c/em\u003e for \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e and found that it co-segregated with \u003cem\u003ePmCWI16926\u003c/em\u003e after genotyping the 2000 F\u003csub\u003e2:3\u003c/sub\u003e families of CWI16926-4Y\u0026times;LDN (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). By integrating sequence information, gene expression patterns after \u003cem\u003eBgt\u003c/em\u003e inoculation, and gene-specific marker analysis, \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e was identified as the key candidate gene for \u003cem\u003ePmCWI16926.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eAnalysis of potential value in MAS\u003c/h3\u003e\n\u003cp\u003eAs a tetraploid wheat, cultivated emmer wheat CWI16926-4Y exhibits distinct agronomic characteristics compared with common wheat, including taller plant height, enhanced tillering capacity, and denser spikes featuring two-grained spikelets (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Although its yield potential is generally lower than that of common wheat, CWI16926-4Y demonstrates superior resistance to powdery mildew, underscoring its breeding value as a genetic donor for resistance improvement.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo facilitate MAS for \u003cem\u003ePmCWI16926\u003c/em\u003e, four co-segregated markers tightly linked to this locus were validated across a diverse panel of 46 elite wheat cultivars/lines representing major wheat-growing regions in China. Among them, \u003cem\u003eYTU2BS-011\u003c/em\u003e and \u003cem\u003eYTU2BS-072\u003c/em\u003e consistently differentiated CWI16926-4Y from all susceptible genotypes. In addition, \u003cem\u003eYTU2BS-045\u003c/em\u003e and \u003cem\u003eYTU2BS-072\u003c/em\u003e were polymorphic in most accessions (95.65% and 78.26%, respectively) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). All markers produced clear polymorphic bands between CWI16926-4Y and majority of the tested genotypes, demonstrating their effectiveness as diagnostic tools for tracking \u003cem\u003ePmCWI16926\u003c/em\u003e in breeding programs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eExploiting emmer wheat for novel\u003c/b\u003e \u003cb\u003ePm\u003c/b\u003e \u003cb\u003egene discovery\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWheat powdery mildew remains one of the most destructive foliar diseases of wheat, severely affecting both yield and quality\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The deployment of host resistance genes is regarded as the most effective, environmentally friendly, and sustainable approach for disease control, thereby reducing dependence on chemical fungicides\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Cultivated emmer wheat (\u003cem\u003eTriticum turgidum\u003c/em\u003e ssp. \u003cem\u003edicoccum\u003c/em\u003e), one of the direct progenitors of common wheat, harbors abundant genetic diversity and serves as an invaluable reservoir of alleles for enhancing disease resistance and stress tolerance\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Its close genetic relationship with common wheat makes it particularly amenable to introgression, as desirable alleles from emmer wheat can be transferred into bread wheat backgrounds through relatively simple breeding schemes\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To date, several \u003cem\u003ePm\u003c/em\u003e genes have been reported from cultivated emmer wheat, including \u003cem\u003ePm4a\u003c/em\u003e and \u003cem\u003ePm50\u003c/em\u003e on chromosome arm 2AL\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ePm49\u003c/em\u003e\u003cb\u003e/\u003c/b\u003e\u003cem\u003eMl5323\u003c/em\u003e on chromosome arm 2BS\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003ePm71\u003c/em\u003e on chromosome arm 6AS\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These findings underscore the potential of emmer wheat as a promising source for novel resistance loci.