Discovery of a novel adult-plant resistance locus conferring broad-spectrum stripe rust immunity in spelt wheat and its suppression in bread wheat

Discovery of a novel adult-plant resistance locus conferring broad-spectrum stripe rust immunity in spelt wheat and its suppression in bread wheat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discovery of a novel adult-plant resistance locus conferring broad-spectrum stripe rust immunity in spelt wheat and its suppression in bread wheat Vincent Fetterley, Jasneet Singh, Jujhar Singh Gill, Samuel Holden, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8991239/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Adult-plant resistance (APR) genes provide durable, non-race-specific resistance against wheat rust diseases. We identified a novel APR locus in the spelt wheat cultivar CDC Silex that confers non-race-specific near-immune adult-plant resistance to stripe rust. The trait segregates as a single dominant gene and bulked segregant analysis sequencing (BSA-seq) mapped the locus, QYr.cbl-5D , to a single 0.31 Mb interval on chromosome 5D, confirming monogenic inheritance. Interestingly, introgression into bread wheat suppresses this resistance, suggesting the presence of a dominant suppressor factor in the bread wheat genome. We generated a scaffold-scale assembly of CDC Silex to annotate the locus and its flanking region on 5D. Within a ± 1 Mb interval flanking the QYr.cbl-5D peak, six genes were identified, of which five had no predicted function and one encodes a C2H2 zinc-finger transcription factor. Future work will aim to clone this APR and its suppressor in bread wheat. Flanking and linked SNPs from this region will enable marker-assisted selection to deploy this APR locus, providing a durable resistance resource to support sustainable wheat breeding and reduce reliance on fungicides. Wheat adult-plant resistance (APR) stripe rust yellow rust BSAseq. Figures Figure 1 Figure 2 Figure 3 Key message A novel non-race-specific adult-plant resistance locus, QYr.cbl-5D , from spelt wheat confers near-immune resistance to stripe rust. The resistance conferred by QYr.cbl-5D is suppressed in bread wheat from an unknown suppressor. Introduction Stripe rust (caused by Puccinia striiformis f. sp. tritici ( Pst )), also known as yellow rust, is a devastating foliar disease of wheat that can reduce yields by over 70% in severe epidemics (Chen 2020 ). Genetic resistance is the most effective and environment-friendly strategy for managing stripe rust or other wheat rusts. Wheat rust resistance genes are typically classified as seedling/all stage resistance (ASR) or adult-plant resistance (APR) based on the growth stage at which it is effective (Brar et al. 2019 ). All-stage resistance (ASR) genes are usually single, race-specific genes that confer complete protection at all growth stages but deploying them singly often leads to “boom-and-bust” cycles as new pathogen races can easily overcome the resistance (Schwessinger 2017 ). In contrast, APR genes express only in older plants (usually after flag leaf expansion) and typically confer partial, non-race-specific resistance. APR often slows pathogen development rather than completely stopping it via hypersensitive response, which allows some sporulation and thus exerts lower selection pressure on the pathogen to escape recognition by the resistance gene. Because APR genes tend to be quantitative and broad-spectrum, they are generally considered durable compared to single gene ASR. This durability is further enhanced by the pleiotropic effects of several APR genes, which confer broad-spectrum resistance to multiple pathogens, for example, Yr18/Lr34/Sr57 , Yr29/Lr46/Sr58 , and Yr46/Lr67/Sr55 (Kolmer et al. 2015 ; Krattinger et al. 2009 ; Moore et al. 2015 ). To date, only a few adult-plant stripe rust resistance genes have been cloned in wheat and the cloned APR genes differ from the classical R-genes such as NLR (nucleotide-binding leucine-rich repeat) and RLK (receptor like kinase) protein encoding genes (Fu et al. 2009 ; Krattinger et al. 2009 ; Moore et al. 2015 ). For example, Yr18 encodes an ATP-binding cassette (ABC) transporter and Yr46 encodes a sugar transporter (Krattinger et al. 2009 ; Moore et al. 2015 ). Each of these APR genes on its own provides partial resistance, so breeders often tend to pyramid multiple APR genes to achieve a high level of durable additive resistance. Finally, combining APR genes with additive effects can help breed wheat varieties with durable, more effective broad-spectrum rust resistance across different environments (Zelba et al. 2024 ). Spelt wheat ( Triticum aestivum subsp. spelta L.) is a hexaploid subspecies of bread wheat. It has been cultivated as a specialty crop in organic farming systems, because of its tolerance to environmental stressors and lower input requirements (Caballero et al. 2004 ). Spelt typically has lower yield and tougher, tenacious glumes that require dehulling, but it can readily be crossed with modern bread wheat since both share the same hexaploid genome. Spelt has been an important reservoir of resistance genes for bread wheat improvement, having contributed leaf rust resistance genes ( Lr44, Lr65, Lr71 ) and a stripe rust resistance gene Yr5 (Dyck and Sykes, 1994 ; Law 1976 ; Mohler et al. 2012 ; Singh et al. 2013 ). Notably Yr5 represents a rare example of an NLR conferring all-stage resistance effective against all known Pst isolates, so far with no apparent evolution to overcome the resistance (Brar and Kutcher 2016 ; Ghanbarnia et al. 2021 ; Holtz et al. 2013 ; Kumar et al. 2012 ; Liu et al. 2017 ; Wang et al. 2022 ). In this study, we identified a novel APR locus ( QYr.cbl-5D ) effective against stripe rust and derived from the spelt wheat cultivar, CDC Silex, which is nearly immune to stripe rust at the adult-plant stage. Through genetic analysis of crosses with susceptible spelt wheat and bread wheat cultivars, we investigated the inheritance and expression of this resistance and mapped the underlying locus. Our findings reveal a previously uncharacterized stripe rust APR derived from spelt wheat, demonstrating its potential as a valuable source of durable resistance for bread wheat improvement. Materials and methods Plant material A total of 15 bi-parental mapping populations were developed and used for adult-plant stripe rust phenotyping in the present study (Table 1 ). Seven crosses were made using spelt wheat lines CDC Silex (resistant, released cultivar), 10Spelt17 (resistant, an advanced breeding line), CDC Origin (susceptible, released cultivar), and Avocet (susceptible, bread wheat cultivar) as parents. The spelt wheat lines were developed at the Crop Development Centre of University of Saskatchewan. The spelt lines CDC Silex and 10Spelt17 both exhibited near-immune reactions to stripe rust at the adult-plant stage (Fig. 1 a, b), while exhibiting full susceptibility at the seedling stage (data not shown), and were therefore used as resistant parents, whereas CDC Origin, another spelt cultivar, was used as the susceptible spelt parent (Fig. 2 c). Avocet, a bread wheat cultivar with universal susceptibility to stripe rust, was included as a susceptible control in phenotyping studies and as a susceptible bread wheat parent (Fig. 1 c, 2 c). The crosses were made at the University of British Columbia (UBC) South Campus greenhouses during 2021 and 2022. The resulting F 1 plants were grown in one-gallon pots in the greenhouse to produce large F 2 populations. The F 2 plants from each cross were grown, advanced to F 2:3 generation, and phenotyped in Conviron® growth chambers. Two populations from cross between Avocet/CDC Silex (AvSi) and Avocet/10Spelt17 (AvSp) were further advanced to the F 5 generation through single-seed descent to establish recombinant inbred line (RIL) populations. Table 1 Bi-parental populations developed and used for stripe rust phenotyping in the present study. Female parent Male parent Population name Population size AAC Brandon 10Spelt17 BrSp F 1 6 10Spelt17 AAC Brandon SpBr F 1 3 CDC Silex AAC Brandon SiBr F 1 2 CDC Silex CDC Zorba SiZo F 1 2 10Spelt17 CDC Silex SpSi F 1 2 Avocet 10Spelt17 AvSp F 2 134 Avocet CDC Silex AvSi F 2 158 CDC Origin CDC Silex OrSi F 2 138 CDC Silex Avocet SiAv F 2 181 10Spelt17 Avocet SpAv F 2 84 10Spelt17 CDC Silex SpSi F 2 65 CDC Silex CDC Origin SiOr A F 2 138 CDC Silex CDC Origin SiOr B F 2 350 Avocet 10Spelt17 AvSp F 2:3 133 Avocet CDC Silex AvSi F 2:3 128 CDC Silex Avocet SiAv F 2:3 156 10Spelt17 Avocet SpAv F 2:3 81 10Spelt17 CDC Silex SpSi F 2:3 65 Avocet 10Spelt17 AvSp F 5 112 Avocet CDC Silex AvSi F 5 85 After mapping the resistance locus, F 1 plants were generated by crossing the resistant spelt lines with Canada Western Red Spring (CWRS) wheat cultivars AAC Brandon (carrying Yr18 ), as well as with the moderately susceptible spelt cultivar CDC Zorba (Table 1 ). Stripe rust phenotyping Indoor phenotyping Plants were grown in Conviron® growth chambers under a 21/16°C day/night (16 h/8 h) regime. CDC Silex and 10Spelt17 were susceptible to all tested Pst isolates at the seedling stage (data not shown), stripe rust resistance was evaluated at the adult-plant stage, when the flag leaf had fully emerged (Z49–Z69; Zadoks et al. 1974 ). CDC Silex and 10Spelt17 exhibited consistent immunity to stripe rust in inoculated (with multiple races) field stripe rust nurseries over years (Fig. 1 ), therefore, we assumed the resistance as non-race-specific. For inoculation, Pst urediniospores were suspended in mineral oil (Novec 7100™) and sprayed onto the flag and penultimate leaves using a compressed-air sprayer. Inoculated plants were incubated in a dew chamber at 10°C, 100% relative humidity, and complete darkness for 24–36 h to promote spore germination and initial infection. Plants were then transferred back to the growth chambers and maintained under a 16/11°C Day/night (16 h/8 h) cycle. Disease assessments were conducted when the susceptible control Avocet displayed full susceptibility (infection type (IT) = 9) usually at 14–16 days post inoculation (Fig. 2 c), following the 0–9 scale described by Line and Qayoum (1992) and further described by Gill et al. ( 2025 ). To minimize observer bias, all plants were rated by a single observer. Unless otherwise stated, inoculations were performed using an in-house Pst admixture named “BC Mix”, representing a mixture of stripe rust races collected from susceptible wheat plants in the UBC, Vancouver stripe rust nursery over the 2020 growing season. Phenotyping in the field nursery : Field phenotyping was conducted at the Totem Plant Science Field Station (UBC, Vancouver) during the summers of 2022 and 2023. The AvSp F 2:3 and AvSi F 2:3 families were evaluated in 2022, while the SiAv F 2:3 , SpAv F 2:3 , AvSi F 5 , AvSp F 5 , and SpSi F 2:3 populations were assessed in 2023. Each F 2:3 family was planted as 30 cm rows, spaced 30 cm apart between families and 45 cm between rows, following an augmented design where parental lines served as checks and were planted every 15–20 plots. The F 5 RILs were planted as hill plots with identical spacing. The susceptible cultivar Avocet was used as a border to enhance disease pressure and positive infection