Identification and characterization of the powdery mildew resistance gene in spelt accession Lsy-93 

Identification and characterization of the powdery mildew resistance gene in spelt accession Lsy-93 | 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 Research Article Identification and characterization of the powdery mildew resistance gene in spelt accession Lsy-93 Nina Sun, Jiatong Li, Jiansheng Lu, Ningning Yu, Hongxing Xu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6722793/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Aug, 2025 Read the published version in BMC Plant Biology → Version 1 posted 10 You are reading this latest preprint version Abstract Background Powdery mildew, a widespread fungal disease caused by Blumeria graminis f. sp. tritici ( Bgt ), seriously threatens the yield and quality of wheat. The most effective and sustainable approach to disease control is utilizing resistance genes and unraveling their underlying molecular mechanisms. Spelt ( Triticum aestivum ssp. Spelta , 2n = 6x = 42, AABBDD), an ancient hexaploid wheat subspecies, has emerged as a valuable genetic resource for enhancing powdery mildew resistance in modern wheat breeding programs. Results Spelt accession Lsy-93 demonstrated resistance against powdery mildew at the whole-growth stage. Genetic analysis revealed that this resistance is conferred by a single dominant gene, tentatively designated as PmLsy-93 . Bulked segregant RNA sequencing (BSR-seq) and molecular markers positioned PmLsy-93 within a 1.5 cM interval flanked by markers S93-2 and S93-46 . Nine genes in this interval were associated with disease resistance and were considered as the candidate genes of PmLsy-93 . Furthermore, a total of 3,140 differentially expressed genes (DEGs) were identified between the two bulks, with 2,214 down-regulated and 916 up-regulated genes relative to the susceptible bulk. The integration of gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis underscores the multifaceted roles of these DEGs in plant defense, stress response, and metabolic regulation. Then, six genes, encoding disease resistance protein, serine threonine-protein kinase, or protein kinase domain, were induced with Bgt invasion via qRT-PCR. Three closely linked or co-segregated markers L93-293 , CIT02g-20 and L93-277 were confirmed to be available for marker-assisted selection (MAS) of PmLsy-93 in breeding programs. Conclusions This study successfully pinpointed critical genetic loci and candidate genes associated with powdery mildew resistance in the spelt wheat accession Lsy-93. The results provide valuable insights into plant-pathogen defense mechanisms and lay an important foundation for subsequent molecular breeding efforts to enhance crop disease resistance. Spelt wheat Powdery mildew Gene mapping GO KEGG MAS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Common wheat ( Triticum aestivum L.) is the world’s second most widely cultivated crop, serves as a staple food for 35% of the global population and contributes 20% of the calories and protein[1]. However, diseases such as powdery mildew, wheat rust, and fusarium head blight threaten wheat yield and quality. Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici ( Bgt ), is a globally devastating disease prevalent in major wheat-growing countries, severely reducing wheat yield and quality [2]. Host resistance remains the most effective strategy to reduce yield losses caused by crop diseases. However, genetic changes in pathogen populations often render existing resistance (R) genes ineffective [3]. Therefore, identification and exploitation of resistance genes represent the cornerstone of developing crop varieties with durable resistance [4]. Despite extensive research into wheat and its wild relatives, over 100 Pm genes/alleles ( Pm1 - Pm71 ) have been identified, but the number of those that have been cloned and comprehensively characterized to date remains rather limited [5]. Among these Pm genes, the majority encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins, with a smaller number coding for kinases and other protein types [6]. Pm1 , Pm2 , Pm3 , Pm8 , Pm17 , Pm5 , Pm12 , Pm21 , Pm41 , Pm60 and Pm69 have been experimentally verified to encode NBS-LRR proteins. Pm24 ( WTK3 ) and WTK4 encode tandem kinase proteins [7]. Pm4 encodes a chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions [8]. For other protein type, Pm38/Yr18/Lr34/Sr57 encode a putative ATP-binding cassette transporter and Pm46 / Yr46 / Lr67 / Sr55 encoding hexose transporter [9, 10]. Common wheat is a polyploid species containing three analogous genomes (97% identical among homoeologous genes), this characteristic severely hampers elite gene cloning and mining the resistance genes [11]. Bulked segregant RNA sequencing (BSR-Seq) analysis is an effective method by sequencing RNA from a mixture of cells or tissues [12]. It is widely used to identify candidate resistance genes against diseases and study overall transcriptome profiles. Based on the distribution characteristics of putative SNP loci across 21 wheat chromosomes via BSR-Seq, the powdery mildew resistance gene PmSGD was predicted to reside in the 240–250 Mb interval of chromosome 7B.[13]. Through Gene Ontology (GO) analysis of W762, Qian et al. (2024) identified 3,653 differentially expressed genes (DEGs) between the resistant and susceptible bulks [14]. Ma et al. (2021) identified six disease-related genes as critical candidates to unravel the molecular mechanisms of resistance by GO, clusters of orthologous group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis [15]. Spelt ( T . aestivum ssp. Spelta , 2n = 6x = 42, AABBDD), a hexaploid wheat (2n = 6x = 42, AABBDD) widely cultivated in Europe until the early 20th century, remains a critical genetic reservoir for enhancing modern wheat cultivars [16]. Many disease resistance genes have been identified in Spelta, such as Pm1d , MlHubel , Yr5 , Yr10 , Lr65 and Lr71 [17]. Spelt accession Lsy-93 demonstrated resistance against powdery mildew at the whole-growth stage. To understand the resistance mechanism, our study aimed to (i) assess the resistance to powdery mildew; (ii) explore the key candidate gene(s) in Lsy-93; (iii) use BSR-Seq combined with qRT-PCR to analyze regulatory genes. Results Evaluation and genetic inheritance of powdery mildew resistance in Lsy-93 Upon inoculation with the Bgt isolate E09, Lsy-93 did not produce any visible symptoms, scoring an infection type (IT) 0, while Tainong 18 (TN18) exhibited abundant sporulation, and hence scoring an IT 4 (Fig. 1a; Fig. 1b). Ten F 1 plants of Lsy-93 × TN18 showed similar symptom as Lsy-93, suggesting dominant inheritance pattern for the seedling resistance to the Bgt isolate E09. Among 253 inoculated F 2 plants, 188 ones were resistant and 65 ones were susceptible, fitting a ratio of 3:1 for Mendel monogenic segregation (χ 2 = 0.06, P = 0.79). To confirm the genotype of the resistant F 2 plants and also revalidate the phenotype of the F 2 plants, the 253 F 2 plants were transplanted in the field after scoring to produce the corresponding F 2:3 families. The harvested F 2:3 families segregated with 61 (homozygous resistant) : 124 (segregating) : and 65 (homozygous susceptible), also fitting a ratio of 1:2:1 for monogenic inheritance (χ 2 = 0.14, P = 0.93) (Fig. 1a; Table 1). In conclusion, the resistance to this Bgt isolate E09 was determined by single dominant resistance gene, tentatively designated PmLsy-93 . Table1 Genetic analysis of resistance to the Blumeria graminis f. sp. tritici ( Bgt ) isolate E09 based on the cross of Lsy-93 × Tainong 18 Parent and Cross Generation Observed ratio Expected ratio χ 2 P Lsy-93 R P R : S = 10 : 0 Tainong 18 S P R : S = 0 : 10 Lsy-93 × Tainong 18 F1 F1 R : S = 10 : 0 Lsy-93 × Tainong 18 F2 F2 R : S = 188 : 65 3 : 1 0.06 0.79 Lsy-93 × Tainong 18 F2:3 F2:3 HR : Seg : HS = 61 : 124 : 65 1 : 2 : 1 0.14 0.93 Note: R P : Resistant parent, S P : Susceptible parent, HR: Homozygous resistant, Seg: Segregating, HS: Homozygous susceptible SNP calling and screening of candidate intervals High-quality sequencing data were obtained by performing BSR-Seq analysis on the resistant and susceptible bulks.The resistant bulk generated 20.513 Gb of clean reads with a Q30 score >93% and GC content of 43–73%. The susceptible bulk produced 27.825 Gb of clean data, showing a Q30 score >94% and GC content of 41–72%. Alignment results indicated 137,400,008 and 186,646,126 reads from the resistant and susceptible bulks successfully mapped to the Chinese Spring reference genome (Ref v2.1), respectively. These high-quality sequences were deemed suitable for downstream analyses. To reduce background noise, original ED values were quartic-transformed and used as correlation values, with a Loess curve applied for fitting. The threshold was determined using the quantile method, which entails arranging all fitted values in ascending order and designating the ED values of SNP markers exceeding the 99% as the threshold during the screening process. Using this threshold, the ED algorithm identified only one estimated candidate region situated at the terminal end of chromosome arm 2BL. (Fig. 2a; Fig. 2b; Table S1). This result shows that PmLsy-93 resides on chromosome arm 2BL. Molecular mapping of PmLsy-93 and prediction of candidate genes Based on the BSR-Seq results, we further validated the candidate interval by screening 12 known markers surrounding the region of interest on chromosome arm 2BL and developing eight SSR and INDEL markers derived from the BSR-Seq data. Eleven markers, including L43177 , L11500 , L93-293 , CIT02g-20 , S93-2 , S93-46 , L93-277 , CIT02g-4 , L93-21 , CINAU140 and L93-260 (Table S2), showed polymorphism between resistant and susceptible parents as well as bulks. Subsequently, based on the genotyping results of these markers for the 250 F 2:3 families, a genetic linkage map for PmLsy-93 was constructed. (Fig. 3a). According to the linkage analysis results, PmLsy-93 was mapped to a 1.5 cM interval flanked by markers S93-2 and S93-46 (Fig. 3a; Fig. 3b). According to the gene annotation results, a total of 75 genes with high confidence were identified in this interval. Among them, six genes were related to disease resistance and regarded as candidate genes for PmLsy-93 . Discovery and analysis of DEGs BSR-Seq analysis identified 3,140 DEGs between the two bulks, including 2,214 down-regulated and 926 up-regulated genes compared with