\u003c/p\u003e\u003cp\u003eIn this study, the cultivated emmer wheat accession CWI16926-4Y displayed high resistance to all 15 tested \u003cem\u003eBgt\u003c/em\u003e isolates at the seedling stage. Genetic analysis revealed that the resistance is controlled by a single dominant gene, designated \u003cem\u003ePmCWI16926\u003c/em\u003e, which was finely mapped to a 590 kb physical interval (21.70-22.29 Mb) on chromosome arm 2BS referred to the durum wheat cv. Svevo genome. Expression profiling further indicated that \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e is the most likely candidate resistance gene, highlighting both the potential of emmer wheat germplasm for broadening the genetic basis of powdery mildew resistance and the potential of \u003cem\u003ePmCWI16926\u003c/em\u003e for further wheat improvement.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparison of\u003c/b\u003e \u003cb\u003ePmCWI16926\u003c/b\u003e \u003cb\u003ewith known powdery mildew resistance genes on 2BS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe short arm of chromosome 2B harbors a well-documented cluster of powdery mildew resistance loci. To clarify whether \u003cem\u003ePmCWI16926\u003c/em\u003e corresponds to a previously reported gene or represents a novel locus, we compared its physical position (21.17\u0026ndash;21.76 Mb in the WEW cv. Zavitan (v2.0) reference genome) with nine known loci mapped to 2BS. These include \u003cem\u003ePm68\u003c/em\u003e (20.73\u0026ndash;22.24 Mb)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ePm49/Ml5323\u003c/em\u003e (22.20\u0026ndash;25.47 Mb)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eMlIW170\u003c/em\u003e (26.41\u0026ndash;27.25 Mb)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ePmL962\u003c/em\u003e (7.03\u0026ndash;23.09 Mb)\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003epmWE99\u003c/em\u003e (distal terminal\u0026ndash;118.92 Mb)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eMlIW39\u003c/em\u003e (21.95\u0026ndash;22.24 Mb)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eMlWE74\u003c/em\u003e (25.48\u0026ndash;26.28 Mb)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePm26\u003c/em\u003e was originally derived from the wild emmer accession TTD140 and later identified to be the same gene as \u003cem\u003eMlIW170\u003c/em\u003e. Based on the uniform WEW Zavitan v2.0 reference genome positions, \u003cem\u003ePmCWI16926\u003c/em\u003e (21.17\u0026ndash;21.76 Mb) can be clearly distinguished from eight loci, but overlaps partially with \u003cem\u003ePm68\u003c/em\u003e (20.73\u0026ndash;22.24 Mb). Through fine-scale genetic mapping, we delineated \u003cem\u003ePmCWI16926\u003c/em\u003e to a compact 0.59 Mb interval (21.70-22.29 Mb) in the durum wheat cv. Svevo reference genome, while \u003cem\u003ePm68\u003c/em\u003e occupied a substantially larger 1.78 Mb genomic segment (21.59\u0026ndash;23.37 Mb) on 2BS chromosome. Importantly, comparative phenotyping with 15 distinct \u003cem\u003eBgt\u003c/em\u003e isolates revealed distinct resistance spectra between \u003cem\u003ePmCWI16926\u003c/em\u003e and \u003cem\u003ePm68\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, the genetic interval comparisons and phenotypic differences strongly indicate that \u003cem\u003ePmCWI16926\u003c/em\u003e is distinct from all nine previously reported loci on 2BS. This discovery expands the repertoire of resistance genes contributed by emmer wheat and highlights the evolutionary diversification of the 2BS resistance cluster. While several NLR-like candidates were identified within the fine-mapped interval, the precise molecular identity of \u003cem\u003ePmCWI16926\u003c/em\u003e remains to be determined. Nevertheless, its broad-spectrum and stable resistance makes it a valuable genetic resource for durable powdery mildew resistance breeding in wheat.\u003c/p\u003e\n\u003ch3\u003eAnalysis and identification of pivotal candidate gene(s)\u003c/h3\u003e\n\u003cp\u003eWithin the mapped interval of \u003cem\u003ePmCWI16926\u003c/em\u003e on chromosome arm 2BS, two candidate genes (\u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e and \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e) were identified based on their annotation and expression profiles following \u003cem\u003eBgt\u003c/em\u003e inoculation. \u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e showed transient induction after \u003cem\u003eBgt\u003c/em\u003e inoculation, but its stronger activation in the susceptible background suggests a possible role as a pathogen-responsive or susceptibility-related gene rather than a resistance determinant. In