control. The nursery was not inoculated as southern British Columbia has high natural stripe rust disease pressure (Brar et al. 2019 ). Disease scoring was performed once Avocet reached near full susceptibility on the flag leaf. F 2:3 families were classified as homozygous resistant (Hr), segregating (Seg), or homozygous susceptible (Hs), whereas F 5 RILs were rated for infection response as resistant (R), moderately resistant (MR), moderately susceptible (MS), or susceptible (S) according to Roelfs et al. ( 1992 ) and Gill et al. ( 2025 ). Disease severity (0–100%) was estimated using the modified Cobb’s scale (Gill et al. 2025 ; Peterson et al. 1948 ). Adult-plant responses were recorded twice over a 10-day period following the initial scoring. Statistical analysis Segregation of stripe rust resistance in F 2 population was evaluated using chi-square (χ 2 ) goodness-of-fit tests by classifying plants as resistant (IT = 0–6) or susceptible (IT = 7–9) and comparing observed counts with expected Mendelian ratios. For F 2:3 populations, families were classified as homozygous resistant (Hr), segregating (Seg), or homozygous susceptible (Hs), and χ 2 tests were used to assess deviation from expected segregation ratios. Differences in mean F 2 infection types between populations among F 2:3 family classes were assessed using analysis of variance (ANOVA). Statistical significance was declared at P <0.05. Whole-genome re-sequencing and bulk-segregant analysis To decipher the genetics of APR in spelt wheat, additional F 1 plants derived from the cross between CDC Origin (susceptible spelt) and CDC Silex (resistant spelt) were advanced to the F 2 generation for phenotyping and BSA-seq. Two F 2:3 populations were developed and phenotyped using the indoor stripe rust phenotyping protocol described above: the first in 2022, consisting of 138 F₂ lines, and the second in 2024, comprising 350 F₂ lines, from which a total of 29 highly resistant (IT = 0–3) and 21 highly susceptible (IT = 7–9) plants were selected and used in the resistant (R) and susceptible (S) bulks, respectively. Genomic DNA was extracted from susceptible and resistant F₂ plants, along with both parental lines, using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. DNA quality was assessed using Nanodrop spectrophotometry and agarose gel electrophoresis, quantified with a Qubit fluorometer (Thermo Fisher Scientific, Waltham, USA), and normalized to 1 ng/µL. Tagmentation was conducted using the Illumina Tagment DNA TDE1 Enzyme and Buffer Kit (Illumina, San Diego, USA) with 1 µL of diluted DNA (1 µL DNA, 3.3964 µL ddH 2 O, 0.504 µL TD Buffer, and 0.0996 µL TDE1). After 15 minutes incubation at 55°C, libraries were indexed with Nextera i7 and i5 adapters, each with unique combinations. The reaction mixture included 2 µL of i7/i5 index mix (2.5 µM each), 12.5 µL of NEB 2× Taq Master Mix, and 5.5 µL of ddH₂O. Thermocycling was performed under the following conditions: 72°C for 3 min, 95°C for 1 min, 18 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 3 min, followed by a final extension at 72°C for 3 min. Library quality was verified by agarose gel electrophoresis and quantified using a Qubit fluorometer. Libraries were normalized to 6 ng/µL, and 10 µL from each were pooled to form the R and S bulks, with corresponding parental libraries added to both pools. Each bulk (180 µL) was cleaned using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and eluted in 30 µL of ddH₂O. For size selection, 80 µL ddH₂O was added to 20 µL of each bulk, followed by 50 µL of SPRI beads (0.5×) (Beckman Coulter Life Sciences, Brea, USA). After 10 minutes of binding, the supernatant was transferred to a second tube containing 30 µL of SPRI beads (0.8× total), incubated for another 10 minutes, and then washed twice with freshly prepared 70% ethanol. The size-selected, bulked libraries were eluted in 30 µL ddH₂O, normalized to 3 ng/µL, and sequenced on the NovaSeq X Plus platform (Novogene, Tianjin, China). Sequencing generated 7,236,354 to 96,109,886 reads per individual in the R bulk and 20,470,792 to 95,254,716 reads per individual in the S bulk. Based on total mapped reads and assuming a genome size of 15.5 Gb, the R and S bulks achieved approximately 14.4× and 9.9× effective genome coverage, respectively. Reads were aligned to the spelt reference genome PI190962 (Walkowiak et al. 2020 ) and to an in-house HiFi whole-genome assembly of CDC Silex (C. Pozniak, unpublished) using Burrows–Wheeler Aligner (BWA) (Li and Durbin 2010 ). Variants were called using the Genome Analysis Toolkit (GATK) pipeline (Van der Auwera et al. 2013 ). Initial variant calling against the spelt reference genome PI190962 identified 84.4 million variant sites in the R bulk (79.8 million SNPs and 4.66 million InDels) and 65.5 million variant sites in the S bulk (62.1 million SNPs and 3.49 million InDels) ( Table S1 ). Transition-to-transversion (Ts/Tv) ratios were 2.78 and 2.96 in the R and S bulks, respectively, and multiallelic sites numbered 79.5 million in R and 61.9 million in S ( Table S1 ) . Variant calling of the combined R and S bulks yielded 68,271,461 SNPs. To obtain reliable and informative markers for BSA-seq, a series of stringent filtering steps were applied. SNPs were first filtered based on reference allele frequency (0.22≤ REF_FRQ ≤ 0.78) to remove loci with extreme allele frequencies unlikely to be informative for segregation-based mapping, eliminating 51,594,971 SNPs. Variants with total read depth 100 were also removed to minimize false positives arising from repetitive regions or misaligned reads, eliminating 37,541 SNPs ( Table S2 ). Additional quality filtering removed SNPs with per-sample read depth < 7 (447,062 SNPs) and genotype quality (GQ) 30 between bulks were excluded (46,092 SNPs) to reduce bias caused by uneven sequencing coverage ( Table S2 ). After all filtering steps, 1,417,555 high-confidence SNPs remained and were used for ΔSNP-index calculation in the R package QTLseqR (Mansfeld and Grumet 2018 ). Annotation of the physical region carrying QYr.cbl-5D To characterize the physical region underlying QYr.cbl-5D the in-house scaffold-level genome assembly of the resistant parent CDC Silex was used (C. Pozniak, unpublished). A 50-Mb genomic interval flanking the QTL peak was extracted and annotated using the BRAKER pipeline (Hoff et al. 2016 ), with publicly available wheat RNA-seq (RanSeq) data used as transcriptomic evidence to support gene prediction. Results Stripe rust resistance in CDC Silex and 10Spelt17 is allelic and non-race-specific CDC Silex and 10Spelt17 lines were developed by the bread wheat breeding program at CDC, University of Saskatchewan. Spelt wheat germplasm in the program is limited to a small number of founder lines as it is not the main focus of the program. Both these spelt wheat lines share a common spelt wheat landrace parent, PI 348771, in their pedigrees (Table 2 ). The landrace PI 348771 is believed to be the source of stripe rust resistance in CDC Silex and 10Spelt17. Due to shared parent in the pedigree, our hypothesis was that the resistance in CDC Silex and 10Spelt17 is identical or allelic. To test the hypothesis, we performed an allelism test by crossing CDC Silex and 10Spelt17 and phenotyping the F 1 s and F 2 progeny (n = 65) with stripe rust. All 65 SpSi F 2 plants from the cross exhibited resistance (IT = 0–3) to stripe rust when phenotyped in growth chambers. Similarly, the corresponding F 2:3 families, evaluated in the 2023 stripe rust nursery under natural infection, consistently displayed resistance. These findings confirm that the stripe rust resistance in 10Spelt17 and CDC Silex is conferred by either the same gene or complementary alleles of the same gene, likely inherited from their common ancestor, PI 348771. Therefore, for further experiments, we focused on crosses derived from CDC Silex. Table 2 Pedigree of the spelt wheat accessions used in the study. Accession Pedigree CDC Silex PI 348771/Oberkulmer 10Spelt17 00Spelt5/00Spelt20 00Spelt5 PI 348771/Oberkulmer 00Spelt20 PI 348771/Oberkulmer CDC Origin RL5407/Winter spelt (F5)//PGR8801 CDC Zorba RL5407/Common winter spelt The APR in CDC Silex and 10Spelt17 is described as non-race-specific because the lines were consistently rated R (IT = 0–1) (Fig. 1 , 2 c) in non-inoculated field stripe rust nurseries in Saskatoon, Saskatchewan and Vancouver, British Columbia between 2016–2025 (data not shown). The southern British Columbia is a hotspot of stripe rust in North America, and the region harbors many diverse Pst races (Brar et al. 2019 ; Chen 2005; Holden et al. 2023). Consistent adult-plant resistance in these field nurseries suggest that the APR carried by CDC Silex and 10Spelt17 is non-race-specific. Genetics and mapping of APR in spelt wheat cv. CDC Silex To understand the genetic regulation of the APR, CDC Silex was crossed with a susceptible spelt wheat parent CDC Origin. The F 2 progeny from the cross (CDC Origin/CDC Silex (OrSi A )) were initially phenotyped in 2022 (Fig. 2 a). These experiments indicated a single dominant gene regulating the APR, with 128 resistant and 10 susceptible plants. To confirm this, a large set of F 2s from the same cross (OrSi B ) were screened for resistance to stripe rust at the adult plant stage (Fig. 2 a). The progeny segregation confirmed to the expected 3:1 ratio for a single dominant gene (χ² = 0.86, df = 1, P = 0.35), indicating that APR in CDC Silex is governed by a single dominant locus (Table 3 ). Table 3 Chi-square goodness-of-fit test for segregation ratios expected under one- or two-gene models conferring dominant resistance in F₂ populations inoculated with a mixture of Puccinia striiformis f. sp. tritici races (BC Mix). Population Expected Ratio (R:S) Expected Observed Chi-Square P-value OrSi B 03:01 262.5 : 87.5 270 : 80 0.86 0.35 (n = 350) 15:01 328.1 : 21.9 270 : 80 163.9 < 0.001 AvSi 03:01 100.5:33.5 86:48:00 8.3682 < 0.01 (n = 134) 15:01 125.6:8.4 86:48:00 199.98 < 0.01 AvSp 03:01 118.5:39.5 126:32:00 1.8987 0.1682 (n = 158) 15:01 148.1:9.9 126:32:00 52.876 < 0.01 To map the physical location providing resistance, BSAseq was performed. Whole-genome resequencing of the resistant (R) and susceptible (S) bulks generated high-quality datasets with effective genome coverages of 14.4× and 9.9×, respectively. Alignment to the PI190962 spelt reference genome identified 84.4 million and 65.5 million variant sites in the R and S bulks, respectively, including both SNPs and InDels ( Table S1 ). Transition-to-transversion (Ts/Tv) ratios of 2.78 (R) and 2.96 (S) indicated high-confidence variant calls consistent with expected values in wheat. A substantial proportion of sites were multiallelic (79.5 million in R and 61.9 million in S) ( Table S1 ), reflecting the expected allelic diversity and segregation within the pooled F 2 populations. Collectively, these findings confirmed a high-quality variant detection and appropriate heterozygosity levels for F 2 -derived bulks. A total of 68,271,461 SNPs were identified in the combined dataset of the resistant (R) and susceptible (S) bulks. After stringent quality filtering (see Materials and Methods), 1,417,555 high-confidence SNPs were retained for downstream ΔSNP-index analysis ( Table S2 ). A single major locus ( QYr.cbl-5D ) was mapped on chromosome (5D) (Fig. 3 a; Table 4 ), spanning from 139.12 Mb to 139.42 Mb with a total length of approximately 0.31 Mb and comprising 17 SNPs (Fig. 3 b; Table 4 ). The region showed an average tricube ΔSNP-index of − 0.53, with a peak tricube ΔSNP-index of − 0.55 at position 139,421,704 bp, indicating strong enrichment of the susceptible allele in this genomic interval (Table 4 ). This pattern suggests the presence of a putative locus contributing to stripe rust resistance or susceptibility in the studied population. Table 4 Summary of loci identified in BSA-seq analysis. Locus Chr Start (bp) End (bp) Length(bp) No of Linked SNPs Peak tricubeΔ (SNP-index) Position of Peak (bp) Avg. tricube Δ(SNP-index) QYr.cbl-5D 5D 139115840 139421704 305864 17 -0.5487 139421704 -0.5275 QYr.cbl-5D-1 5D 141313106 153597143 12284037 2 0.5337 153597143 0.476 QTLs were detected based on tricube Δ(SNP-index) values calculated from resistant and susceptible bulks in the F₂ population. Significant regions were identified at α = 0.05 To minimize reference-assembly bias and to account for potential structural variation or sequence divergence associated with the resistant parent, the bulks were additionally aligned to an in-house developed scaffold-level genome assembly of the resistant parent, CDC Silex. Using the same filtering criteria as described above, 334,671 high-confidence SNPs were retained from an initial set of 41,658,484 SNPs and used for ΔSNP-index estimation in QTLseqR. This analysis also identified a single major peak on chromosome (5D) ( Fig. S1 a; Table 4 ). The locus ( QYr.cbl-5D-1 ) spanned approximately 12 Mb, from 141.13 Mb to 153.59 Mb, and was supported by two SNPs passing the applied thresholds ( Fig. S1 b; Table 4 ). The difference in the physical span of the QTL interval between the two analyses is primarily attributable to differences in SNP density and marker resolution. Alignment to the PI190962 reference genome yielded approximately 1.4 million high-confidence SNPs genome-wide, enabling finer mapping resolution and delimiting the QTL to a narrow ~ 0.31 Mb interval. In contrast, alignment to the scaffold-level CDC Silex assembly resulted in approximately 0.3 million high-confidence SNPs, substantially reducing local marker density and consequently broadening the QTL interval to ~ 12 Mb. Analysis of the QTL locus in PI190962 and CDC Silex Although the physical coordinates of the QTL differed between analyses performed against the PI190962 reference genome and the CDC Silex assembly, gene content comparison revealed strong synteny between the two intervals. Notably, the same set of annotated genes present in ± 1 Mb window of the QYr.cbl-5D at ~ 139 Mb in PI190962 was located at ~ 146 Mb in the CDC Silex assembly ( Table S3 ). This indicates that an orthologous physical segment was consistently enriched in both analyses, and that the observed positional shift reflects assembly-specific differences in physical coordinate placement rather than a biologically distinct locus. Within ± 1 Mb region flanking the QYr.cbl-5D , a total of six genes were detected, of which five had no predicted function and one gene, TraesTSP5D01G127300 , encodes a fusion protein with HSP domain fused to a C2H2 zinc-finger transcription factor (TF) ( Table S3; Fig. 3 c). Notably, TraesTSP5D01G127300 is the only gene located within the core 0.31 Mb QTL interval. This gene encodes an 1,812 bp transcript with three exons. The first exon codes for a DnaJ HSP domain, while the second and third exons encode a C2H2 zinc-finger transcription factor (Fig. 3 c). An identical copy of the TraesTSP5D01G127300 gene is present in the wheat D-genome donor, Aegilops tauschii ( AET5Gv20276300 ) and bread wheat ( TraesCS5D02G116700 ), demonstrating that this fused domain is evolutionary conserved. In A. tauschii , the orthologue gene is reported to have six alternate transcripts ( Table S4 ). This may be true in spelt wheat as well but remains untested due to the limited availability of genomics resources in the species. The public expression databases on stripe rust in wheat indicate that this gene is upregulated after 24 hours of infection in two independent datasets ( Fig. S3d, e ), further suggesting a role in the immune response. Suppression of QYr.cbl-5D in bread wheat At the beginning of the project, we chose bread wheat cv. Avocet as susceptible parent to develop mapping populations because Avocet is universally used in stripe rust mapping studies and that the bread and spelt wheats are readily crossable. Therefore, CDC Silex and 10Spelt17, were crossed with Avocet. The F 1 plants derived from these crosses consistently exhibited susceptibility, with infection types of 7–8 when challenged with Pst BC Mix or single isolates: W034 and W003, (Brar and Kutcher 2016 ) regardless of whether the resistant spelt was used as the female (cytoplasmic) or male (pollen) parent. Conversely, all F 1 plants from the spelt x spelt cross between CDC Silex (resistant) and CDC Origin (susceptible) were resistant. This led us to the use of susceptible spelt wheat CDC Origin in our aforementioned studies. To determine if the susceptibility is genotype specific or not, we also crossed CDC Silex and 10Spelt17 with a hard red spring wheat cv. AAC Brandon (carries Yr18/Lr34 ). Similarly, AAC Brandon/10Spelt17 or AAC Brandon/CDC Silex (n = 6) F 1 adult-plants showed variable infection responses ranging from 2–6. In general, F 1 plants from bread wheat x spelt wheat crosses displayed resistance patterns similar to the bread wheat parent, whereas ‘10Spelt17/CDC Silex’ (n = 2) F 1s showed complete resistance (IT = 0–1). Crosses between resistant and either intermediate (CDC Zorba) or susceptible (CDC Origin) spelt accessions also produced fully resistant F 1 plants (IT = 0–3). Despite inconclusive phenotypes of F 1s , we also screened F 2 s and F 2:3 families from Avocet/CDC Silex and Avocet/10Spelt17 crosses. In AvSi F 2 population, among 134 individuals, 86 were resistant (IT = 0–6) and 48 were susceptible (IT = 7–9) (Fig. 2 a). The higher frequency of resistant plants indicated dominant resistance, although the segregation ratio did not fit expected Mendelian ratios for one (χ² = 8.37, P < 0.01) or two (χ² = 199.98, P < 0.01) genes (Table 3 ). Notably, only two F 2 individuals displayed resistance levels comparable to the resistant parent (IT = 2) which was likely a disease escape. The AvSp F 2 population exhibited a similar trend, with 126 resistant and 32 susceptible individuals (Fig. 2 a), fitting a single dominant gene model (χ² = 1.90, P = 0.17) (Table 3 ). As in AvSi, only a few plants (n = 3) showed resistance somewhat comparable (IT = 3–4) to the resistant parent in the AvSp population. This was in contrast to the pattern seen in the F 2 population of spelt x spelt crosses (OrSi A & OrSi B ), where most of the resistant plants showed resistance comparable to the resistant parent (Fig. 2 a), thus, indicating suppression of QYr.cbl-5D in bread wheat background. To determine the zygosity of resistance loci, seeds from AvSi and AvSp F 2 individuals were advanced and evaluated as F 2:3 families. Among 133 AvSp F 2:3 families, 44 were homozygous resistant (Hr), 46 segregating (Seg), and 43 homozygous susceptible (Hs). This distribution deviated significantly from ratios expected for one (1Hr:2Seg:1Hs; χ² = 12.7, P < 0.01) or two genes (1Hr:14Seg:1Hs; χ² = 340.5, P < 0.01) (Table 5 ). A similar pattern was observed for the AvSi F 2:3 families, with 19 Hr, 38 Seg, and 71 Hs families, also deviating from Mendelian expectations (1 gene: χ² = 63.4, P < 0.01; 2 genes: χ² = 560.1, P < 0.01) (Table 5 ). Table 5 Chi-square goodness-of-fit test for expected ratio for one or two resistance gene conferring resistance for F 2:3 populations. All populations were phenotyped under natural infection in stripe rust field nursery during the 2022 (AvSi, AvSp) or the 2023 (SiAv, SpAv) growing season. AvSi: Avocet/CDC Silex; AvSp: Avocet/10Spelt17; SiAv: CDC Silex/Avocet; SpAv:10Spelt17/Avocet. Population Expected Ratio (Hr:Seg:Hs) Expected Observed Chi-Square P-value AvSi 1:2:1 32:64:32 19:38:71 63.4 < 0.01 (n = 128) 1:14:1 8:112:8 19:38:71 560.1 < 0.01 AvSp 1:2:1 33:67:33 44:46:43 12.7 < 0.01 (n = 133) 1:14:1 8:117:8 44:46:43 340.5 < 0.01 SiAv 1:2:1 39:78:39 24:86:46 7.9 < 0.05 (n = 156) 1:14:1 11:136:11 24:86:46 174.3 < 0.01 SpAv 1:2:1 20:41:20 27:43:11 6.6 < 0.05 (n = 81) 1:14:1 5:71:5 27:43:11 113.0 < 0.01 Phenotypes in the F 2:3 populations were not strongly predictive/correlated of their respective F 2 parent ratings (Fig. 2 b). Mean F 2 ratings differed significantly between AvSi and AvSp (F = 14.4, P < 0.01), averaging 5.88 (SD = 1.36) and 5.25 (SD = 1.38), respectively. Within populations, F 2 plants classified as Hr or Seg produced significantly lower mean ITs than Hs individuals, though Hr and Seg did not differ significantly. For AvSp, no significant difference in F 2 ITs was observed between categories, while in AvSi, Hs F 2:3 families were derived from significantly more susceptible F 2 plants. Only 31.8% (14/44) of AvSp Hr families and 31.6% (6/19) of AvSi Hr families originated from resistant F 2 plants. The reciprocal F 2:3 populations, SiAv and SpAv, consisted of 24 Hr, 86 Seg, and 46 Hs and 27 Hr, 43 Seg, and 11 Hs families, respectively. None of these fit expected ratios for single or two-gene models. Finally, 112 AvSp and 85 AvSi F 2:5 RILs were phenotyped in field nurseries in 2023. However, even the F 5 generation did not fit to a single-gene model. The consistent partial suppression of QYr.cbl-5D in the bread wheat background observed across different bread wheat genotypes, reciprocal crosses and population stages, from segregating F 2 to inbred F 5 , indicates the presence of a suppressor factor or gene in the bread wheat genome. QYr.cbl-5D is a novel gene and does not overlap with any known loci QYr.cbl-5D was mapped to the short arm of chromosome 5D and delimited to a physical interval centered at ~ 139 Mb on the reference genome. Examination of this region revealed no overlap with any previously reported stripe rust resistance genes or quantitative trait loci on chromosome 5D. Among the known loci on this chromosome, Yr70 , a high-temperature adult-plant resistance gene, is located at approximately 8.86 Mb on chromosome arm 5DS (Bansal et al. 2017), which is physically distant from the QYr.cbl-5D interval (Fig. 3 c). Yr40 , an alien introgression from Aegilops umbellulata conferring race-specific all-stage resistance, has been genetically mapped to chromosome 5D (Kuraparthy et al. 2009 ); however, its precise physical position on the wheat reference genome remains undefined due to the non-collinearity of the alien introgressed chromatin. Discussion Spelt wheat is a proven reservoir of diverse rust resistance genes which can be used in bread wheat improvement, having contributed major loci such as Yr5 and several leaf rust genes (Dyck and Sykes 1994 ; Law 1976 ; Mohler et al. 2012 ; Singh et al. 2013 ). Continued mining of spelt germplasm is therefore well justified, particularly as stripe rust populations remain dynamic and widely distributed in major wheat-growing regions, including Canada (Brar et al. 