the susceptible bulk (Fig. 4a). GO analysis indicated that the DEGs were mainly related to three functional domains. In molecular function, key activities included manganese ion binding, oxidoreductase activity, heme binding, and hydrolase activity acting on glycosyl bonds, which are critical for enzymatic reactions and metabolic regulation. In cellular component, enriched terms such as extracellular region and cell wall macromolecule catabolic process highlighted the involvement of DEGs in extracellular interactions and cell wall remodeling, which are vital for pathogen defense and structural integrity. In biological process, DEGs were linked to processes like response to water, response to biotic stimulus, and chitin catabolic process, indicating their roles in stress adaptation and pathogen resistance (Fig. 4b; Table S3). Furthermore, according to KEGG pathway enrichment analysis, the DEGs were significantly enriched in metabolic and signaling pathways associated with plant defense and stress tolerance. The MAPK signaling pathway-plant and plant-pathogen interaction pathways were enriched, highlighting their pivotal roles in defense mechanisms against pathogens. Additionally, glutathione metabolism and photosynthesis-antenna proteins were identified, emphasizing DEGs involved in mitigating oxidative damage during infection and involvement in energy production and light harvesting, respectively (Fig. 4c; Table S4). The integration of GO and KEGG analyses underscores the multifaceted roles of DEGs in plant defense, stress response, and metabolic regulation. Validation of disease resistance-related genes in spelt accession Lsy-93 via qRT-PCR To further identify candidate genes, DEGs within the candidate interval of 710.73 to 721.07 Mb were analyzed. A total of 123 DEGs were found in this interval, and they were considered the essential candidates for powdery mildew resistance in accession Lsy-93. Expression patterns of six DEGs in the resistant parent Lsy-93 and susceptible parent TN18 were analyzed at various time points (hpi) after inoculation with Bgt isolate E09. All genes were found to be induced by Bgt invasion. The transcription levels of TraesCS2B0361276100 (encoding disease resistance protein) and TraesCS2B0361276200 (encoding disease resistance protein) reached peak transcript levels at 0.5 hpi in resistant parent Lsy-93. The remaining four genes TraesCS2B03G1285300 (encoding serine threonine-protein kinase), TraesCS2B0361283800 (encoding serine threonine-protein kinase), TraesCS2B0361284900 (encoding serine threonine-protein kinase) and TraesCS2B0361290400 (encoding protein kinase domain) reached their peak transcript levels at 24 hpi or even later. Additionally, most of these genes displayed significantly higher expression in the early stage of inoculation in Lsy-93 compared to TN18 (Fig. 5; Table S2), suggesting potential regulatory mechanisms. Screening and confirmation of molecular markers for MAS To assess the potential of the linked markers in MAS, 46 susceptible elite wheat cultivars/lines were tested with markers L93-293 , CIT02g-20 and L93-277 . All these markers amplified polymorphic bands between Lsy-93 and most of wheat accessions, displaying their widely effective potential in different wheat backgrounds (Fig. 6; Table S5). Hence, when the PmLsy-93 was transferred into these genotypes, the corresponding markers could be employed to accelerate the transfer of PmLsy-93 in wheat breeding practices. Discussion Spelt, an ancient hexaploid wheat subspecies, has emerged as a valuable genetic resource for enhancing powdery mildew resistance in modern wheat breeding programs. As a crop historically cultivated in Europe, spelt possesses a rich reservoir of disease resistance genes that have been largely lost in elite wheat cultivars due to intense selection for agronomic traits.[ 18 ]. Of particular interest is its potential contribution to combating powdery mildew. Spelt accession Lsy-93 showed resistance against powdery mildew across all growth stages. Through genetic mapping in this study, a dominant Pm gene, provisionally designated PmLsy-93 , was identified to confer this resistance. Using BSR-Seq and genetic markers, PmLsy-93 was mapped to a 10.34 Mb physical interval (710.73-721.07 Mb) on chromosome arm 2BL referred to Chinese spring reference genome (Ref v2.1). Six formally designated Pm genes, including Pm6 from T. timopheevii , have been reported to located on chromosome arm 2BL, with diverse gene donors contributing to this locus. [ 19 ], Pm33 from T. persicum Vav. [ 20 ], Pm51 from a Thinopyrum ponticum introgression line [ 21 ], Pm52 from Chinese wheat cultivar Liangxing 99 [ 22 ], Pm63 from Iranian wheat landrace PI 628024 [ 23 ] and Pm64 from wild emmer [ 24 ]. Additionally, several temporarily named genes have been also identified, such as PmQ , MlZec1 , MlAB10 , PmKN0816 , PmJM809 and so on. Compared with those documented genes, PmLsy-93 (710.73-721.07 Mb) was derived from spelt, which have a unique source. Furthermore, PmLsy-93 could be clearly distinguished from four of them: Pm6 (698.3-699.2 Mb), Pm33 (773.2-784.3 Mb), Pm52 (581.0-585.0 Mb) and Pm64 (699.2-710.3 Mb). It overlapped with Pm51 (709.8-739.4 Mb) and Pm63 (710.3-723.4 Mb). In the future, clarifying their allelism relationships through allelism tests and cloning these genes will be necessary. Previously studies have identified several major Pm genes and quantitative trait loci (QTLs) in spelt that confer either race-specific or broad-spectrum resistance, including notable examples such as Pm54 and PmTm4 [ 25 , 26 ]. These genetic elements often trigger defense mechanisms like cell wall reinforcement and hypersensitive responses, providing multiple layers of protection against pathogen invasion [ 27 ].In this study, a total of 3,140 DEGs were detected between the two bulks. Compared with the susceptible bulk, 2,214 genes were downregulated and 926 were upregulated. The GO and KEGG enrichment analyses provide comprehensive insights into the functional roles and metabolic pathways associated with the DEGs in response to biotic stress. The GO analysis revealed that DEGs are predominantly involved in molecular functions which are critical for enzymatic reactions and stress responses. Additionally, cellular components like the extracellular region and cell wall-related processes suggest active remodeling during pathogen defense. Biological processes, including response to biotic stimuli and chitin catabolism, further emphasize the activation of defense mechanisms against pathogens [ 28 ]. The KEGG analysis highlighted key pathways linked to plant immunity. The enrichment of MAPK signaling and plant-pathogen interaction pathways underscores the importance of signal transduction in defense responses [ 29 ]. Furthermore, glutathione metabolism indicates a role in mitigating oxidative stress, while photosynthesis-related pathways suggest a balance between defense and energy metabolism [ 30 ]. These findings were consistent with previously studies, including PmW762 , PmJM23 and PmL709 [31– 33 ]. Looking forward, spelt accessions will continue to play a critical role in sustainable wheat disease management systems. The ultimate goal of mining disease-resistant genes is to apply them in wheat breeding and production. In this study, PmLsy-93 is derived from spelt wheat, which shares the A, B and D genomes with common wheat. Spelt has maintained greater allelic diversity in resistance-related genes due to its adaptation to diverse environments and lower selection pressure. Compared with the alien species of wheat without any of A, B, D genomes, it can be easily integrated in wheat genome without linkage drags [ 34 ]. Therefore, PmLsy-93 has high potential in wheat breeding to combat powdery mildew. MAS now enables more precise transfer of target genes while minimizing linkage drag. To promote the transfer of PmLsy-93 by means of MAS, three PCR-based and co-dominant markers were screened to be available for efficiently tracing PmLsy-93 once it has been introduced into those susceptible cultivars/breeding lines. Furthermore, the strategic integration of spelt genetic resources with resistance loci from wild relatives may offer a robust, multi-layered defense system against rapidly evolving pathogen populations. With the climate change and intensive agricultural practices, the conservation and exploitation of genetic diversity in ancient wheat varieties such as spelt is emerging as a critical component of global food security strategies. Conclusion In this study, a single dominant Pm gene PmLsy-93 was identified in the spelt accession Lsy-93. The results provide valuable insights into plant-pathogen defense mechanisms and lay an important foundation for subsequent molecular breeding efforts to enhance crop disease resistance. Materials and methods Plant materials Spelt wheat Lsy-93 is sourced from Prof. Hongxing Xu, Henan University, Kaifeng, China. Because Lsy-93 displayed continuous resistance to powdery mildew across all growth stages, it was selected as the resistant parent for hybridization with the susceptible parent TN 18. And F 1 , F 2 and F 2:3 progenies were produced for genetic and BSR-Seq analysis. The wheat cultivar TN 18 was also planted as a susceptible control. Phenotypic evaluation The population F 1 , F 2 , and F 2:3 progenies of the cross Lsy-93 × TN 18 was planted in the 128-cell rectangular trays, and 5–8 seeds were sown in each cell, which were inoculated with Bgt isolate E09 for genetic analysis. The incubator was set at 20℃ for 14 hours daily and 18℃ for 10 hours nightly. Upon reaching the two-leaf stage, seedlings underwent phenotypic observations. When observing phenotypes, ITs 0–4 scale was used. Ratings from 0 to 2 indicated resistance, while ratings of 3 and 4 signified susceptibility [35]. The evaluation procedure was repeated three times to guarantee the reliability of the results. For F 2:3 families, at least 20 seeds per family were planted and assessed. At the same time, TN 18 were randomly distributed and planted in each tray. Genetic analysis For genetic analysis, 10 F₁, 253 F₂, and 252 F₂:₃ families (approximately 20 seedlings per family) were planted and inoculated with Bgt isolate E09 for resistance phenotyping. The Chi-squared test ( χ ²) was used to check goodness-of-fit to expected the Mendelian segregation ratio for monogenic inheritance. Microscopic evaluation of powdery mildew resistance responses To additionally