contrast, \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e was rapidly and strongly upregulated in CWI16926-4Y, peaking at nearly 13-fold at 12 hpi, whereas its expression remained basal in controls. This pathogen-inducible profile resembles known resistance genes that trigger defense upon infection. Sequence analysis further reinforced the candidacy of \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e. Four unique nucleotide polymorphisms were detected in CWI16926-4Y but not in any of the susceptible accessions (HXH, KN199, LDN, TN18) or in genotypes carrying previously reported 2BS resistance loci (\u003cem\u003ePm26, pm42, Pm68, MlIW170\u003c/em\u003e, and \u003cem\u003ePmXQ-0508\u003c/em\u003e). These polymorphisms caused non-synonymous substitutions (R\u0026rarr;H, S\u0026rarr;S, S\u0026rarr;G, D\u0026rarr;N), generating a distinct haplotype that may underlie the resistance phenotype of CWI16926-4Y.\u003c/p\u003e\u003cp\u003eBoth \u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e and \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e encode NLR-type proteins. Recent reports of cloned resistance genes such as \u003cem\u003ePm26\u003c/em\u003e\u003csup\u003e17\u003c/sup\u003e, \u003cem\u003ePm6SI\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eYr87/Lr85\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003e have highlighted the crucial role and unique mechanisms of non-canonical NLR proteins or NLR pairs in plant immunity. Collectively, these findings suggest that \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e is the most likely candidate for \u003cem\u003ePmCWI16926\u003c/em\u003e, with \u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e potentially acting as a secondary or co-regulatory component. Functional validation, including transgenic overexpression or gene silencing, will be essential to confirm the causal role of \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e and/or \u003cem\u003eTRITD2Bv1G010130\u003c/em\u003e in conferring resistance.\u003c/p\u003e\n\u003ch3\u003eDevelopment of molecular markers for wheat breeding\u003c/h3\u003e\n\u003cp\u003eThe ultimate goal of identifying disease resistance genes is to enhance wheat production by providing genetic resources for disease management strategies\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. To facilitate the practical use of \u003cem\u003ePmCWI16926\u003c/em\u003e in breeding, we developed four co-segregated markers and one gene-specific KASP marker, which can serve as robust tools for MAS breeding. Given that emmer wheat is one of the direct progenitors of common wheat and can easily hybridize to generate fertile offspring, \u003cem\u003ePmCWI16926\u003c/em\u003e holds considerable potential for transfer into common wheat to improve resistance. Moreover, further exploration of the line CWI16926-4Y may uncover additional favorable genes that could be harnessed for wheat genetic improvement. Importantly, the development of tightly linked molecular markers not only accelerates the deployment of \u003cem\u003ePmCWI16926\u003c/em\u003e in breeding but also provides a foundation for pyramiding this gene with other resistance loci to achieve durable and broad-spectrum resistance.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials\u003c/h2\u003e\u003cp\u003eThe cultivated emmer wheat accession CWI16926-4Y (CIMMYT, accession CWI16926-4Y, kindly provided by Dr. Hongxing Xu, Henan University), exhibited stable powdery mildew resistance throughout all growth stages, while durum wheat accession LDN was highly susceptible. CWI16926-4Y was crossed with LDN to generate F\u003csub\u003e1\u003c/sub\u003e, F\u003csub\u003e2\u003c/sub\u003e and F\u003csub\u003e2:3\u003c/sub\u003e generations for determining the inheritance of the resistance to powdery mildew in CWI16926-4Y and mapping the \u003cem\u003ePm\u003c/em\u003e gene(s) in CWI16926-4Y. The wheat cultivar TN18 served as a susceptible control during powdery mildew phenotyping. For comparative analyses, wheat accessions harboring \u003cem\u003ePm26\u003c/em\u003e (TTD140), \u003cem\u003epm42\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eB05329\u003cb\u003e)\u003c/b\u003e, \u003cem\u003ePm68\u003c/em\u003e (TRI1796), \u003cem\u003eMlIW170\u003c/em\u003e (IW170), \u003cem\u003epmWE99\u003c/em\u003e (WE99) and \u003cem\u003ePmXQ-0508\u003c/em\u003e (XQ-0508) were included to evaluate their responses to \u003cem\u003eBgt\u003c/em\u003e isolates. In addition, 46 susceptible wheat cultivars/breeding lines from different wheat producing regions in China were used to analyze the candidate gene and to evaluate the availability of the closely linked markers for MAS.