2019 ; Holden et al. 2025 ). QYr.cbl-5D confers a near-immune adult-plant response with non-race-specific, broad-spectrum effectiveness against stripe rust. If successfully expressed in bread wheat, this locus could complement APR genes already deployed in Canadian wheat breeding, such as Yr18/Lr34 and Yr46/Lr67 , to further strengthen durable stripe rust resistance. A key finding of this study is that resistance identified in a donor background did not manifest predictably following introgression into elite germplasm. The reduced expression of the spelt-derived APR in bread wheat backgrounds is most consistent with epistasis, potentially involving a dominant suppressor present in common wheat. Suppressors of rust resistance have been documented previously and can mask otherwise effective loci, complicating both genetic analysis and breeding deployment (Hiebert et al. 2020 ; Jin et al. 2023 ; Wu et al. 2015 ). Such interactions are particularly relevant in hexaploid wheat, where homeologous genes and regulatory networks can buffer or override allele effects. These observations highlight the importance of evaluating resistance alleles directly in target breeding backgrounds and of identifying genetic modifiers that constrain their expression (Poland et al. 2009; Wu et al. 2015 ). The candidate gene content in the QYr.cbl-5D locus supports a resistance mechanism consistent with an emerging theme in adult-plant resistance: unusual or non-canonical genes providing strong, durable resistance through an unknown mechanism that is not traditional extracellular or intracellular recognition by receptor kinase or NLR proteins. No candidate gene in the region matches these expectations for R-gene predicted domain structure and function. A putative candidate encodes a fusion protein containing a DnaJ/HSP domain and a plant-specific C2H2 zinc-finger domain, suggesting roles in protein homeostasis and transcriptional regulation rather than classical immune receptor-mediated recognition. This interpretation is consistent with the observation that several cloned APR genes encode non-canonical proteins, including transporters such as Lr34/Yr18 and Lr67/Yr46 (Krattinger et al. 2009 ; Moore et al. 2015 ). Transcriptional regulators are increasingly recognized as key components of wheat-rust interactions, with both positive and negative effects on disease outcomes (Zheng et al. 2023 ; Huang et al. 2024 ). Notably, a C2H2 zinc-finger transcription factors, TaZFP8-5B , was shown to negatively regulate stripe rust resistance as its overexpression led to increased fungal infection in inoculated plants (Huang et al. 2024 ), demonstrating that members of this family can modulate wheat-rust interactions. Also, the presence of a conserved ortholog of the putative candidate gene in bread wheat and Aegilops tauschii suggests that functional differences may arise from allele-specific variation or regulatory context rather than gene presence alone. From a breeding perspective, this study highlights both the opportunity and the complexity of exploiting non-elite germplasm for durable disease resistance. The QYr.cbl-5D represents a potentially valuable addition to the stripe rust resistance portfolio, though its practical utility will depend on understanding and overcoming suppression in bread wheat backgrounds. Declarations Funding The funding for this research was provided by Saskatchewan Ministry of Agriculture (Project ID: ADF20200205), Saskatchewan Wheat Development Commission (Project ID: 173-201127), Alberta Grains (Project ID: 2021AWC100B), and Manitoba Crop Alliance (Project ID: MCA2184). Author Contribution GSB conceived the idea and supervised the project. GSB and VF developed the mapping populations. VF and JSG performed the stripe rust phenotyping. VF, ML extracted DNA and prepared the libraries. VF and JS performed bioinformatic and other data analyses. VF, JS, and GSB wrote the manuscript. VF and JS prepared figures and tables. JE, CJP generated the CDC Silex genome assembly. CJP and PJH made significant intellectual contributions, provided the spelt wheat lines, and the genome assembly of CDC Silex. All authors reviewed and approved the final manuscript. Acknowledgement The first author (VF) of the paper acknowledges various scholarships and awards that he has received during his tenure of M.Sc. degree program. The authors acknowledge the support and help received from members of the Cereal Breeding Lab (CBL). Data Availability Data are included in the article and its supplementary files with an exception of genome assembly of CDC Silex . References Brar GS, Kutcher HR (2016) Race characterization of Puccinia striiformis f. sp. tritici , the cause of wheat stripe rust, in Saskatchewan and southern Alberta, Canada, and virulence comparison with races from the United States. Plant Dis 100:1744–1753. Brar GS, Fetch T, McCallum BD, Hucl PJ, Kutcher HR (2019) Virulence dynamics and breeding for resistance to stripe, stem, and leaf rust in Canada since 2000. 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Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26:589–595. Liu T, Wan A, Liu D, Chen X (2017) Changes of races and virulence genes in Puccinia striiformis f. sp. tritici in the United States from 1968 to 2009. Plant Dis 101:1522–1532. Mansfeld BN, Grumet R (2018) QTLseqr: an R package for bulk segregant analysis with next-generation sequencing. Plant Genome 11:e180006. Metzger RJ, Silbaugh BA (1970) Inheritance of resistance to stripe rust and its association with brown glume color in Triticum aestivum L. ‘PI 178383’. Crop Sci 10:567–568. Mohler V, Singh D, Singrün C, Park RF (2012) Characterization and mapping of Lr65 in spelt wheat ‘ Altgold Rotkorn’ . Plant Breed 131:252–257. Moore JW, Herrera-Foessel S, Lan C, Schnippenkoetter W, Ayliffe M, Huerta-Espino J, Lillemo M, Viccars L, Milne R, Periyannan S, Kong X (2015) A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat. Nat Genet 47:1494–1498. Peterson RF, Campbell AB, Hannah AE (1948) A diagrammatic scale for estimating rust intensity on leaves and stems of cereals. Can J Res 26:496–500. Roelfs AP, Singh RP, Saari EE (1992) Rust diseases of wheat: concepts and methods of disease management. CIMMYT, Mexico. Schwessinger B (2017) Fundamental wheat stripe rust research in the 21st century. New Phytol 213:1625–1631. Singh D, Mohler V, Park RF (2013) Discovery, characterisation and mapping of wheat leaf rust resistance gene Lr71 . Euphytica 190:131–136. Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E (2013) From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline. Curr Protoc Bioinformatics 43:11.10.1–11.10.33. Walkowiak S, Gao L, Monat C, et al. 2020. Multiple wheat genomes reveal global variation in modern breeding. Nature. 588:277–283. Wang M, Wan A, Chen X (2022) Race characterization of Puccinia striiformis f. sp. tritici in the United States from 2013 to 2017. Plant Dis 106:1462–1473. Wu L, Xia X, Rosewarne GM, Zhu H, Li S, Zhang Z, He Z (2015) Stripe rust resistance gene Yr18 and its suppressor gene in Chinese wheat landraces. Plant Breed 134:634–640. Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the growth stages of cereals. Weed Res 14:415–421. Zelba O, Wilderspin S, Hubbard A, Nellist CF, Mortensen AK, Schulz P, Huerta-Espino J, Singh R, Sørensen CK (2024) The adult plant resistance genes Yr18 , Yr29 and Yr46 in spring wheat showed significant effects against important yellow rust races under north-west European field conditions. Euphytica 220:107. Zheng P, Liu M, Pang L, Sun R, Yao M, Wang X, Kang Z, Liu J (2023) Stripe rust effector Pst21674 compromises wheat resistance by targeting transcription factor TaASR3 . Plant Physiol 193:2806–2824. Supplementary Figures and Tables Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.tif Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 May, 2026 Reviews received at journal 17 Mar, 2026 Reviewers agreed at journal 09 Mar, 2026 Reviewers invited by journal 09 Mar, 2026 Editor assigned by journal 07 Mar, 2026 Submission checks completed at journal 28 Feb, 2026 First submitted to journal 27 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-8991239","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":604674227,"identity":"8eb6c382-9934-4a8e-88ff-58c2273798b8","order_by":0,"name":"Vincent Fetterley","email":"","orcid":"","institution":"University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"","lastName":"Fetterley","suffix":""},{"id":604674229,"identity":"1a46fc05-b440-4516-8814-3d89f6a09ac2","order_by":1,"name":"Jasneet Singh","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Jasneet","middleName":"","lastName":"Singh","suffix":""},{"id":604674230,"identity":"b766495d-e350-4a28-b9cb-ab70f5d285f5","order_by":2,"name":"Jujhar Singh Gill","email":"","orcid":"","institution":"University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Jujhar","middleName":"Singh","lastName":"Gill","suffix":""},{"id":604674231,"identity":"11b0ae44-a409-400a-8a62-f946102d3f8a","order_by":3,"name":"Samuel Holden","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Holden","suffix":""},{"id":604674236,"identity":"4d899bd7-2c07-4f65-ae36-fac28a3c99c7","order_by":4,"name":"Meng Li","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Li","suffix":""},{"id":604674237,"identity":"2abbfd8d-61db-4574-ad20-581225d668bc","order_by":5,"name":"Jennifer Ens","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Ens","suffix":""},{"id":604674238,"identity":"35c62b7a-70bf-4edc-82cd-5774a96cb4f3","order_by":6,"name":"Pierre Jan Hucl","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Pierre","middleName":"Jan","lastName":"Hucl","suffix":""},{"id":604674240,"identity":"1ca6966f-15fc-4dcd-8482-ff46a64c8253","order_by":7,"name":"Curtis Jerry Pozniak","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Curtis","middleName":"Jerry","lastName":"Pozniak","suffix":""},{"id":604674241,"identity":"df69e800-07f9-48b9-ba59-24eb624ffd15","order_by":8,"name":"Gurcharn Singh Brar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYJACZjApASIqGBj4JEjTcoaBgQ3EOEC0FsY2IrTIR+Q+/FxQcy+PQbr34efKeYfl2aSbj33+UMMgz9+AXYvhjXRj6RnHiosZZI4bS57ddtiwTeZY8owDxxgMZ+CwynBGGhszD1tCYoNEGoNk47bDjG0SOcYMB9gYEnC5DqLlH1gL88/GOYft2yTyPzMc+MeQII9Di7wEUAtvG1gLm2Rjw+FEoC3MDAfbGBIMcGgx4HnGLM3bl5AI9AKbZcOx9GQgw5jhbJ+E4UZctrSnMX7m+ZaQ2C/dxnyzocbatl+6+TFDxTcbeTlctsDE2dAkcKcB+QacUqNgFIyCUTAKoAAAzwNU2HdE+Q0AAAAASUVORK5CYII=","orcid":"","institution":"University of Alberta","correspondingAuthor":true,"prefix":"","firstName":"Gurcharn","middleName":"Singh","lastName":"Brar","suffix":""}],"badges":[],"createdAt":"2026-02-27 19:39:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8991239/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8991239/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104517347,"identity":"9c594f44-6eeb-4e41-83f1-e5dfb091bced","added_by":"auto","created_at":"2026-03-12 17:57:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50754461,"visible":true,"origin":"","legend":"\u003cp\u003eStripe rust on resistant spelt (a: CDC Silex; b: 10Spelt17) and susceptible Avocet (c) lines due to natural infection in the UBC Totem Field stripe rust nursery during the 2023 field season. For all three lines, the infection shown is typical of what is observed under natural and controlled conditions, for all stripe rust isolate.