analyze hyphal morphology post-invasion by Bgt isolate E09, 10 seedlings Lsy-93 and 10 seedlings TN 18 were planted separately. At 0, 0.5, 2, 4, 12, 24, 48, and 72 hours post inoculation (hpi) after Bgt invasion, 2 cm seedling leaf samples were collected from Lsy-93 and TN 18. Samples were fixed using Carnoy's fixative (anhydrous alcohol-glacial acetic acid, 3:1, v/v) immediately and remained at an environmental temperature of 37℃ for 24 hours. Before observation under the microscope, the samples were stained in 5 mL of 0.6% (w/v) Coomassie blue solution for 5 min and then wash the leaves with distilled water to remove the reagent residue [36]. Finally, an Axioscope 5 microscope (ZEISS, Oberkochen, Germany) was used for sample analysis. RNA extraction and BSR-Seq library preparation At 10 days post inoculation (dpi), leaf tissues were sampled (10 plants per family) from 30 homozygous resistant and 30 homozygous susceptible F 2:3 lines. Equal aliquots of these samples were combined to generate resistant (R) and susceptible (S) bulks, from which total RNA was extracted using the RNA Simple Total RNA Kit (Tiangen, Beijing, China). To purify mRNA from total RNA samples, oligo(dT) beads were added to the mixture, leveraging the specific binding between the poly-A tails of mRNA and the beads. The mixture was then gently shaken and incubated at 18–20°C for 15–30 minutes. Subsequently, the beads were washed with a washing solution to remove non-mRNA components such as rRNA and tRNA. Before this purification step, RNA quality was initially evaluated by agarose gel electrophoresis, spectrophotometry, and other methods. Following successful mRNA purification, fragmentation was achieved using either chemical reagents or enzymatic digestion. After fragmentation, cDNA synthesis was carried out using reverse transcriptase in conjunction with random primers. The obtained cDNA was subjected to end repair and adapter ligation, after which the ligation products were amplified by PCR to generate the final sequencing library. Subsequently, the library was quantified and its quality was assessed to ensure it met the requirements for sequencing.[37]. Sequencing and data analysis The sequencing was carried out on the Illumina HiSeq4000 platform (Illumina HiSeq4000) at Tcuni Bioscience (Chengdu, China). At Tcuni Bioscience, Large quantities of sequencing data were produced as short-read sequences. The raw data pre-processing steps included read quality score assessment, low-quality reads elimination, and adapter sequence trimming. Subsequently, the cleaned data were aligned to the Chinese Spring 2.1 reference genome to prepare for subsequent analysis using the STAR software. [38]. Determination of the candidate interval and genetic mapping of the target Pm gene To identify the target interval, the following procedures were implemented. Initially, SNP calling for the resistant and susceptible bulks was carried out using the software packages GATK v4.2.3.0 and SAMtools v1.17 [39]. This was followed by the quantification of allele frequencies for each SNP across the two bulks. Following the calculation of SNP index values using MutMap, the ΔSNP index for each SNP was defined as: ΔSNP index = SNP index (resistant) – SNP index (susceptible) [40-41]. Subsequently, the linkage between SNPs and the target gene(s) was determined using Bayesian analysis integrated with the Euclidean distance (ED) algorithm. Potential candidate intervals were then identified according to previously reported procedures. [37]. Following the identification of the candidate interval, molecular markers within it were utilized to map the target gene. Polymorphic genetic markers were applied to genotype the F 2:3 families from the Lsy-93 and TN 18 cross for mapping. Subsequently, phenotypic and genotypic data were collected, followed by linkage analysis using Mapmaker 3.0b [42]. A LOD score threshold of 3.0 was applied. And the genetic map of the Pm gene in Lsy-93 was constructed using Mapdraw v2.1 [43]. DEGs analysis associated with the powdery mildew resistance in Lsy-93 After BSR-Seq, software like HTSeq v2.0.3 or featureCounts v2.0.1 estimated transcript abundances mapped to each gene to quantify candidate gene expression levels. Statistical comparisons between experimental groups were made using EBSeq v3.5 to identify DEGs with a fold change ≥ 2 and FDR (false discovery rate) < 0.01. For functional characterization of the identified DEGs, annotation was conducted using GO and KEGG databases, combined with an R package specifically designed for analyzing DEGs. [44]. Finally, an enrichment analysis of DEGs in terms of defined pathways, cell structures, or molecular activities provided insights into the role of candidate genes. qRT-PCR qRT-PCR was performed to validate the expression of DEGs potential linked to disease resistance within the target interval. At 0, 0.5, 2, 4, 12, 24, 48, and 72 hpi, leaves with a length of 3-5 cm of Lsy-93 and TN 18 were collected, respectively. Total RNA was isolated from samples using the RNAsimple Total RNA Kit (Tiangen, Beijing, China). Primers were designed using Primer5 software. Approximately 2 µg of RNA was reverse transcribed into cDNA by employing a FastQuant RT Kit (Tiangen, Beijing, China). qRT-PCR reactions were prepared with SYBR Premix Ex Taq (Takara, China) and conducted on a Bio-Rad CFX Connect real-time PCR system (BIO-RAD, Hercules, USA) following standard protocols. The TaActin gene was chosen as the reference gene. Gene expression levels were finally calculated using the 2 −∆∆Ct method [45]. Each sample underwent triplicate testing. Evaluation of linked markers applied in MAS Markers that amplify different bands in the tested wheat genotypes and Lsy-93 can be identified as suitable for MAS. Through the detection of 46 elite wheat cultivars/lines collected from major wheat-producing areas in China, three closely linked markers suitable for MAS were screened out. Abbreviations Bgt Blumeria graminis f. sp. Tritici NBS-LRR Nucleotide-binding site leucine-rich repeat BSR-Seq Bulked segregant RNA sequencing SNP Single nucleotide polymorphism DEGs Differentially expressed genes GO Gene ontology COG Clusters of orthologous groups KEGG Kyoto encyclopedia of genes and genomes IT Infection type ED Euclidean distance SSR Simple sequence repeat marker hpi Hours post-inoculation TN18 Tainong 18 MAS Marker-assisted selection QTL Quantitative trait loci dpi Days post inoculation FDR False discovery rate qRT-PCR Real-time quantitative PCR Declarations Author contribution PM, YJ and HZ conceived the research. HZ, YJ, JL, JL, NY, HX, QS, TY, JZ, LL performed the experiments and collected data. NS, JL and JL performed data analyses. NY developed the experimental materials. HZ, YJ and PM wrote and revised the manuscript. All authors read and approved the final manuscript. Funding This research was financially supported by Key R&D Program of Shandong Province (2024LZGC001), National Modern Wheat Industry Technology System, Yantai Comprehensive Experimental Station project (CARS-03-62), National Natural Science Foundation of China (32301923), Natural Science Foundation of Shandong Province, China (ZR2023QC203) and Henan Province Natural Science Foundation (232300420003). Data Availability The datasets generated and analysed during the current study are available in the National Center for Biotechnology Information repository, SRA: SUB15322822. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no conflicts of interest. Author details Yantai Academy of Agricultural Sciences, Yantai 265500, China Nina Sun, Jiansheng Lu, Qingpeng Sun, Tangyu Yuan, Linzhi Li, Huanchun Zhang Yantai Key Laboratory of Characteristic Agricultural Biological Resources Conservation and Germplasm Innovative Utilization, College of Life Sciences, Yantai University, Yantai 264005, China Jiatong Li, Ningning Yu, Jiadong Zhang, Yuli Jin, Pengtao Ma School of Life Sciences, Henan University, Kaifeng 475004, China Hongxing Xu Corresponding authors Correspondence to Pengtao Ma, Yuli Jin or Huanchun Zhang References Zörb C, Ludewig U, Hawkesford MJ. Perspective on wheat yield and quality with reduced nitrogen supply. Trends Plant Sci. 2018;23(11):1029–1037. G. A. BENNETT F. Resistance to powdery mildew in wheat: a review of its use in agriculture and breeding programmes. Plant Physiol. 1984;33:279–300. Jin Y, Han G, Zhang W, Bu B, Zhao Y, Wang J, Liu R, Yang H, Xu H, Ma P. 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Supplementary Files TableS1.xlsx Table S1 SNPs between the resistant and susceptible bulks of Lsy-93 × Tainong 18 (TN18) TableS2.xlsx Table S2 Primers for the mapping of PmLsy-93 and qRT-PCR of the six selected differentially expressed genes (DEGs) TableS3.xlsx Table S3 Differentially expressed genes (DEGs) between the resistant and susceptible bulks of Lsy-93 × Tainong 18 (TN18) TableS4.xlsx Table S4 Information about the detailed kyoto encyclopedia of genes and genomes (KEGG) pathway for differentially expressed genes (DEGs) TableS5.xlsx Table S5 Validation of diagnostic/closely linked markers L93-293 , CIT02g-20 and L93-277 on 46 wheat cultivars/breeding lines for marker-assisted selection breeding. Note: "-" indicates that the markers did not amplify the polymorphic products linked to PmLsy-93 in corresponding cultivars/breeding lines, while "+" shows the opposite results uncroppedgelsandblotsimages.zip Cite Share Download PDF Status: Published Journal Publication published 18 Aug, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 19 Jun, 2025 Reviews received at journal 18 Jun, 2025 Reviews received at journal 04 Jun, 2025 Reviewers agreed at journal 04 Jun, 2025 Reviewers agreed at journal 03 Jun, 2025 Reviewers invited by journal 03 Jun, 2025 Editor invited by journal 28 May, 2025 Editor assigned by journal 26 May, 2025 Submission checks completed at journal 26 May, 2025 First submitted to journal 22 May, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6722793","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466675252,"identity":"59337c14-f0b6-4e24-b788-aa1904cad9e7","order_by":0,"name":"Nina Sun","email":"","orcid":"","institution":"Yantai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nina","middleName":"","lastName":"Sun","suffix":""},{"id":466675253,"identity":"01e00498-57b3-471d-a095-bc9812114a15","order_by":1,"name":"Jiatong Li","email":"","orcid":"","institution":"Yantai