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePowdery mildew resistance phenotyping\u003c/h2\u003e\u003cp\u003eTo evaluate adult-stage resistance to powdery mildew, CWI16926-4Y was inoculated with a mixture of \u003cem\u003eBgt\u003c/em\u003e isolates, together with naturally occurring \u003cem\u003eBgt\u003c/em\u003e isolates collected from wheat fields in northern China during the 2022\u0026ndash;2024 cropping seasons. Field experiments were arranged in a randomized complete block design with three replications. Each plot consisted of three rows, 2 m in length, with 25 cm between rows. The highly susceptible cultivar TN18 was planted as spreader rows surrounding each plot to ensure uniform disease pressure. Disease reactions were assessed after heading using a 0\u0026ndash;9 infection scale, in which scores of 0\u0026ndash;4 denoted resistance and 5\u0026ndash;9 denoted susceptibility\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Assessments were performed twice at one-week intervals to confirm consistency of the evaluations.\u003c/p\u003e\u003cp\u003eFifteen \u003cem\u003eBgt\u003c/em\u003e isolates with distinct virulence spectra were collected from different wheat-growing provinces in China and used for resistance evaluation under controlled conditions. All \u003cem\u003eBgt\u003c/em\u003e isolates were maintained and propagated on the susceptible TN18 seedlings. For inheritance analysis, the F\u003csub\u003e1\u003c/sub\u003e, F\u003csub\u003e2\u003c/sub\u003e and F\u003csub\u003e2:3\u003c/sub\u003e progenies of the cross CWI16926-4Y\u0026times;LDN, together with their parents, were inoculated with \u003cem\u003eBgt\u003c/em\u003e isolate E09 for assessment of response to powdery mildew. In addition, 15 other \u003cem\u003eBgt\u003c/em\u003e isolates were employed to assess the seedling reaction patterns of CWI16926-4Y, LDN, and wheat genotypes with the reported \u003cem\u003ePm\u003c/em\u003e genes on chromosome arm 2BS, using TN18 as susceptible control. For each isolate, five seeds per genotype were sown in separate trays to prevent cross-contamination. Seedlings were grown under controlled conditions (24\u0026deg;C/14 h light and 18\u0026deg;C/10 h dark; relative humidity\u0026thinsp;\u0026gt;\u0026thinsp;60%). At the two-leaf stage, plants were inoculated with freshly increased conidiospores on the TN18 seedlings, and then placed in the dark environment with relative humidity above 60% at 18℃ for 24 h, and then moved back to the greenhouse. After about 14 days post-inoculation (dpi), the phenotypes were divided into two categories, resistant (R, IT 0\u0026ndash;2) and susceptible (S, IT 3\u0026ndash;4). Assessments were performed in three independent replications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGenetic analysis\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eBgt\u003c/em\u003e isolate E09 was employed to inoculate 10 F\u003csub\u003e1\u003c/sub\u003e plants, 207 F\u003csub\u003e2\u003c/sub\u003e individuals, and their corresponding F\u003csub\u003e2:3\u003c/sub\u003e families (20 seeds for each family), together with the parents CWI16926-4Y and LDN, at the seedling stage for genetic analysis. A goodness-of-fit analysis of these phenotype data was performed to determine the deviation of the observed phenotypic data and the theoretical segregation ratios by chi-squared (χ\u003csup\u003e2\u003c/sup\u003e) test on SPSS 16.0 (SPSS Inc., Chicago, IL, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eBSR-Seq and data analysis\u003c/h2\u003e\u003cp\u003eAt 10 dpi with the \u003cem\u003eBgt\u003c/em\u003e isolate E09, equal amounts of leaf tissue from 30 homozygous resistant and 30 homozygous susceptible F\u003csub\u003e2:3\u003c/sub\u003e plants of CWI16926-4Y\u0026times;LDN were pooled to construct R and S bulks for RNA isolation. The two contrasting bulks were subjected to BSR-Seq (Chengdu Tcuni Technology, Chengdu, China). Raw reads were trimmed to remove adapters and low-quality