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8991239/v1/e1a435052bdb770dedce237e.png"},{"id":104517345,"identity":"6226ff1c-b28a-47f3-9046-dad50842be84","added_by":"auto","created_at":"2026-03-12 17:57:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12203818,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Distribution of stripe rust infection type in the bi-parental F\u003csub\u003e2\u003c/sub\u003e populations screened in this study. For all populations, an admixture of \u003cem\u003ePst\u003c/em\u003e races was used for inoculation. (b) To investigate inheritance of resistance in bread wheat background, the resistant spelt by Avocet populations were evaluated as F\u003csub\u003e3\u003c/sub\u003e families. Infection type of the F\u003csub\u003e2 \u003c/sub\u003eplants were evaluated based on the 0-9 scale, while the F\u003csub\u003e3\u003c/sub\u003e plants were evaluated based on disease severity (Gill et al. 2025; Line and Qayoum 1992; Roelfs et al. 1992). (c) Stripe rust infection phenotypes on leaves of the resistant line CDC Silex and the susceptible lines CDC Origin and Avocet at 14–16 days post-inoculation.\u003c/p\u003e\n\u003cp\u003eAvSi: Avocet/CDC Silex; AvSp: Avocet/10Spelt17; OrSi: CDC Origin/CDC Silex; Hr: Homozygous Resistant; Seg: Segregating; Hs: Homozygous Susceptible.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8991239/v1/63d7e8a983e1cf259c559044.png"},{"id":104517344,"identity":"9749f8ab-47c7-4fde-a170-4040b93e8992","added_by":"auto","created_at":"2026-03-12 17:57:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3671681,"visible":true,"origin":"","legend":"\u003cp\u003eMapping and candidate gene identification for the spelt-derived stripe rust resistance locus on chromosome 5D. (a) Genome-wide tricube ΔSNP index plot generated from bulked segregant analysis (BSA) of R and S F₂ bulks in the CDC Origin/CDC Silex population. A clear peak on chromosome 5D indicates the resistance-associated region. (b) Zoomed-in view of the 5D ΔSNP index plot showing the major peak corresponding to the resistance locus. (c) Annotated genes in ±1MB region of the identified 5D locus. Domain structure of putative candidate gene (\u003cem\u003eTraesTSP5D01G127300\u003c/em\u003e) encoding a C2H2 zinc- transcription factor. (d \u0026amp; e) Expression pattern of \u003cem\u003eTraesCS5D02G116700\u003c/em\u003e, bread wheat orthologue of\u003cem\u003eTraesTSP5D01G127300,\u003c/em\u003e in response to stripe rust infection at different time points in two independent studies.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8991239/v1/615ef5573b341988b11118d7.png"},{"id":104782049,"identity":"8960fcee-2ad8-4faf-b0cc-a03f2e726df3","added_by":"auto","created_at":"2026-03-17 07:56:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":61750271,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8991239/v1/6b97bdc9-7338-47af-a150-e7be0b19b39f.pdf"},{"id":104517348,"identity":"3adc6ae2-6c97-428f-b826-bba5eb495526","added_by":"auto","created_at":"2026-03-12 17:57:25","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":30816712,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8991239/v1/aab0b1119dcd89f4d45e5bdb.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discovery of a novel adult-plant resistance locus conferring broad-spectrum stripe rust immunity in spelt wheat and its suppression in bread wheat","fulltext":[{"header":"Key message","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eA novel non-race-specific adult-plant resistance locus,\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eQYr.cbl-5D\u003cem\u003e, from spelt wheat confers near-immune resistance to stripe rust. The resistance conferred by\u0026nbsp;\u003c/em\u003eQYr.cbl-5D\u003cem\u003e\u0026nbsp;is suppressed in bread wheat from an unknown suppressor.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eStripe rust (caused by \u003cem\u003ePuccinia striiformis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e (\u003cem\u003ePst\u003c/em\u003e)), also known as yellow rust, is a devastating foliar disease of wheat that can reduce yields by over 70% in severe epidemics (Chen \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Genetic resistance is the most effective and environment-friendly strategy for managing stripe rust or other wheat rusts. Wheat rust resistance genes are typically classified as seedling/all stage resistance (ASR) or adult-plant resistance (APR) based on the growth stage at which it is effective (Brar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). All-stage resistance (ASR) genes are usually single, race-specific genes that confer complete protection at all growth stages but deploying them singly often leads to \u0026ldquo;boom-and-bust\u0026rdquo; cycles as new pathogen races can easily overcome the resistance (Schwessinger \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast, APR genes express only in older plants (usually after flag leaf expansion) and typically confer partial, non-race-specific resistance. APR often slows pathogen development rather than completely stopping it via hypersensitive response, which allows some sporulation and thus exerts lower selection pressure on the pathogen to escape recognition by the resistance gene. Because APR genes tend to be quantitative and broad-spectrum, they are generally considered durable compared to single gene ASR. This durability is further enhanced by the pleiotropic effects of several APR genes, which confer broad-spectrum resistance to multiple pathogens, for example, \u003cem\u003eYr18/Lr34/Sr57\u003c/em\u003e, \u003cem\u003eYr29/Lr46/Sr58\u003c/em\u003e, and \u003cem\u003eYr46/Lr67/Sr55\u003c/em\u003e (Kolmer et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Krattinger et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Moore et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo date, only a few adult-plant stripe rust resistance genes have been cloned in wheat and the cloned APR genes differ from the classical R-genes such as NLR (nucleotide-binding leucine-rich repeat) and RLK (receptor like kinase) protein encoding genes (Fu et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Krattinger et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Moore et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For example, \u003cem\u003eYr18\u003c/em\u003e encodes an ATP-binding cassette (ABC) transporter and \u003cem\u003eYr46\u003c/em\u003e encodes a sugar transporter (Krattinger et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Moore et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Each of these APR genes on its own provides partial resistance, so breeders often tend to pyramid multiple APR genes to achieve a high level of durable additive resistance. Finally, combining APR genes with additive effects can help breed wheat varieties with durable, more effective broad-spectrum rust resistance across different environments (Zelba et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpelt wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e subsp. \u003cem\u003espelta\u003c/em\u003e L.) is a hexaploid subspecies of bread wheat. It has been cultivated as a specialty crop in organic farming systems, because of its tolerance to environmental stressors and lower input requirements (Caballero et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Spelt typically has lower yield and tougher, tenacious glumes that require dehulling, but it can readily be crossed with modern bread wheat since both share the same hexaploid genome. Spelt has been an important reservoir of resistance genes for bread wheat improvement, having contributed leaf rust resistance genes (\u003cem\u003eLr44, Lr65, Lr71\u003c/em\u003e) and a stripe rust resistance gene \u003cem\u003eYr5\u003c/em\u003e (Dyck and Sykes, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Law \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Mohler et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Notably \u003cem\u003eYr5\u003c/em\u003e represents a rare example of an NLR conferring all-stage resistance effective against all known \u003cem\u003ePst\u003c/em\u003e isolates, so far with no apparent evolution to overcome the resistance (Brar and Kutcher \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ghanbarnia et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Holtz et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we identified a novel APR locus (\u003cem\u003eQYr.cbl-5D\u003c/em\u003e) effective against stripe rust and derived from the spelt wheat cultivar, CDC Silex, which is nearly immune to stripe rust at the adult-plant stage. Through genetic analysis of crosses with susceptible spelt wheat and bread wheat cultivars, we investigated the inheritance and expression of this resistance and mapped the underlying locus. Our findings reveal a previously uncharacterized stripe rust APR derived from spelt wheat, demonstrating its potential as a valuable source of durable resistance for bread wheat improvement.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eA total of 15 bi-parental mapping populations were developed and used for adult-plant stripe rust phenotyping in the present study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Seven crosses were made using spelt wheat lines CDC Silex (resistant, released cultivar), 10Spelt17 (resistant, an advanced breeding line), CDC Origin (susceptible, released cultivar), and Avocet (susceptible, bread wheat cultivar) as parents. The spelt wheat lines were developed at the Crop Development Centre of University of Saskatchewan. The spelt lines CDC Silex and 10Spelt17 both exhibited near-immune reactions to stripe rust at the adult-plant stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b), while exhibiting full susceptibility at the seedling stage (data not shown), and were therefore used as resistant parents, whereas CDC Origin, another spelt cultivar, was used as the susceptible spelt parent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Avocet, a bread wheat cultivar with universal susceptibility to stripe rust, was included as a susceptible control in phenotyping studies and as a susceptible bread wheat parent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The crosses were made at the University of British Columbia (UBC) South Campus greenhouses during 2021 and 2022. The resulting F\u003csub\u003e1\u003c/sub\u003e plants were grown in one-gallon pots in the greenhouse to produce large F\u003csub\u003e2\u003c/sub\u003e populations. The F\u003csub\u003e2\u003c/sub\u003e plants from each cross were grown, advanced to F\u003csub\u003e2:3\u003c/sub\u003e generation, and phenotyped in Conviron\u0026reg; growth chambers. Two populations from cross between Avocet/CDC Silex (AvSi) and Avocet/10Spelt17 (AvSp) were further advanced to the F\u003csub\u003e5\u003c/sub\u003e generation through single-seed descent to establish recombinant inbred line (RIL) populations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBi-parental populations developed and used for stripe rust phenotyping in the present study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale parent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale parent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePopulation name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePopulation size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAAC Brandon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrSp F\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAC Brandon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpBr F\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAC Brandon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSiBr F\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Zorba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSiZo F\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpSi F\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAvSp F\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e134\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAvSi F\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Origin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOrSi F\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSiAv F\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpAv F\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpSi F\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Origin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSiOr\u003csup\u003eA\u003c/sup\u003e F\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Origin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSiOr\u003csup\u003eB\u003c/sup\u003e F\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAvSp F\u003csub\u003e2:3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e133\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAvSi F\u003csub\u003e2:3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSiAv F\u003csub\u003e2:3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpAv F\u003csub\u003e2:3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpSi F\u003csub\u003e2:3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAvSp F\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvocet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAvSi F\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter mapping the resistance locus, F\u003csub\u003e1\u003c/sub\u003e plants were generated by crossing the resistant spelt lines with Canada Western Red Spring (CWRS) wheat cultivars AAC Brandon (carrying \u003cem\u003eYr18\u003c/em\u003e), as well as with the moderately susceptible spelt cultivar CDC Zorba (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStripe rust phenotyping\u003c/h3\u003e\n\u003cp\u003e \u003cstrong\u003eIndoor phenotyping\u003c/strong\u003e \u003cp\u003ePlants were grown in Conviron\u0026reg; growth chambers under a 21/16\u0026deg;C day/night (16 h/8 h) regime. CDC Silex and 10Spelt17 were susceptible to all tested \u003cem\u003ePst\u003c/em\u003e isolates at the seedling stage (data not shown), stripe rust resistance was evaluated at the adult-plant stage, when the flag leaf had fully emerged (Z49\u0026ndash;Z69; Zadoks et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). CDC Silex and 10Spelt17 exhibited consistent immunity to stripe rust in inoculated (with multiple races) field stripe rust nurseries over years (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), therefore, we assumed the resistance as non-race-specific. For inoculation, \u003cem\u003ePst\u003c/em\u003e urediniospores were suspended in mineral oil (Novec 7100\u0026trade;) and sprayed onto the flag and penultimate leaves using a compressed-air sprayer. Inoculated plants were incubated in a dew chamber at 10\u0026deg;C, 100% relative humidity, and complete darkness for 24\u0026ndash;36 h to promote spore germination and initial infection. Plants were then transferred back to the growth chambers and maintained under a 16/11\u0026deg;C Day/night (16 h/8 h) cycle. Disease assessments were conducted when the susceptible control Avocet displayed full susceptibility (infection type (IT)\u0026thinsp;=\u0026thinsp;9) usually at 14\u0026ndash;16 days post inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), following the 0\u0026ndash;9 scale described by Line and Qayoum (1992) and further described by Gill et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To minimize observer bias, all plants were rated by a single observer. Unless otherwise stated, inoculations were performed using an in-house \u003cem\u003ePst\u003c/em\u003e admixture named \u0026ldquo;BC Mix\u0026rdquo;, representing a mixture of stripe rust races collected from susceptible wheat plants in the UBC, Vancouver stripe rust nursery over the 2020 growing season.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePhenotyping in the field nursery\u003c/b\u003e: Field phenotyping was conducted at the Totem Plant Science Field Station (UBC, Vancouver) during the summers of 2022 and 2023. The AvSp F\u003csub\u003e2:3\u003c/sub\u003e and AvSi F\u003csub\u003e2:3\u003c/sub\u003e families were evaluated in 2022, while the SiAv F\u003csub\u003e2:3\u003c/sub\u003e, SpAv F\u003csub\u003e2:3\u003c/sub\u003e, AvSi F\u003csub\u003e5\u003c/sub\u003e, AvSp F\u003csub\u003e5\u003c/sub\u003e, and SpSi F\u003csub\u003e2:3\u003c/sub\u003e populations were assessed in 2023. Each F\u003csub\u003e2:3\u003c/sub\u003e family was planted as 30 cm rows, spaced 30 cm apart between families and 45 cm between rows, following an augmented design where parental lines served as checks and were planted every 15\u0026ndash;20 plots. The F\u003csub\u003e5\u003c/sub\u003e RILs were planted as hill plots with identical spacing. The susceptible cultivar Avocet was used as a border to enhance disease pressure and positive infection control. The nursery was not inoculated as southern British Columbia has high natural stripe rust disease pressure (Brar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Disease scoring was performed once Avocet reached near full susceptibility on the flag leaf. F\u003csub\u003e2:3\u003c/sub\u003e families were classified as homozygous resistant (Hr), segregating (Seg), or homozygous susceptible (Hs), whereas F\u003csub\u003e5\u003c/sub\u003e RILs were rated for infection response as resistant (R), moderately resistant (MR), moderately susceptible (MS), or susceptible (S) according to Roelfs et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) and Gill et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Disease severity (0\u0026ndash;100%) was estimated using the modified Cobb\u0026rsquo;s scale (Gill et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Peterson et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1948\u003c/span\u003e). Adult-plant responses were recorded twice over a 10-day period following the initial scoring.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSegregation of stripe rust resistance in F\u003csub\u003e2\u003c/sub\u003e population was evaluated using chi-square (χ\u003csup\u003e2\u003c/sup\u003e) goodness-of-fit tests by classifying plants as resistant (IT\u0026thinsp;=\u0026thinsp;0\u0026ndash;6) or susceptible (IT\u0026thinsp;=\u0026thinsp;7\u0026ndash;9) and comparing observed counts with expected Mendelian ratios. For F\u003csub\u003e2:3\u003c/sub\u003e populations, families were classified as homozygous resistant (Hr), segregating (Seg), or homozygous susceptible (Hs), and χ\u003csup\u003e2\u003c/sup\u003e tests were used to assess deviation from expected segregation ratios. Differences in mean F\u003csub\u003e2\u003c/sub\u003e infection types between populations among F\u003csub\u003e2:3\u003c/sub\u003e family classes were assessed using analysis of variance (ANOVA). Statistical significance was declared at \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWhole-genome re-sequencing and bulk-segregant analysis\u003c/h3\u003e\n\u003cp\u003eTo decipher the genetics of APR in spelt wheat, additional F\u003csub\u003e1\u003c/sub\u003e plants derived from the cross between CDC Origin (susceptible spelt) and CDC Silex (resistant spelt) were advanced to the F\u003csub\u003e2\u003c/sub\u003e generation for phenotyping and BSA-seq.\u0026nbsp;Two F\u003csub\u003e2:3\u003c/sub\u003e populations were developed and phenotyped using the indoor stripe rust phenotyping protocol described above: the first in 2022, consisting of 138 F₂ lines, and the second in 2024, comprising 350 F₂ lines, from which a total of 29 highly resistant (IT\u0026thinsp;=\u0026thinsp;0\u0026ndash;3) and 21 highly susceptible (IT\u0026thinsp;=\u0026thinsp;7\u0026ndash;9) plants were selected and used in the resistant (R) and susceptible (S) bulks, respectively.\u003c/p\u003e \u003cp\u003e Genomic DNA was extracted from susceptible and resistant F₂ plants, along with both parental lines, using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s instructions. DNA quality was assessed using Nanodrop spectrophotometry and agarose gel electrophoresis, quantified with a Qubit fluorometer (Thermo Fisher Scientific, Waltham, USA), and normalized to 1 ng/\u0026micro;L. Tagmentation was conducted using the Illumina Tagment DNA TDE1 Enzyme and Buffer Kit (Illumina, San Diego, USA) with 1 \u0026micro;L of diluted DNA (1 \u0026micro;L DNA, 3.3964 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO, 0.504 \u0026micro;L TD Buffer, and 0.0996 \u0026micro;L TDE1). After 15 minutes incubation at 55\u0026deg;C, libraries were indexed with Nextera i7 and i5 adapters, each with unique combinations. The reaction mixture included 2 \u0026micro;L of i7/i5 index mix (2.5 \u0026micro;M each), 12.5 \u0026micro;L of NEB 2\u0026times; Taq Master Mix, and 5.5 \u0026micro;L of ddH₂O. Thermocycling was performed under the following conditions: 72\u0026deg;C for 3 min, 95\u0026deg;C for 1 min, 18 cycles of 95\u0026deg;C for 10 s, 55\u0026deg;C for 20 s, and 72\u0026deg;C for 3 min, followed by a final extension at 72\u0026deg;C for 3 min. Library quality was verified by agarose gel electrophoresis and quantified using a Qubit fluorometer. Libraries were normalized to 6 ng/\u0026micro;L, and 10 \u0026micro;L from each were pooled to form the R and S bulks, with corresponding parental libraries added to both pools. Each bulk (180 \u0026micro;L) was cleaned using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and eluted in 30 \u0026micro;L of ddH₂O. For size selection, 80 \u0026micro;L ddH₂O was added to 20 \u0026micro;L of each bulk, followed by 50 \u0026micro;L of SPRI beads (0.5\u0026times;) (Beckman Coulter Life Sciences, Brea, USA). After 10 minutes of binding, the supernatant was transferred to a second tube containing 30 \u0026micro;L of SPRI beads (0.8\u0026times; total), incubated for another 10 minutes, and then washed twice with freshly prepared 70% ethanol. The size-selected, bulked libraries were eluted in 30 \u0026micro;L ddH₂O, normalized to 3 ng/\u0026micro;L, and sequenced on the NovaSeq X Plus platform (Novogene, Tianjin, China).\u003c/p\u003e \u003cp\u003eSequencing generated 7,236,354 to 96,109,886 reads per individual in the R bulk and 20,470,792 to 95,254,716 reads per individual in the S bulk. Based on total mapped reads and assuming a genome size of 15.5 Gb, the R and S bulks achieved approximately 14.4\u0026times; and 9.9\u0026times; effective genome coverage, respectively.