University","correspondingAuthor":false,"prefix":"","firstName":"Jiatong","middleName":"","lastName":"Li","suffix":""},{"id":466675254,"identity":"a1cf3600-abbf-42fa-98a9-7d5379893f24","order_by":2,"name":"Jiansheng Lu","email":"","orcid":"","institution":"Yantai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jiansheng","middleName":"","lastName":"Lu","suffix":""},{"id":466675255,"identity":"0e598d38-9715-47a6-9c0c-ae0e18964cce","order_by":3,"name":"Ningning Yu","email":"","orcid":"","institution":"Yantai University","correspondingAuthor":false,"prefix":"","firstName":"Ningning","middleName":"","lastName":"Yu","suffix":""},{"id":466675256,"identity":"1aca3f32-74c9-450b-9f36-c680b6ffd26c","order_by":4,"name":"Hongxing Xu","email":"","orcid":"","institution":"Henan University","correspondingAuthor":false,"prefix":"","firstName":"Hongxing","middleName":"","lastName":"Xu","suffix":""},{"id":466675257,"identity":"b355e0da-a89e-44bd-8895-9267f0cf683f","order_by":5,"name":"Qingpeng Sun","email":"","orcid":"","institution":"Yantai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qingpeng","middleName":"","lastName":"Sun","suffix":""},{"id":466675258,"identity":"faeea8b7-1ac8-43d3-995a-429b3186faf5","order_by":6,"name":"Tangyu Yuan","email":"","orcid":"","institution":"Yantai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tangyu","middleName":"","lastName":"Yuan","suffix":""},{"id":466675259,"identity":"1536910e-df2e-4c8c-a5fd-09c2e2c07e27","order_by":7,"name":"Jiadong Zhang","email":"","orcid":"","institution":"Yantai University","correspondingAuthor":false,"prefix":"","firstName":"Jiadong","middleName":"","lastName":"Zhang","suffix":""},{"id":466675260,"identity":"fcc8753f-a158-4d8e-8c3e-3fb03b5184e0","order_by":8,"name":"Linzhi Li","email":"","orcid":"","institution":"Yantai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Linzhi","middleName":"","lastName":"Li","suffix":""},{"id":466675261,"identity":"5edbe3f9-4e9d-40ab-853e-23f53b07316c","order_by":9,"name":"Huanchun Zhang","email":"","orcid":"","institution":"Yantai Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Huanchun","middleName":"","lastName":"Zhang","suffix":""},{"id":466675262,"identity":"5812eed6-0bcc-4a2b-84df-3c6d9fc04264","order_by":10,"name":"Yuli Jin","email":"","orcid":"","institution":"Yantai University","correspondingAuthor":false,"prefix":"","firstName":"Yuli","middleName":"","lastName":"Jin","suffix":""},{"id":466675263,"identity":"1d732927-1b26-442a-936a-25a4f23d1c44","order_by":11,"name":"Pengtao Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYLCCDxDKgEjlbAwMjDMgqknQwsxDkhZz+e7Ez7ZtfxIb2Ju3STDU3CGsxbKNd7N0bptBYgPPsTIJhmPPCGsxOMa7AaJFIsdMgrHhMFFaNv+2BGmRf0O8lm3SjGBbeIjUYtmWu82y55yxcRtPWrFFwjEitJgzn91840eZnGw/++GNNz7UEOMwEMHIBo4fBoYEwhpg0feHGKWjYBSMglEwYgEApEQ1aMXcj3gAAAAASUVORK5CYII=","orcid":"","institution":"Yantai University","correspondingAuthor":true,"prefix":"","firstName":"Pengtao","middleName":"","lastName":"Ma","suffix":""}],"badges":[],"createdAt":"2025-05-22 08:23:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6722793/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6722793/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07040-5","type":"published","date":"2025-08-18T16:29:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84069472,"identity":"c0b84caf-5711-426b-a73c-0f3e982f4cff","added_by":"auto","created_at":"2025-06-06 11:55:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7895262,"visible":true,"origin":"","legend":"\u003cp\u003e(a). Responses of resistant parent Lsy-93, susceptible parent Tainong 18 (TN18), and part of F\u003csub\u003e2:3\u003c/sub\u003e plants at 14 days post inoculation (dpi) with \u003cem\u003eBlumeria graminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003eBgt\u003c/em\u003e) isolate E09; (b). Infection process of \u003cem\u003eBgt \u003c/em\u003eisolate E09 on leaves of Lsy-93 and TN18. Wheat leaf samples were taken at different times post inoculation (hpi) for Coomassie blue staining. Scale bar, 100 μm\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/a2173264ed5768eaa184e2f8.png"},{"id":84068775,"identity":"d1d2fed1-2ed3-4d0f-ad84-4c5426e3a5df","added_by":"auto","created_at":"2025-06-06 11:47:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3453102,"visible":true,"origin":"","legend":"\u003cp\u003eCandidate interval analysis of \u003cem\u003ePmLsy-93\u003c/em\u003e. (a). SNPs distribution on 21 wheat chromosomes between resistant and susceptible bulks of Lsy-93 and Tainong 18 (TN18); (b). Euclidean distance (ED) analysis of the candidate region on 21wheat chromosomes\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/f1f086f903bf128c4b4b2c83.png"},{"id":84069473,"identity":"9505d90e-4482-444a-8550-334942585073","added_by":"auto","created_at":"2025-06-06 11:55:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5070901,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular mapping of \u003cem\u003ePmLsy-93. \u003c/em\u003e(a). Genetic linkage map of \u003cem\u003ePmLsy-93\u003c/em\u003e; (b). Amplification patterns of \u003cem\u003ePmLsy-93\u003c/em\u003e-linked markers S93-2 and S93-46 in genotyping the resistant parent Lsy-93, susceptible parent Tainong 18, and randomly selected F\u003csub\u003e2:3\u003c/sub\u003e families of Lsy-93×Tainong 18. Lane M: pUC19/MspI; 1: Lsy-93; 2: Tainong 18; 3-7: homozygous resistant F\u003csub\u003e2:3\u003c/sub\u003e families; 8-12: heterozygous F\u003csub\u003e2:3\u003c/sub\u003e families; 13-17: homozygous susceptible F\u003csub\u003e2:3\u003c/sub\u003e families. The white arrows were used to indicate the polymorphic bands linked to \u003cem\u003ePmLsy-93\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/d7870f10d9b992588ea34582.png"},{"id":84068770,"identity":"142afe3e-3a30-4dc7-b452-09c2112ab782","added_by":"auto","created_at":"2025-06-06 11:47:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6716661,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution and functional classification of differentially expressed genes (DEGs) based on the bulked segregant RNA sequencing (BSR-Seq) of the resistant and susceptible bulks of Lsy-93 × Tainong 18. (a). The distribution of the DEGs using a volcano plot; (b) A Gene Ontology (GO) analysis of the DEGs; (c) A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/83ff26b240002e5ec826c809.png"},{"id":84068773,"identity":"8114f0f5-7892-4996-ad42-50829dd1c13b","added_by":"auto","created_at":"2025-06-06 11:47:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":749892,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of \u003cem\u003eTraesCS2B03G1285300\u003c/em\u003e,\u003cem\u003e TraesCS2B0361276100\u003c/em\u003e, \u003cem\u003eTraesCS2B0361276200\u003c/em\u003e, \u003cem\u003eTraesCS2B0361283800\u003c/em\u003e, \u003cem\u003eTraesCS2B0361284900\u003c/em\u003e and \u003cem\u003eTraesCS2B0361290400 \u003c/em\u003ein Lsy-93 and Tainong 18 (TN18) at 0, 0.5, 2, 4, 12, 24, 48 and 72 hours post inoculation (hpi) following the invasion of \u003cem\u003eBlumeria graminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003eBgt\u003c/em\u003e) isolate E09. Error bars represent standard deviation (SD) based on three independent repeats. Asterisks indicate significant differences (\u003cem\u003et\u003c/em\u003e tests) between Lsy-93 and Tainong 18 at each time point (\u003cem\u003e* P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e** P \u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e*** P \u003c/em\u003e\u0026lt; 0.001, \u003cem\u003e**** P \u003c/em\u003e\u0026lt;0.0001,\u003cem\u003e \u003c/em\u003ens: not significant); the sphemplasts indicate biological replicates; \u003cem\u003eTaActin\u003c/em\u003e was used as the internal control\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/e6b48cd1e85f32fbfde379f1.png"},{"id":84068772,"identity":"0b87404b-e402-4cca-93d6-c33590dfb7c2","added_by":"auto","created_at":"2025-06-06 11:47:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":651013,"visible":true,"origin":"","legend":"\u003cp\u003eAmplification patterns of \u003cem\u003ePmLsy-93\u003c/em\u003e-linked markers L93-293 and CIT02g-20 in Lsy-93 and 14 susceptible wheat cultivars/lines to powdery mildew. M: pUC19/MspI; 1: Lsy-93; 2: Yannong 15; 3: Yannong 17; 4: Yannong 30; 5: Yannong 161; 6: Yannong 191; 7: Yannong 836; 8: Yannong 5158; 9: Saidemai 16; 10: Shengmai 116; 11: Luomai 013; 12: Taishan 6039; 13: Xumai 1108; 14: Henong 974; The white arrows indicate the polymorphic bands in Lsy-93\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/3a4532334574f3af5dd02e4a.png"},{"id":89847214,"identity":"9fa0448b-4c54-4fce-8bfd-b8d23f23868d","added_by":"auto","created_at":"2025-08-25 16:42:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":31015733,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/c03b5119-28e5-4b21-8605-51700ea81af7.pdf"},{"id":84068778,"identity":"51707d57-365f-458c-bb7a-b7468ba4cd8d","added_by":"auto","created_at":"2025-06-06 11:47:06","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5661528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1 \u003c/strong\u003eSNPs between the resistant and susceptible bulks of Lsy-93 × Tainong 18 (TN18)\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/3fb07fd32b65901c3c3eec4b.xlsx"},{"id":84070900,"identity":"f07bf2c2-57c2-4e26-a5c1-4369726042b0","added_by":"auto","created_at":"2025-06-06 12:11:06","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11292,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2\u003c/strong\u003e Primers for the mapping of \u003cem\u003ePmLsy-93\u003c/em\u003e and qRT-PCR of the six selected differentially expressed genes (DEGs)\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/8a69548444921da5ddb25ecb.xlsx"},{"id":84068779,"identity":"2cb15cbe-5848-4641-836a-746031177e29","added_by":"auto","created_at":"2025-06-06 11:47:06","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":410094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3 \u003c/strong\u003eDifferentially expressed genes (DEGs) between the resistant and susceptible bulks of Lsy-93 × Tainong 18 (TN18)\u003c/p\u003e","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/c6b4cf28869648a1f8640f2e.xlsx"},{"id":84068766,"identity":"6e3faefe-7d80-41c4-af77-ad943f84f7a6","added_by":"auto","created_at":"2025-06-06 11:47:06","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":30070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4 \u003c/strong\u003eInformation about the detailed kyoto encyclopedia of genes and genomes (KEGG) pathway for differentially expressed genes (DEGs)\u003c/p\u003e","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/cad705a9e19b6dc8dc6cc46f.xlsx"},{"id":84068776,"identity":"e6c1553d-c648-4d37-b644-74de7851e03a","added_by":"auto","created_at":"2025-06-06 11:47:06","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S5 \u003c/strong\u003eValidation of diagnostic/closely linked markers \u003cem\u003eL93-293\u003c/em\u003e, \u003cem\u003eCIT02g-20 \u003c/em\u003eand \u003cem\u003eL93-277 \u003c/em\u003eon 46 wheat cultivars/breeding lines for marker-assisted selection breeding. Note: \"-\" indicates that the markers did not amplify the polymorphic products linked to \u003cem\u003ePmLsy-93\u003c/em\u003ein corresponding cultivars/breeding lines, while \"+\" shows the opposite results\u003c/p\u003e","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/2114a7247e68c9de4734d4c4.xlsx"},{"id":84068780,"identity":"9e0fa021-abbc-4bc9-a8cb-be635552076a","added_by":"auto","created_at":"2025-06-06 11:47:07","extension":"zip","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":8853764,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedgelsandblotsimages.zip","url":"https://assets-eu.researchsquare.com/files/rs-6722793/v1/eb17462c9bee323e8b4e0093.