sequences using Trimmomatic v0.38\u003csup\u003e45\u003c/sup\u003e, and high-quality reads were aligned to the durum wheat cv. Svevo reference genome via STAR v2.5.1b\u003csup\u003e46\u003c/sup\u003e on the WheatOmics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://202.194.139.32\u003c/span\u003e\u003cspan address=\"https://202.194.139.32\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). PCR duplicates were removed, and split reads spanning introns were retained for variant calling with GATK v4.2.3.0\u003csup\u003e47\u003c/sup\u003e. Variants were filtered using BCFtools v1.9\u003csup\u003e48\u003c/sup\u003e with thresholds of QUAL\u0026thinsp;\u0026gt;\u0026thinsp;30 and DP\u0026thinsp;\u0026ge;\u0026thinsp;5. To identify candidate genomic intervals, single nucleotide polymorphisms (SNPs) and small InDels were analyzed using the varBScore algorithm\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Euclidean distance (ED) values of SNPs were further filtered with a quantile-based cutoff, retaining the top 1% to minimize noise from low-density regions\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Markers with varBScore\u0026thinsp;\u0026gt;\u0026thinsp;2.0 \u0026times; 10⁹ were considered significantly associated with the target locus. Additional association statistics, including the ΔSNP index, G\u0026prime;, and ED⁴, were calculated using a 10 Mb sliding window with 1 Mb increments. All analyses were performed using the WheatGmap online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.wheatmap.org\u003c/span\u003e\u003cspan address=\"https://www.wheatmap.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e51\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eMolecular marker development and gene mapping\u003c/h2\u003e\u003cp\u003eAfter delimiting the candidate interval(s), SSRs, InDels, and SNPs between R and S bulks within the target region(s) were hunted for designing polymorphic markers. SSR and InDel primers were designed using the software WEKits (v1.0.2) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/GP-sir/wekits/releases\u003c/span\u003e\u003cspan address=\"https://github.com/GP-sir/wekits/releases\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Primer 5. To develop KASP markers, 200 bp sequences flanking each target SNP were extracted using Polymarker (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.polymarker.info/\u003c/span\u003e\u003cspan address=\"https://www.polymarker.info/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Newly developed and previously reported markers from homologous regions were screened for polymorphisms between the resistant and susceptible parents as well as the two contrasting bulks. Polymorphic markers were subsequently used to genotype the F\u003csub\u003e2:3\u003c/sub\u003e families derived from CWI16926-4Y\u0026times;LDN for gene mapping. Markers co-segregating with \u003cem\u003ePmCWI16926\u003c/em\u003e were further applied to fine mapping with a larger population comprising 2000 F\u003csub\u003e2:3\u003c/sub\u003e families. All the technical procedures, including reaction mixture, DNA amplification, and polymorphic marker visualization, were performed according to Wu \u003cem\u003eet al\u003c/em\u003e. (2022) with minor modifications. The markers used for mapping are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Linkage relationship between the markers and the \u003cem\u003ePm\u003c/em\u003e gene(s) in CWI16926-4Y was determined by Mapmaker 3.0b with a logarithm of odds (LOD) score of 3.0 as the threshold.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eMicro-collinearity analysis of the candidate interval\u003c/h2\u003e\u003cp\u003eMicro-collinearity analysis among different genomes was performed using the Triticeae-Gene Tribe database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://wheat.cau.edu.cn/TGT\u003c/span\u003e\u003cspan address=\"http://wheat.cau.edu.cn/TGT\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The nearest flanking markers of \u003cem\u003ePmCWI16926\u003c/em\u003e were used to extract homologous genomic regions from wild emmer v1.0, durum wheat cv. Svevo, hexaploid wheat cv. Chinese Spring v1.0, Fielder, Mace, and Jagger on WheatOmics 1.