\u003c/p\u003e \u003cp\u003eReads were aligned to the spelt reference genome PI190962 (Walkowiak et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and to an in-house HiFi whole-genome assembly of CDC Silex (C. Pozniak, unpublished) using Burrows\u0026ndash;Wheeler Aligner (BWA) (Li and Durbin \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Variants were called using the Genome Analysis Toolkit (GATK) pipeline (Van der Auwera et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Initial variant calling against the spelt reference genome PI190962 identified 84.4\u0026nbsp;million variant sites in the R bulk (79.8\u0026nbsp;million SNPs and 4.66\u0026nbsp;million InDels) and 65.5\u0026nbsp;million variant sites in the S bulk (62.1\u0026nbsp;million SNPs and 3.49\u0026nbsp;million InDels) (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Transition-to-transversion (Ts/Tv) ratios were 2.78 and 2.96 in the R and S bulks, respectively, and multiallelic sites numbered 79.5\u0026nbsp;million in R and 61.9\u0026nbsp;million in S (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eVariant calling of the combined R and S bulks yielded 68,271,461 SNPs. To obtain reliable and informative markers for BSA-seq, a series of stringent filtering steps were applied. SNPs were first filtered based on reference allele frequency (0.22\u0026le; REF_FRQ\u0026thinsp;\u0026le;\u0026thinsp;0.78) to remove loci with extreme allele frequencies unlikely to be informative for segregation-based mapping, eliminating 51,594,971 SNPs. Variants with total read depth\u0026thinsp;\u0026lt;\u0026thinsp;25 were excluded to remove low-confidence sites with insufficient sampling across the ~\u0026thinsp;50 individuals represented in each bulk, eliminating 12,320,121 SNPs. SNPs with total read depth\u0026thinsp;\u0026gt;\u0026thinsp;100 were also removed to minimize false positives arising from repetitive regions or misaligned reads, eliminating 37,541 SNPs (\u003cb\u003eTable S2\u003c/b\u003e). Additional quality filtering removed SNPs with per-sample read depth\u0026thinsp;\u0026lt;\u0026thinsp;7 (447,062 SNPs) and genotype quality (GQ)\u0026thinsp;\u0026lt;\u0026thinsp;80 (2,408,119 SNPs). Finally, loci exhibiting a read depth difference\u0026thinsp;\u0026gt;\u0026thinsp;30 between bulks were excluded (46,092 SNPs) to reduce bias caused by uneven sequencing coverage (\u003cb\u003eTable S2\u003c/b\u003e). After all filtering steps, 1,417,555 high-confidence SNPs remained and were used for ΔSNP-index calculation in the R package QTLseqR (Mansfeld and Grumet \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnnotation of the physical region carrying\u003c/b\u003e \u003cb\u003eQYr.cbl-5D\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo characterize the physical region underlying \u003cem\u003eQYr.cbl-5D\u003c/em\u003e the in-house scaffold-level genome assembly of the resistant parent CDC Silex was used (C. Pozniak, unpublished). A 50-Mb genomic interval flanking the QTL peak was extracted and annotated using the BRAKER pipeline (Hoff et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), with publicly available wheat RNA-seq (RanSeq) data used as transcriptomic evidence to support gene prediction.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStripe rust resistance in CDC Silex and 10Spelt17 is allelic and non-race-specific\u003c/h2\u003e \u003cp\u003eCDC Silex and 10Spelt17 lines were developed by the bread wheat breeding program at CDC, University of Saskatchewan. Spelt wheat germplasm in the program is limited to a small number of founder lines as it is not the main focus of the program. Both these spelt wheat lines share a common spelt wheat landrace parent, PI 348771, in their pedigrees (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The landrace PI 348771 is believed to be the source of stripe rust resistance in CDC Silex and 10Spelt17. Due to shared parent in the pedigree, our hypothesis was that the resistance in CDC Silex and 10Spelt17 is identical or allelic. To test the hypothesis, we performed an allelism test by crossing CDC Silex and 10Spelt17 and phenotyping the F\u003csub\u003e1\u003c/sub\u003es and F\u003csub\u003e2\u003c/sub\u003e progeny (n\u0026thinsp;=\u0026thinsp;65) with stripe rust. All 65 SpSi F\u003csub\u003e2\u003c/sub\u003e plants from the cross exhibited resistance (IT\u0026thinsp;=\u0026thinsp;0\u0026ndash;3) to stripe rust when phenotyped in growth chambers. Similarly, the corresponding F\u003csub\u003e2:3\u003c/sub\u003e families, evaluated in the 2023 stripe rust nursery under natural infection, consistently displayed resistance. These findings confirm that the stripe rust resistance in 10Spelt17 and CDC Silex is conferred by either the same gene or complementary alleles of the same gene, likely inherited from their common ancestor, PI 348771. Therefore, for further experiments, we focused on crosses derived from CDC Silex.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePedigree of the spelt wheat accessions used in the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePedigree\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Silex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePI 348771/Oberkulmer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10Spelt17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e00Spelt5/00Spelt20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e00Spelt5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePI 348771/Oberkulmer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e00Spelt20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePI 348771/Oberkulmer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Origin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRL5407/Winter spelt (F5)//PGR8801\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDC Zorba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRL5407/Common winter spelt\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe APR in CDC Silex and 10Spelt17 is described as non-race-specific because the lines were consistently rated R (IT\u0026thinsp;=\u0026thinsp;0\u0026ndash;1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) in non-inoculated field stripe rust nurseries in Saskatoon, Saskatchewan and Vancouver, British Columbia between 2016\u0026ndash;2025 (data not shown). The southern British Columbia is a hotspot of stripe rust in North America, and the region harbors many diverse \u003cem\u003ePst\u003c/em\u003e races (Brar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen 2005; Holden et al. 2023). Consistent adult-plant resistance in these field nurseries suggest that the APR carried by CDC Silex and 10Spelt17 is non-race-specific.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenetics and mapping of APR in spelt wheat cv. CDC Silex\u003c/h3\u003e\n\u003cp\u003eTo understand the genetic regulation of the APR, CDC Silex was crossed with a susceptible spelt wheat parent CDC Origin. The F\u003csub\u003e2\u003c/sub\u003e progeny from the cross (CDC Origin/CDC Silex (OrSi\u003csup\u003eA\u003c/sup\u003e)) were initially phenotyped in 2022 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). These experiments indicated a single dominant gene regulating the APR, with 128 resistant and 10 susceptible plants. To confirm this, a large set of F\u003csub\u003e2s\u003c/sub\u003e from the same cross (OrSi\u003csup\u003eB\u003c/sup\u003e) were screened for resistance to stripe rust at the adult plant stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The progeny segregation confirmed to the expected 3:1 ratio for a single dominant gene (χ\u0026sup2; = 0.86, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.35), indicating that APR in CDC Silex is governed by a single dominant locus (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChi-square goodness-of-fit test for segregation ratios expected under one- or two-gene models conferring dominant resistance in F₂ populations inoculated with a mixture of \u003cem\u003ePuccinia striiformis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e races (BC Mix).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExpected Ratio (R:S)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExpected\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObserved\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChi-Square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrSi\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e03:01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e262.5 : 87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e270 : 80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;350)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15:01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e328.1 : 21.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e270 : 80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e163.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvSi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e03:01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.5:33.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86:48:00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.3682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;134)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15:01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e125.6:8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86:48:00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e199.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvSp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e03:01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118.5:39.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e126:32:00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.8987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1682\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;158)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15:01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e148.1:9.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e126:32:00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo map the physical location providing resistance, BSAseq was performed. Whole-genome resequencing of the resistant (R) and susceptible (S) bulks generated high-quality datasets with effective genome coverages of 14.4\u0026times; and 9.9\u0026times;, respectively. Alignment to the PI190962 spelt reference genome identified 84.4\u0026nbsp;million and 65.5\u0026nbsp;million variant sites in the R and S bulks, respectively, including both SNPs and InDels (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Transition-to-transversion (Ts/Tv) ratios of 2.78 (R) and 2.96 (S) indicated high-confidence variant calls consistent with expected values in wheat. A substantial proportion of sites were multiallelic (79.5\u0026nbsp;million in R and 61.9\u0026nbsp;million in S) (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), reflecting the expected allelic diversity and segregation within the pooled F\u003csub\u003e2\u003c/sub\u003e populations. Collectively, these findings confirmed a high-quality variant detection and appropriate heterozygosity levels for F\u003csub\u003e2\u003c/sub\u003e-derived bulks. A total of 68,271,461 SNPs were identified in the combined dataset of the resistant (R) and susceptible (S) bulks. After stringent quality filtering (see Materials and Methods), 1,417,555 high-confidence SNPs were retained for downstream ΔSNP-index analysis (\u003cb\u003eTable S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eA single major locus (\u003cem\u003eQYr.cbl-5D\u003c/em\u003e) was mapped on chromosome (5D) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea; Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), spanning from 139.12 Mb to 139.42 Mb with a total length of approximately 0.31 Mb and comprising 17 SNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb; Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The region showed an average tricube ΔSNP-index of \u0026minus;\u0026thinsp;0.53, with a peak tricube ΔSNP-index of \u0026minus;\u0026thinsp;0.55 at position 139,421,704 bp, indicating strong enrichment of the susceptible allele in this genomic interval (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This pattern suggests the presence of a putative locus contributing to stripe rust resistance or susceptibility in the studied population.