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification and characterization of the powdery mildew resistance gene in spelt accession Lsy-93 ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCommon wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) is the world\u0026rsquo;s second most widely cultivated crop, serves as a staple food for 35% of the global population and contributes 20% of the calories and protein[1]. However, diseases such as powdery mildew, wheat rust, and fusarium head blight threaten wheat yield and quality. Wheat powdery mildew, caused by \u003cem\u003eBlumeria graminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003eBgt\u003c/em\u003e), is a globally devastating disease prevalent in major wheat-growing countries, severely reducing wheat yield and quality [2]. Host resistance remains the most effective strategy to reduce yield losses caused by crop diseases. However, genetic changes in pathogen populations often render existing resistance (R) genes ineffective [3]. Therefore, identification and exploitation of resistance genes represent the cornerstone of developing crop varieties with durable resistance [4].\u003c/p\u003e\n\u003cp\u003eDespite extensive research into wheat and its wild relatives, over 100 \u003cem\u003ePm\u003c/em\u003e genes/alleles (\u003cem\u003ePm1\u003c/em\u003e - \u003cem\u003ePm71\u003c/em\u003e) have been identified, but the number of those that have been cloned and comprehensively characterized to date remains rather limited [5].\u0026nbsp;Among these \u003cem\u003ePm\u0026nbsp;\u003c/em\u003egenes, the majority encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins, with a smaller number coding for kinases and other protein types [6]. \u003cem\u003ePm1\u003c/em\u003e, \u003cem\u003ePm2\u003c/em\u003e, \u003cem\u003ePm3\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Pm8\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Pm17\u003c/em\u003e, \u003cem\u003ePm5\u003c/em\u003e, \u003cem\u003ePm12\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Pm21\u003c/em\u003e, \u003cem\u003ePm41\u003c/em\u003e, \u003cem\u003ePm60\u003c/em\u003e and \u003cem\u003ePm69\u003c/em\u003e have been experimentally verified to encode NBS-LRR proteins. \u003cem\u003ePm24\u0026nbsp;\u003c/em\u003e(\u003cem\u003eWTK3\u003c/em\u003e) and \u003cem\u003eWTK4\u003c/em\u003e encode tandem kinase proteins [7]. \u003cem\u003ePm4\u0026nbsp;\u003c/em\u003eencodes a chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions [8]. For other protein type, \u003cem\u003ePm38/Yr18/Lr34/Sr57\u0026nbsp;\u003c/em\u003eencode a putative ATP-binding cassette transporter and\u0026nbsp;\u003cem\u003ePm46\u003c/em\u003e/\u003cem\u003eYr46\u003c/em\u003e/\u003cem\u003eLr67\u003c/em\u003e/\u003cem\u003eSr55\u003c/em\u003e encoding hexose transporter [9, 10].\u003c/p\u003e\n\u003cp\u003eCommon wheat is a polyploid species containing three analogous genomes (97% identical among homoeologous genes), this characteristic severely hampers elite gene cloning and mining the resistance genes [11]. Bulked segregant RNA sequencing (BSR-Seq) analysis is an effective method by sequencing RNA from a mixture of cells or tissues [12]. It is widely used to identify candidate resistance genes against diseases and study overall transcriptome profiles. Based on the distribution characteristics of putative SNP loci across 21 wheat chromosomes via BSR-Seq, the powdery mildew resistance gene \u003cem\u003ePmSGD\u003c/em\u003e was predicted to reside in the 240\u0026ndash;250 Mb interval of chromosome 7B.[13]. Through Gene Ontology (GO) analysis of W762, Qian et al. (2024) identified 3,653 differentially expressed genes (DEGs) between the resistant and susceptible bulks [14]. Ma et al. (2021) identified six disease-related genes as critical candidates to unravel the molecular mechanisms of resistance by GO, clusters of orthologous group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis [15].\u003c/p\u003e\n\u003cp\u003eSpelt (\u003cem\u003eT\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;aestivum\u003c/em\u003e ssp. \u003cem\u003eSpelta\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003e2n = 6x = 42, AABBDD), a hexaploid wheat (2n = 6x = 42, AABBDD) widely cultivated in Europe until the early 20th century, remains a critical genetic reservoir for enhancing modern wheat cultivars [16]. Many disease resistance genes have been identified in Spelta, such as \u003cem\u003ePm1d\u003c/em\u003e, \u003cem\u003eMlHubel\u003c/em\u003e, \u003cem\u003eYr5\u003c/em\u003e, \u003cem\u003eYr10\u003c/em\u003e, \u003cem\u003eLr65\u003c/em\u003e and \u003cem\u003eLr71\u003c/em\u003e [17]. Spelt accession Lsy-93 demonstrated resistance against powdery mildew at the whole-growth stage. To understand the resistance mechanism, our study aimed to (i) assess the resistance to powdery mildew; (ii) explore the key candidate gene(s) in Lsy-93; (iii) use BSR-Seq combined with qRT-PCR to analyze regulatory genes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEvaluation and genetic inheritance of powdery mildew resistance in Lsy-93\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon inoculation with the \u003cem\u003eBgt\u003c/em\u003e isolate E09, Lsy-93 did not produce any visible symptoms, scoring an infection type (IT) 0, while Tainong 18 (TN18) exhibited abundant sporulation, and hence scoring an IT 4 (Fig. 1a; Fig. 1b). Ten F\u003csub\u003e1\u003c/sub\u003e plants of Lsy-93 \u0026times; TN18 showed similar symptom as Lsy-93, suggesting dominant inheritance pattern for the seedling resistance to the \u003cem\u003eBgt\u0026nbsp;\u003c/em\u003eisolate E09. Among 253 inoculated F\u003csub\u003e2\u003c/sub\u003e plants, 188 ones were resistant and 65 ones were susceptible, fitting a ratio of 3:1 for Mendel monogenic segregation (\u0026chi;\u003csup\u003e2\u003c/sup\u003e = 0.06, \u003cem\u003eP\u003c/em\u003e = 0.79). To confirm the genotype of the resistant F\u003csub\u003e2\u003c/sub\u003e plants and also revalidate the phenotype of the F\u003csub\u003e2\u003c/sub\u003e plants, the 253 F\u003csub\u003e2\u003c/sub\u003e plants were transplanted in the field after scoring to produce the corresponding F\u003csub\u003e2:3\u003c/sub\u003e families. The harvested F\u003csub\u003e2:3\u003c/sub\u003e families segregated with 61 (homozygous resistant) : 124 (segregating) : and 65 (homozygous susceptible), also fitting a ratio of 1:2:1 for monogenic inheritance (\u0026chi;\u003csup\u003e2\u003c/sup\u003e = 0.14, \u003cem\u003eP\u003c/em\u003e = 0.93) (Fig. 1a; Table 1). In conclusion, the resistance to this \u003cem\u003eBgt\u003c/em\u003e isolate E09 was determined by single dominant resistance gene, tentatively designated \u003cem\u003ePmLsy-93\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable1\u0026nbsp;\u003c/strong\u003eGenetic analysis of resistance to the \u003cem\u003eBlumeria graminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003eBgt\u003c/em\u003e) isolate E09 based on the cross of Lsy-93\u0026thinsp;\u0026times;\u0026thinsp;Tainong 18\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"101%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParent and Cross\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGeneration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eObserved ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExpected ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003eLsy-93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eR\u003csub\u003eP\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003eR : S = 10 : 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003eTainong 18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eS\u003csub\u003eP\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003eR : S = 0 : 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003eLsy-93 \u0026times; Tainong 18 F1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003eR : S = 10 : 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003eLsy-93 \u0026times; Tainong 18 F2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003eR : S = 188 : 65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e3 : 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.79\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 27px;\"\u003e\n \u003cp\u003eLsy-93 \u0026times; Tainong 18 F2:3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eF2:3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003eHR : Seg : HS = 61 : 124 : 65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e1 : 2 : 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: R\u003csub\u003eP\u003c/sub\u003e: Resistant parent, S\u003csub\u003eP\u003c/sub\u003e: Susceptible parent, HR: Homozygous resistant, Seg: Segregating, HS: Homozygous susceptible\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSNP calling and screening of candidate intervals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-quality sequencing data were obtained by performing BSR-Seq analysis on the resistant and susceptible bulks.The resistant bulk generated 20.513 Gb of clean reads with a Q30 score \u0026gt;93% and GC content of 43\u0026ndash;73%. The susceptible bulk produced 27.825 Gb of clean data, showing a Q30 score \u0026gt;94% and GC content of 