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://202.194.139.32\u003c/span\u003e\u003cspan address=\"https://202.194.139.32\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Sequences with the highest identity to the target genes were retained for comprehensive collinearity analysis. Gene annotations on the database EnsemblPlants (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://plants.ensembl.org/index.html\u003c/span\u003e\u003cspan address=\"http://plants.ensembl.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and UniProt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to explore potential disease resistance genes with homologous regions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCandidate gene screening and quantitative real-time PCR (qRT-PCR) analysis\u003c/h2\u003e\u003cp\u003eAfter fine mapping, high-confidence genes within the region were annotated using the durum cv. Svevo reference genome on the WheatOmics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://202.194.139.32/\u003c/span\u003e\u003cspan address=\"http://202.194.139.32/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To examine their expression, qRT-PCR was conducted on leaf samples of CWI16926-4Y and LDN collected at 0, 0.5, 2, 4, 12, 24, 48 and 72 hpi with \u003cem\u003eBgt\u003c/em\u003e isolate E09. Similarly, expression patterns of the candidate genes between infected and non-infected controls were further analyzed. Total RNA was extracted from each sample using the TRIzol reagent (Invitrogen, Shanghai, China), and qRT-PCR assays were performed on the Bio-Rad CFX Connect Real-Time PCR detection system (Bio-Rad, Hercules, USA). Relative quantitation of the transcript levels was calculated using the 2\u003csup\u003e\u0026minus;∆∆CT\u003c/sup\u003e method\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, with the \u003cem\u003eTaActin\u003c/em\u003e gene as normalization control. The real-time PCR reactions were performed in triplicate for each sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMicroscopic observation of fungal structures\u003c/h2\u003e\u003cp\u003eTo monitor fungal invasion, seedlings of CWI16926-4Y and LDN were inoculated with \u003cem\u003eBgt\u003c/em\u003e isolate E09. Leaf segments (2 cm) were collected at 0, 4, 8, 12, 24, 48, and 72 hpi, fixed in Carnoy\u0026rsquo;s fixative (ethanol: glacial acetic acid, 3:1, v/v) for 24 h, and then stained with 0.6% (w/v) Coomassie brilliant blue solution for 5 min. Excess dye was removed by rinsing with distilled water, and samples were observed using an Axioscope 5 microscope (ZEISS, Oberkochen, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of linked markers for MAS\u003c/h2\u003e\u003cp\u003eTo assess their suitability of markers for MAS, closely linked markers were screened in CWI16926-4Y and 46 elite wheat cultivars/lines collected from major wheat-growing regions across China. Markers showing clear polymorphisms between CWI16926-4Y and the tested genotypes were considered effective for MAS and applicable for tracking the target \u003cem\u003ePm\u003c/em\u003e gene in diverse breeding backgrounds.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eStatistics and reproducibility\u003c/h2\u003e\u003cp\u003eThe specific number (n) of replicates employed in each experiment was provided in the corresponding figure legends. In this study, student\u0026prime;s \u003cem\u003et\u003c/em\u003e-test was used for statistical analysis of intersample differences by Graphpad Prism5 software. A goodness-of-fit analysis of phenotype data of F\u003csub\u003e2\u003c/sub\u003e and F\u003csub\u003e2:3\u003c/sub\u003e generations was performed by chi-squared (χ\u003csup\u003e2\u003c/sup\u003e) test were used for statistical analysis on SPSS 16.0 (SPSS Inc., Chicago, IL, USA). All quantitative data was expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). The sample size of the qRT-PCR experiments in this article was set to three biological replicates. Statistically significant differences are denoted in graphs with * standing for \u003cem\u003eP\u003c/em\u003e-value, where * refers to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and **** to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll source data supporting the findings of this study are available within the paper and its supplementary information files. The genomic data for DNA sequence and gene analyzed in this study was obtained from the WheatOmics platform (https://202.194.139.32). All formatted data required to reproduce the results are available from the corresponding authors by request.