41\u0026ndash;72%. Alignment results indicated 137,400,008 and 186,646,126 reads from the resistant and susceptible bulks successfully mapped to the Chinese Spring reference genome (Ref v2.1), respectively. These high-quality sequences were deemed suitable for downstream analyses. To reduce background noise, original ED values were quartic-transformed and used as correlation values, with a Loess curve applied for fitting. The threshold was determined using the quantile method, which entails arranging all fitted values in ascending order and designating the ED values of SNP markers exceeding the 99% as the threshold during the screening process. Using this threshold, the ED algorithm identified only one estimated candidate region situated at the terminal end of chromosome arm 2BL. (Fig. 2a; Fig. 2b; Table S1). This result shows that \u003cem\u003ePmLsy-93\u003c/em\u003e resides on chromosome arm 2BL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular mapping of \u003cem\u003ePmLsy-93\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eand prediction of candidate genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the BSR-Seq results, we further validated the candidate interval by screening 12 known markers surrounding the region of interest on chromosome arm 2BL and developing eight SSR and INDEL markers derived from the BSR-Seq data. Eleven markers, including \u003cem\u003eL43177\u003c/em\u003e, \u003cem\u003eL11500\u003c/em\u003e, \u003cem\u003eL93-293\u003c/em\u003e, \u003cem\u003eCIT02g-20\u003c/em\u003e, \u003cem\u003eS93-2\u003c/em\u003e, \u003cem\u003eS93-46\u003c/em\u003e, \u003cem\u003eL93-277\u003c/em\u003e, \u003cem\u003eCIT02g-4\u003c/em\u003e, \u003cem\u003eL93-21\u003c/em\u003e, \u003cem\u003eCINAU140\u003c/em\u003e and \u003cem\u003eL93-260\u003c/em\u003e (Table S2), showed polymorphism between resistant and susceptible parents as well as bulks. Subsequently, based on the genotyping results of these markers for the 250 F\u003csub\u003e2:3\u003c/sub\u003e families, a genetic linkage map for \u003cem\u003ePmLsy-93\u003c/em\u003e was constructed. (Fig. 3a). According to the linkage analysis results, \u003cem\u003ePmLsy-93\u003c/em\u003e was mapped to a 1.5 cM interval flanked by markers \u003cem\u003eS93-2\u003c/em\u003e and \u003cem\u003eS93-46\u003c/em\u003e (Fig. 3a; Fig. 3b). According to the gene annotation results, a total of 75 genes with high confidence were identified in this interval. Among them, six genes were related to disease resistance and regarded as candidate genes for \u003cem\u003ePmLsy-93\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscovery and analysis of DEGs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBSR-Seq analysis identified 3,140 DEGs between the two bulks, including 2,214 down-regulated and 926 up-regulated genes compared with the susceptible bulk (Fig. 4a). GO analysis indicated that the DEGs were mainly related to three functional domains. In molecular function, key activities included manganese ion binding, oxidoreductase activity, heme binding, and hydrolase activity acting on glycosyl bonds, which are critical for enzymatic reactions and metabolic regulation. In cellular component, enriched terms such as extracellular region and cell wall macromolecule catabolic process highlighted the involvement of DEGs in extracellular interactions and cell wall remodeling, which are vital for pathogen defense and structural integrity. In biological process, DEGs were linked to processes like response to water, response to biotic stimulus, and chitin catabolic process, indicating their roles in stress adaptation and pathogen resistance (Fig. 4b; Table S3). Furthermore, according to KEGG pathway enrichment analysis, the DEGs were significantly enriched in metabolic and signaling pathways associated with plant defense and stress tolerance. The MAPK signaling pathway-plant and plant-pathogen interaction pathways were enriched, highlighting their pivotal roles in defense mechanisms against pathogens. Additionally, glutathione metabolism and photosynthesis-antenna proteins were identified, emphasizing DEGs involved in mitigating oxidative damage during infection and involvement in energy production and light harvesting, respectively (Fig. 4c; Table S4). The integration of GO and KEGG analyses underscores the multifaceted roles of DEGs in plant defense, stress response, and metabolic regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of disease resistance-related genes in spelt accession Lsy-93 via qRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further identify candidate genes, DEGs within the candidate interval of 710.73 to 721.07 Mb were analyzed. A total of 123 DEGs were found in this interval, and they were considered the essential candidates for powdery mildew resistance in accession Lsy-93. Expression patterns of six DEGs in the resistant parent Lsy-93 and susceptible parent TN18 were analyzed at various time points (hpi) after inoculation with \u003cem\u003eBgt\u003c/em\u003e isolate E09. All genes were found to be induced by \u003cem\u003eBgt\u003c/em\u003e invasion. The transcription levels of \u003cem\u003eTraesCS2B0361276100\u003c/em\u003e (encoding disease resistance protein) and \u003cem\u003eTraesCS2B0361276200\u0026nbsp;\u003c/em\u003e(encoding disease resistance protein) reached peak transcript levels at 0.5 hpi in resistant parent Lsy-93. The remaining four genes \u003cem\u003eTraesCS2B03G1285300\u0026nbsp;\u003c/em\u003e(encoding serine threonine-protein kinase), \u003cem\u003eTraesCS2B0361283800\u0026nbsp;\u003c/em\u003e(encoding serine threonine-protein kinase), \u003cem\u003eTraesCS2B0361284900\u0026nbsp;\u003c/em\u003e(encoding serine threonine-protein kinase) and \u003cem\u003eTraesCS2B0361290400\u0026nbsp;\u003c/em\u003e(encoding protein kinase domain)\u003cem\u003e\u0026nbsp;\u003c/em\u003ereached their peak transcript levels at 24 hpi or even later. Additionally, most of these genes displayed significantly higher expression in the early stage of inoculation in Lsy-93 compared to TN18 (Fig. 5; Table S2), suggesting potential regulatory mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening and confirmation of molecular markers for MAS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the potential of the linked markers in MAS, 46 susceptible elite wheat cultivars/lines were tested with markers \u003cem\u003eL93-293\u003c/em\u003e,\u003cem\u003e\u0026nbsp;CIT02g-20\u0026nbsp;\u003c/em\u003eand \u003cem\u003eL93-277\u003c/em\u003e. All these markers amplified polymorphic bands between Lsy-93 and most of wheat accessions, displaying their widely effective potential in different wheat backgrounds (Fig. 6; Table S5). Hence, when the \u003cem\u003ePmLsy-93\u003c/em\u003e was transferred into these genotypes, the corresponding markers could be employed to accelerate the transfer of \u003cem\u003ePmLsy-93\u003c/em\u003e in wheat breeding practices.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSpelt, an ancient hexaploid wheat subspecies, has emerged as a valuable genetic resource for enhancing powdery mildew resistance in modern wheat breeding programs. As a crop historically cultivated in Europe, spelt possesses a rich reservoir of disease resistance genes that have been largely lost in elite wheat cultivars due to intense selection for agronomic traits.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Of particular interest is its potential contribution to combating powdery mildew. Spelt accession Lsy-93 showed resistance against powdery mildew across all growth stages. Through genetic mapping in this study, a dominant \u003cem\u003ePm\u003c/em\u003e gene, provisionally designated \u003cem\u003ePmLsy-93\u003c/em\u003e, was identified to confer this resistance. Using BSR-Seq and genetic markers, \u003cem\u003ePmLsy-93\u003c/em\u003e was mapped to a 10.34 Mb physical interval (710.73-721.07 Mb) on chromosome arm 2BL referred to Chinese spring reference genome (Ref v2.1). Six formally designated \u003cem\u003ePm\u003c/em\u003e genes, including \u003cem\u003ePm6\u003c/em\u003e from \u003cem\u003eT. timopheevii\u003c/em\u003e, have been reported to located on chromosome arm 2BL, with diverse gene donors contributing to this locus. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], \u003cem\u003ePm33\u003c/em\u003e from \u003cem\u003eT. persicum Vav.\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003ePm51\u003c/em\u003e from a \u003cem\u003eThinopyrum ponticum\u003c/em\u003e introgression line [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], \u003cem\u003ePm52\u003c/em\u003e from Chinese wheat cultivar Liangxing 99 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], \u003cem\u003ePm63\u003c/em\u003e from Iranian wheat landrace PI 628024 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and \u003cem\u003ePm64\u003c/em\u003e from wild emmer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, several temporarily named genes have been also identified, such as \u003cem\u003ePmQ\u003c/em\u003e, \u003cem\u003eMlZec1\u003c/em\u003e, \u003cem\u003eMlAB10\u003c/em\u003e, \u003cem\u003ePmKN0816\u003c/em\u003e, \u003cem\u003ePmJM809\u003c/em\u003e and so on. Compared with those documented genes, \u003cem\u003ePmLsy-93\u003c/em\u003e (710.73-721.07 Mb) was derived from spelt, which have a unique source. Furthermore, \u003cem\u003ePmLsy-93\u003c/em\u003e could be clearly distinguished from four of them: \u003cem\u003ePm6\u003c/em\u003e (698.3-699.2 Mb), \u003cem\u003ePm33\u003c/em\u003e (773.2-784.3 Mb), \u003cem\u003ePm52\u003c/em\u003e (581.0-585.0 Mb) and \u003cem\u003ePm64\u003c/em\u003e (699.2-710.3 Mb). It overlapped with \u003cem\u003ePm51\u003c/em\u003e (709.8-739.4 Mb) and \u003cem\u003ePm63\u003c/em\u003e (710.3-723.4 Mb). In the future, clarifying their allelism relationships through allelism tests and cloning these genes will be necessary.\u003c/p\u003e \u003cp\u003ePreviously studies have identified several major \u003cem\u003ePm\u003c/em\u003e genes and quantitative trait loci (QTLs) in spelt that confer either race-specific or broad-spectrum resistance, including notable examples such as \u003cem\u003ePm54\u003c/em\u003e and \u003cem\u003ePmTm4\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These genetic elements often trigger defense mechanisms like cell wall reinforcement and hypersensitive responses, providing multiple layers of protection against pathogen invasion [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].In this study, a total of 3,140 DEGs were detected between the two bulks. Compared with the susceptible bulk, 2,214 genes were downregulated and 926 were upregulated. The GO and KEGG enrichment analyses provide comprehensive insights into the functional roles and metabolic pathways associated with the DEGs in response to biotic stress. The GO analysis revealed that DEGs are predominantly involved in molecular functions which are critical for enzymatic reactions and stress responses. Additionally, cellular components like the extracellular region and cell wall-related processes suggest active remodeling during pathogen defense. Biological processes, including response to biotic stimuli and chitin catabolism, further emphasize the activation of defense mechanisms against pathogens [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The KEGG analysis highlighted key pathways linked to plant immunity. The enrichment of MAPK signaling and plant-pathogen interaction pathways underscores the importance of signal transduction in defense responses [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Furthermore, glutathione metabolism indicates a role in mitigating oxidative stress, while photosynthesis-related pathways suggest a balance between defense and energy metabolism [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These findings were consistent with previously studies, including \u003cem\u003ePmW762\u003c/em\u003e, \u003cem\u003ePmJM23\u003c/em\u003e and \u003cem\u003ePmL709\u003c/em\u003e [31\u0026ndash; \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLooking forward, spelt accessions will continue to play a critical role in sustainable wheat disease management systems. The ultimate goal of mining disease-resistant genes is to apply them in wheat breeding and production. In this study, \u003cem\u003ePmLsy-93\u003c/em\u003e is derived from spelt wheat, which shares the A, B and D genomes with common wheat. Spelt has maintained greater allelic diversity in resistance-related genes due to its adaptation to diverse environments and lower selection pressure. Compared with the alien species of wheat without any of A, B, D genomes, it can be easily integrated in wheat genome without linkage drags [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, \u003cem\u003ePmLsy-93\u003c/em\u003e has high potential in wheat breeding to combat powdery mildew. MAS now enables more precise transfer of target genes while minimizing linkage drag. To promote the transfer of \u003cem\u003ePmLsy-93\u003c/em\u003e by means of MAS, three PCR-based and co-dominant markers were screened to be available for efficiently tracing \u003cem\u003ePmLsy-93\u003c/em\u003e once it has been introduced into those susceptible cultivars/breeding lines. Furthermore, the strategic integration of spelt genetic resources with resistance loci from wild relatives may offer a robust, multi-layered defense system against rapidly evolving pathogen populations. With the climate change and intensive agricultural practices, the conservation and exploitation of genetic diversity in ancient wheat varieties such as spelt is emerging as a critical component of global food security strategies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, a single dominant \u003cem\u003ePm\u003c/em\u003e gene \u003cem\u003ePmLsy-93\u003c/em\u003e was identified in the spelt accession Lsy-93. The results provide valuable insights into plant-pathogen defense mechanisms and lay an important foundation for subsequent molecular breeding efforts to enhance crop disease resistance.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpelt wheat Lsy-93\u0026nbsp;is sourced from Prof. Hongxing Xu, Henan University, Kaifeng, China. Because Lsy-93 displayed continuous resistance to powdery mildew across all growth stages, it was selected as the resistant parent for hybridization with the susceptible parent TN 18. And F\u003csub\u003e1\u003c/sub\u003e, F\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand\u003csub\u003e\u0026nbsp;\u003c/sub\u003eF\u003csub\u003e2:3\u003c/sub\u003e progenies were produced for genetic \u0026nbsp;and BSR-Seq analysis. The wheat cultivar TN 18 was also planted as a susceptible control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotypic evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe population F\u003csub\u003e1\u003c/sub\u003e, F\u003csub\u003e2\u003c/sub\u003e, and F\u003csub\u003e2:3\u003c/sub\u003e progenies of the cross Lsy-93 \u0026times; TN 18 was planted in the 128-cell rectangular trays, and 5\u0026ndash;8 seeds were sown in each cell, which were inoculated with \u003cem\u003eBgt\u003c/em\u003e isolate E09 for genetic analysis. The incubator was set at 20℃ for 14 hours daily and 18℃ for 10 hours nightly. Upon reaching the two-leaf stage, seedlings underwent phenotypic observations. When observing phenotypes, ITs 0\u0026ndash;4 scale was used. Ratings from 0 to 2 indicated resistance, while ratings of 3 and 4 signified susceptibility [35]. The evaluation procedure was repeated three times to guarantee the reliability of the results. For F\u003csub\u003e2:3\u0026nbsp;\u003c/sub\u003efamilies, at least 20 seeds per family were planted and assessed. At the same time, TN 18 were randomly distributed and planted in each tray.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eGenetic analysis\u003c/h3\u003e\n\u003cp\u003eFor genetic analysis, 10 F₁, 253 F₂, and 252 F₂:₃ families (approximately 20 seedlings per family) were planted and inoculated with \u003cem\u003eBgt\u003c/em\u003e isolate E09 for resistance phenotyping. The Chi-squared test (\u003cem\u003e\u0026chi;\u003c/em\u003e\u0026sup2;) was used to check goodness-of-fit to expected the Mendelian segregation ratio for monogenic inheritance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopic evaluation of powdery mildew resistance responses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo additionally analyze hyphal morphology post-invasion by \u003cem\u003eBgt\u003c/em\u003e isolate E09, 10 seedlings Lsy-93 and 10 seedlings TN 18 were planted separately. At 0, 0.5, 2, 4, 12, 24, 48, and 72 hours post inoculation (hpi) after \u003cem\u003eBgt\u003c/em\u003e invasion, 2 cm seedling leaf samples were collected from Lsy-93 and TN 18. Samples were fixed using Carnoy\u0026apos;s fixative (anhydrous alcohol-glacial acetic acid, 3:1, v/v) immediately and remained at an environmental temperature of 37℃ for 24 hours. Before observation under the microscope, the samples were stained in 5 mL of 0.6% (w/v) Coomassie blue solution for 5 min and then wash the leaves with distilled water to remove the reagent residue [36]. Finally, an Axioscope 5 microscope (ZEISS, Oberkochen, Germany) was used for sample analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and BSR-Seq library preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 10 days post inoculation (dpi), leaf tissues were sampled (10 plants per family) from 30 homozygous resistant and 30 homozygous susceptible F\u003csub\u003e2:3\u003c/sub\u003e lines. Equal aliquots of these samples were combined to generate resistant (R) and susceptible (S) bulks, from which total RNA was extracted using the RNA Simple Total RNA Kit (Tiangen, Beijing, China). To purify mRNA from total RNA samples, oligo(dT) beads were added to the mixture, leveraging the specific binding between the poly-A tails of mRNA and the beads. The mixture was then gently shaken and incubated at 18\u0026ndash;20\u0026deg;C for 15\u0026ndash;30 minutes. Subsequently, the beads were washed with a washing solution to remove non-mRNA components such as rRNA and tRNA. Before this purification step, RNA quality was initially evaluated by agarose gel electrophoresis, spectrophotometry, and other methods. Following successful mRNA purification, fragmentation was achieved using either chemical reagents or enzymatic digestion. After fragmentation, cDNA synthesis was carried out using reverse transcriptase in conjunction with random primers. The obtained cDNA was subjected to end repair and adapter ligation, after which the ligation products were amplified by PCR to generate the final sequencing library. Subsequently, the library was quantified and its quality was assessed to ensure it met the requirements for sequencing.[37].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequencing was carried out on the Illumina HiSeq4000 platform (Illumina HiSeq4000) at Tcuni Bioscience (Chengdu, China). At Tcuni Bioscience, Large quantities of sequencing data were produced as short-read sequences. The raw data pre-processing steps included read quality score assessment, low-quality reads elimination, and adapter sequence trimming. Subsequently, the cleaned data were aligned to the Chinese Spring 2.1 reference genome to prepare for subsequent analysis using the STAR software. [38].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of the candidate interval and genetic mapping of the target \u003cem\u003ePm\u003c/em\u003e gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the target interval, the following procedures were implemented. Initially, SNP calling for the resistant and susceptible bulks was carried out using the software packages GATK v4.2.3.0 and SAMtools v1.17 [39]. This was followed by the quantification of allele frequencies for each SNP across the two bulks. Following the calculation of SNP index values using MutMap, the \u0026Delta;SNP index for each SNP was defined as: \u0026Delta;SNP index = SNP index (resistant) \u0026ndash; SNP index (susceptible) [40-41]. Subsequently, the linkage between SNPs and the target gene(s) was determined using Bayesian analysis integrated with the Euclidean distance (ED) algorithm. Potential candidate intervals were then identified according to previously reported procedures. [37].\u003c/p\u003e\n\u003cp\u003eFollowing the identification of the candidate interval, molecular markers within it were utilized to map the target gene. Polymorphic genetic markers were applied to genotype the F\u003csub\u003e2:3\u003c/sub\u003e families from the Lsy-93 and TN 18 cross for mapping. Subsequently, phenotypic and genotypic data were collected, followed by linkage analysis using Mapmaker 3.0b [42]. A LOD score threshold of 3.0 was applied. And the genetic map of the \u003cem\u003ePm\u003c/em\u003e gene in Lsy-93 was constructed using Mapdraw v2.1 [43].