\u003c/p\u003e\u003cp\u003e\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\u003cp\u003eXZX: formal analysis, data curation, investigation, methodology; DML: formal analysis, investigation, methodology, visualization; FTW: formal analysis, investigation, validation; YLJ: data curation, formal analysis, investigation; KW: validation, methodology; NNS: validation, methodology; LZL: validation, methodology; JTL: validation, methodology; YTD: validation, methodology; TZ: validation, methodology; CL: funding acquisition, conceptualization, supervision; GHH: writing-original draft, funding acquisition, conceptualization, methodology, supervision; NNY: writing-review and editing, funding acquisition, conceptualization, supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThis research was supported by Natural Science Foundation of Shandong Province (ZR2023QC292), Natural Science Foundation of Hebei Province (C2023503014), Wheat Industrial Technology System of Shandong Province (SDAIT-01-01), National Natural Science Foundation of China (32301800), and Key Research and Development Project of Jining (2024SHN003).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShewry, P. 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Protoc\u003c/em\u003e. \u003cstrong\u003e3\u003c/strong\u003e, 1101\u0026ndash;1108 (2008).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cultivated emmer wheat, Powdery mildew, PmCWI16926, Fine mapping, Marker-assisted selection","lastPublishedDoi":"10.21203/rs.3.rs-7647818/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7647818/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWheat powdery mildew, caused by \u003cem\u003eBlumeria graminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003eBgt\u003c/em\u003e), severely threatens global wheat production. Cultivated emmer wheat, a direct progenitor of common wheat, harbors rich genetic diversity and represents a valuable source of novel resistance genes. In the present study, the cultivated emmer accession CWI16926-4Y exhibited high-level and broad-spectrum resistance at both seedling and adult plant stages. Genetic analysis revealed that the powdery mildew resistance in CWI16926-4Y is controlled by a single dominant gene, designated \u003cem\u003ePmCWI16926\u003c/em\u003e. Employing bulked segregant RNA analysis combined with high-density mapping, \u003cem\u003ePmCWI16926\u003c/em\u003e was delimited to a 590 kb physical interval (21.70-22.29 Mb) on chromosome 2BS of the durum wheat cv. Svevo genome. Comparative analysis confirmed its distinction from nine previously reported loci on 2BS. Within the interval, two NLR-type genes were identified, with \u003cem\u003eTRITD2Bv1G010140\u003c/em\u003e emerging as the most promising candidate based on pathogen-induced expression and a unique haplotype defined by three nonsynonymous SNPs absent in other resistant and susceptible genotypes. Moreover, four co-segregated markers and a gene-specific marker \u003cem\u003eKASP689-1\u003c/em\u003e were validated, enabling marker-assisted transfer of \u003cem\u003ePmCWI16926\u003c/em\u003e into elite cultivars in breeding. This study expands the repertoire of deployable \u003cem\u003ePm\u003c/em\u003e genes from emmer wheat and provides new genetic tools for durable and precise resistance breeding.\u003c/p\u003e","manuscriptTitle":"Fine mapping and genetic dissection of PmCWI16926, a broad-spectrum powdery mildew resistance gene from cultivated emmer wheat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 08:40:49","doi":"10.21203/rs.3.rs-7647818/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"305b43f6-8a4f-41e6-a752-34a81f821e4f","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55646194,"name":"Biological sciences/Genetics/Agricultural genetics"},{"id":55646195,"name":"Biological sciences/Plant sciences/Plant breeding"}],"tags":[],"updatedAt":"2026-03-18T09:33:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 08:40:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7647818","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7647818","identity":"rs-7647818","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}