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDEGs analysis associated with the powdery mildew resistance in Lsy-93\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter BSR-Seq, software like HTSeq v2.0.3 or featureCounts v2.0.1 estimated transcript abundances mapped to each gene to quantify candidate gene expression levels. Statistical comparisons between experimental groups were made using EBSeq v3.5 to identify DEGs with a fold change \u0026ge; 2 and FDR (false discovery rate) \u0026lt; 0.01. For functional characterization of the identified DEGs, annotation was conducted using GO and KEGG databases, combined with an R package specifically designed for analyzing DEGs. [44]. Finally, an enrichment analysis of DEGs in terms of defined pathways, cell structures, or molecular activities provided insights into the role of candidate genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eqRT-PCR was performed to validate the expression of DEGs potential linked to disease resistance within the target interval. At 0, 0.5, 2, 4, 12, 24, 48, and 72 hpi, leaves with a length of 3-5 cm of Lsy-93 and TN 18 were collected, respectively. Total RNA was isolated from samples using the RNAsimple Total RNA Kit (Tiangen, Beijing, China). Primers were designed using Primer5 software. Approximately 2 \u0026micro;g of RNA was reverse transcribed into cDNA by employing a FastQuant RT Kit (Tiangen, Beijing, China). qRT-PCR reactions were prepared with SYBR Premix Ex Taq (Takara, China) and conducted on a Bio-Rad CFX Connect real-time PCR system (BIO-RAD, Hercules, USA) following standard protocols. The \u003cem\u003eTaActin\u0026nbsp;\u003c/em\u003egene was chosen as the reference gene. Gene expression levels were finally calculated using the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method [45]. Each sample underwent triplicate testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of linked markers applied in MAS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMarkers that amplify different bands in the tested wheat genotypes and Lsy-93 can be identified as suitable for MAS. Through the detection of 46 elite wheat cultivars/lines \u0026nbsp;collected from major wheat-producing areas in China, three closely linked markers suitable for MAS were screened out.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eBgt\u003c/em\u003e \u003cem\u003eBlumeria graminis\u003c/em\u003e f. sp. \u003cem\u003eTritici\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNBS-LRR Nucleotide-binding site leucine-rich repeat\u003c/p\u003e\n\u003cp\u003eBSR-Seq Bulked segregant RNA sequencing\u003c/p\u003e\n\u003cp\u003eSNP Single nucleotide polymorphism\u003c/p\u003e\n\u003cp\u003eDEGs Differentially expressed genes\u003c/p\u003e\n\u003cp\u003eGO Gene ontology\u003c/p\u003e\n\u003cp\u003eCOG Clusters of orthologous groups\u003c/p\u003e\n\u003cp\u003eKEGG Kyoto encyclopedia of genes and genomes\u003c/p\u003e\n\u003cp\u003eIT Infection type\u003c/p\u003e\n\u003cp\u003eED Euclidean distance\u003c/p\u003e\n\u003cp\u003eSSR Simple sequence repeat marker\u003c/p\u003e\n\u003cp\u003ehpi Hours post-inoculation\u003c/p\u003e\n\u003cp\u003eTN18 Tainong 18\u003c/p\u003e\n\u003cp\u003eMAS Marker-assisted selection\u003c/p\u003e\n\u003cp\u003eQTL Quantitative trait loci\u003c/p\u003e\n\u003cp\u003edpi Days post inoculation\u003c/p\u003e\n\u003cp\u003eFDR False discovery rate\u003c/p\u003e\n\u003cp\u003eqRT-PCR Real-time quantitative PCR\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePM, YJ and HZ conceived the research. HZ, YJ, JL, JL, NY, HX, QS, TY, JZ, LL performed the experiments and collected data. NS, JL and JL performed data analyses. NY developed the experimental materials. HZ, YJ and PM wrote and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by Key R\u0026amp;D Program of Shandong Province (2024LZGC001), National Modern Wheat Industry Technology System, Yantai Comprehensive Experimental Station project (CARS-03-62), National Natural Science Foundation of China (32301923), Natural Science Foundation of Shandong Province, China (ZR2023QC203) and Henan Province Natural Science Foundation (232300420003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available in the National Center for Biotechnology Information repository, SRA: SUB15322822.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYantai Academy of Agricultural Sciences, Yantai 265500, China\u003c/p\u003e\n\u003cp\u003eNina Sun, Jiansheng Lu, Qingpeng Sun, Tangyu Yuan, Linzhi Li, Huanchun Zhang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYantai Key Laboratory of Characteristic Agricultural Biological Resources Conservation and Germplasm Innovative Utilization, College of Life Sciences, Yantai University, Yantai 264005, China\u003c/p\u003e\n\u003cp\u003eJiatong Li, Ningning Yu, Jiadong Zhang, Yuli Jin, Pengtao Ma\u003c/p\u003e\n\u003cp\u003eSchool of Life Sciences, Henan University, Kaifeng 475004, China\u003c/p\u003e\n\u003cp\u003eHongxing Xu\u003c/p\u003e\n\u003cp\u003eCorresponding authors\u003c/p\u003e\n\u003cp\u003eCorrespondence to Pengtao Ma, Yuli Jin or Huanchun Zhang\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZ\u0026ouml;rb C, Ludewig U, Hawkesford MJ. 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Hereditas. 2003;25(3):317-321. \u003c/li\u003e\n\u003cli\u003eSherman BT, Hao M, Qiu J, Jiao X, Baseler MW, Lane HC, Imamichi T, Chang W. DAVID: A web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Res. 2022;50(W1):W216\u0026ndash;W221.\u003c/li\u003e\n\u003cli\u003eBourras S, McNally KE, Ben-David R, Parlange F, Roffler S, Praz CR, Oberhaensli S, Menardo F, Stirnweis D, Frenkel Z, et al. Multiple avirulence loci and allele-specific effector recognition control the \u003cem\u003ePm3\u003c/em\u003e race-specific resistance of wheat to powdery mildew. Plant Cell. 2015;27(10):2991\u0026ndash;3012.\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":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Spelt wheat, Powdery mildew, Gene mapping, GO, KEGG, MAS","lastPublishedDoi":"10.21203/rs.3.rs-6722793/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6722793/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003ePowdery mildew, a widespread fungal disease caused by \u003cem\u003eBlumeria graminis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e(\u003cem\u003eBgt\u003c/em\u003e), seriously threatens the yield and quality of wheat. The most effective and sustainable approach to disease control is utilizing resistance genes and unraveling their underlying molecular mechanisms. Spelt (\u003cem\u003eTriticum aestivum\u003c/em\u003e ssp. \u003cem\u003eSpelta\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e2n = 6x = 42, AABBDD),\u003cstrong\u003e an ancient hexaploid wheat subspecies, has emerged as a valuable genetic resource for enhancing powdery mildew resistance in modern wheat breeding programs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e Spelt accession Lsy-93 demonstrated resistance against powdery mildew at the whole-growth stage. Genetic analysis revealed that this resistance is conferred by a single dominant gene, tentatively designated as \u003cem\u003ePmLsy-93\u003c/em\u003e. Bulked segregant RNA sequencing (BSR-seq) and molecular markers positioned \u003cem\u003ePmLsy-93\u003c/em\u003e within a 1.5 cM interval flanked by markers \u003cem\u003eS93-2\u003c/em\u003e and \u003cem\u003eS93-46\u003c/em\u003e. Nine genes in this interval were associated with disease resistance and were considered as the candidate genes of \u003cem\u003ePmLsy-93\u003c/em\u003e. Furthermore, a total of 3,140 differentially expressed genes (DEGs) were identified between the two bulks, with 2,214 down-regulated and 916 up-regulated genes relative to the susceptible bulk. The integration of gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis underscores the multifaceted roles of these DEGs in plant defense, stress response, and metabolic regulation. Then, six genes, encoding disease resistance protein, serine threonine-protein kinase, or protein kinase domain,\u003cem\u003e \u003c/em\u003ewere induced with \u003cem\u003eBgt \u003c/em\u003einvasion via qRT-PCR. Three closely linked or co-segregated markers \u003cem\u003eL93-293\u003c/em\u003e, \u003cem\u003eCIT02g-20\u003c/em\u003e and \u003cem\u003eL93-277\u003c/em\u003e were confirmed to be available for marker-assisted selection (MAS) of \u003cem\u003ePmLsy-93\u003c/em\u003e in breeding programs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eThis study successfully pinpointed critical genetic loci and candidate genes associated with powdery mildew resistance in the spelt wheat accession Lsy-93. The results provide valuable insights into plant-pathogen defense mechanisms and lay an important foundation for subsequent molecular breeding efforts to enhance crop disease resistance.\u003c/p\u003e","manuscriptTitle":"Identification and characterization of the powdery mildew resistance gene in spelt accession Lsy-93 ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-06 11:47:01","doi":"10.21203/rs.3.rs-6722793/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-19T09:40:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-18T14:51:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-05T03:14:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210785921082110166786323948071806579052","date":"2025-06-04T23:08:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70601096125954969092316435272294366094","date":"2025-06-03T14:20:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-03T07:33:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-28T13:49:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-26T14:32:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-26T14:29:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-05-22T08:14:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"005b5999-edbf-4452-8563-cce73cb1eb43","owner":[],"postedDate":"June 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T16:33:59+00:00","versionOfRecord":{"articleIdentity":"rs-6722793","link":"https://doi.org/10.1186/s12870-025-07040-5","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-08-18 16:29:16","publishedOnDateReadable":"August 18th, 2025"},"versionCreatedAt":"2025-06-06 11:47:01","video":"","vorDoi":"10.1186/s12870-025-07040-5","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07040-5","workflowStages":[]},"version":"v1","identity":"rs-6722793","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6722793","identity":"rs-6722793","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}