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of loci identified in BSA-seq analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eChr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStart\u003c/p\u003e \u003cp\u003e(bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEnd\u003c/p\u003e \u003cp\u003e(bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLength(bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo of Linked SNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePeak tricubeΔ (SNP-index)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePosition of Peak (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAvg. tricube Δ(SNP-index)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eQYr.cbl-5D\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e139115840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e139421704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e305864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-0.5487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e139421704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-0.5275\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eQYr.cbl-5D-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e141313106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e153597143\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12284037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e153597143\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.476\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003eQTLs were detected based on tricube Δ(SNP-index) values calculated from resistant and susceptible bulks in the F₂ population. Significant regions were identified at α\u0026thinsp;=\u0026thinsp;0.05\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo minimize reference-assembly bias and to account for potential structural variation or sequence divergence associated with the resistant parent, the bulks were additionally aligned to an in-house developed scaffold-level genome assembly of the resistant parent, CDC Silex. Using the same filtering criteria as described above, 334,671 high-confidence SNPs were retained from an initial set of 41,658,484 SNPs and used for ΔSNP-index estimation in QTLseqR. This analysis also identified a single major peak on chromosome (5D) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea;\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The locus (\u003cem\u003eQYr.cbl-5D-1\u003c/em\u003e) spanned approximately 12 Mb, from 141.13 Mb to 153.59 Mb, and was supported by two SNPs passing the applied thresholds (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb;\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe difference in the physical span of the QTL interval between the two analyses is primarily attributable to differences in SNP density and marker resolution. Alignment to the PI190962 reference genome yielded approximately 1.4\u0026nbsp;million high-confidence SNPs genome-wide, enabling finer mapping resolution and delimiting the QTL to a narrow\u0026thinsp;~\u0026thinsp;0.31 Mb interval. In contrast, alignment to the scaffold-level CDC Silex assembly resulted in approximately 0.3\u0026nbsp;million high-confidence SNPs, substantially reducing local marker density and consequently broadening the QTL interval to ~\u0026thinsp;12 Mb.\u003c/p\u003e\n\u003ch3\u003eAnalysis of the QTL locus in PI190962 and CDC Silex\u003c/h3\u003e\n\u003cp\u003eAlthough the physical coordinates of the QTL differed between analyses performed against the PI190962 reference genome and the CDC Silex assembly, gene content comparison revealed strong synteny between the two intervals. Notably, the same set of annotated genes present in \u0026plusmn;\u0026thinsp;1 Mb window of the \u003cem\u003eQYr.cbl-5D\u003c/em\u003e at ~\u0026thinsp;139 Mb in PI190962 was located at ~\u0026thinsp;146 Mb in the CDC Silex assembly (\u003cb\u003eTable S3\u003c/b\u003e). This indicates that an orthologous physical segment was consistently enriched in both analyses, and that the observed positional shift reflects assembly-specific differences in physical coordinate placement rather than a biologically distinct locus.\u003c/p\u003e \u003cp\u003eWithin \u0026plusmn;\u0026thinsp;1 Mb region flanking the \u003cem\u003eQYr.cbl-5D\u003c/em\u003e, a total of six genes were detected, of which five had no predicted function and one gene, \u003cem\u003eTraesTSP5D01G127300\u003c/em\u003e, encodes a fusion protein with HSP domain fused to a C2H2 zinc-finger transcription factor (TF) (\u003cb\u003eTable S3;\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Notably, \u003cem\u003eTraesTSP5D01G127300\u003c/em\u003e is the only gene located within the core 0.31 Mb QTL interval. This gene encodes an 1,812 bp transcript with three exons. The first exon codes for a DnaJ HSP domain, while the second and third exons encode a C2H2 zinc-finger transcription factor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). An identical copy of the \u003cem\u003eTraesTSP5D01G127300\u003c/em\u003e gene is present in the wheat D-genome donor, \u003cem\u003eAegilops tauschii\u003c/em\u003e (\u003cem\u003eAET5Gv20276300\u003c/em\u003e) and bread wheat (\u003cem\u003eTraesCS5D02G116700\u003c/em\u003e), demonstrating that this fused domain is evolutionary conserved. In \u003cem\u003eA. tauschii\u003c/em\u003e, the orthologue gene is reported to have six alternate transcripts (\u003cb\u003eTable S4\u003c/b\u003e). This may be true in spelt wheat as well but remains untested due to the limited availability of genomics resources in the species. The public expression databases on stripe rust in wheat indicate that this gene is upregulated after 24 hours of infection in two independent datasets (\u003cb\u003eFig. S3d, e\u003c/b\u003e), further suggesting a role in the immune response.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSuppression of\u003c/b\u003e \u003cb\u003eQYr.cbl-5D\u003c/b\u003e \u003cb\u003ein bread wheat\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAt the beginning of the project, we chose bread wheat cv. Avocet as susceptible parent to develop mapping populations because Avocet is universally used in stripe rust mapping studies and that the bread and spelt wheats are readily crossable. Therefore, CDC Silex and 10Spelt17, were crossed with Avocet. The F\u003csub\u003e1\u003c/sub\u003e plants derived from these crosses consistently exhibited susceptibility, with infection types of 7\u0026ndash;8 when challenged with \u003cem\u003ePst\u003c/em\u003e BC Mix or single isolates: W034 and W003, (Brar and Kutcher \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) regardless of whether the resistant spelt was used as the female (cytoplasmic) or male (pollen) parent. Conversely, all F\u003csub\u003e1\u003c/sub\u003e plants from the spelt x spelt cross between CDC Silex (resistant) and CDC Origin (susceptible) were resistant. This led us to the use of susceptible spelt wheat CDC Origin in our aforementioned studies. To determine if the susceptibility is genotype specific or not, we also crossed CDC Silex and 10Spelt17 with a hard red spring wheat cv. AAC Brandon (carries \u003cem\u003eYr18/Lr34\u003c/em\u003e). Similarly, AAC Brandon/10Spelt17 or AAC Brandon/CDC Silex (n\u0026thinsp;=\u0026thinsp;6) F\u003csub\u003e1\u003c/sub\u003e adult-plants showed variable infection responses ranging from 2\u0026ndash;6. In general, F\u003csub\u003e1\u003c/sub\u003e plants from bread wheat x spelt wheat crosses displayed resistance patterns similar to the bread wheat parent, whereas \u0026lsquo;10Spelt17/CDC Silex\u0026rsquo; (n\u0026thinsp;=\u0026thinsp;2) F\u003csub\u003e1s\u003c/sub\u003e showed complete resistance (IT\u0026thinsp;=\u0026thinsp;0\u0026ndash;1). Crosses between resistant and either intermediate (CDC Zorba) or susceptible (CDC Origin) spelt accessions also produced fully resistant F\u003csub\u003e1\u003c/sub\u003e plants (IT\u0026thinsp;=\u0026thinsp;0\u0026ndash;3).\u003c/p\u003e \u003cp\u003eDespite inconclusive phenotypes of F\u003csub\u003e1s\u003c/sub\u003e, we also screened F\u003csub\u003e2\u003c/sub\u003es and F\u003csub\u003e2:3\u003c/sub\u003e families from Avocet/CDC Silex and Avocet/10Spelt17 crosses. In AvSi F\u003csub\u003e2\u003c/sub\u003e population, among 134 individuals, 86 were resistant (IT\u0026thinsp;=\u0026thinsp;0\u0026ndash;6) and 48 were susceptible (IT\u0026thinsp;=\u0026thinsp;7\u0026ndash;9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The higher frequency of resistant plants indicated dominant resistance, although the segregation ratio did not fit expected Mendelian ratios for one (χ\u0026sup2; = 8.37, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) or two (χ\u0026sup2; = 199.98, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) genes (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, only two F\u003csub\u003e2\u003c/sub\u003e individuals displayed resistance levels comparable to the resistant parent (IT\u0026thinsp;=\u0026thinsp;2) which was likely a disease escape. The AvSp F\u003csub\u003e2\u003c/sub\u003e population exhibited a similar trend, with 126 resistant and 32 susceptible individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), fitting a single dominant gene model (χ\u0026sup2; = 1.90, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.17) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As in AvSi, only a few plants (n\u0026thinsp;=\u0026thinsp;3) showed resistance somewhat comparable (IT\u0026thinsp;=\u0026thinsp;3\u0026ndash;4) to the resistant parent in the AvSp population. This was in contrast to the pattern seen in the F\u003csub\u003e2\u003c/sub\u003e population of spelt x spelt crosses (OrSi\u003csup\u003eA\u003c/sup\u003e \u0026amp; OrSi\u003csup\u003eB\u003c/sup\u003e), where most of the resistant plants showed resistance comparable to the resistant parent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), thus, indicating suppression of \u003cem\u003eQYr.cbl-5D\u003c/em\u003e in bread wheat background.\u003c/p\u003e \u003cp\u003eTo determine the zygosity of resistance loci, seeds from AvSi and AvSp F\u003csub\u003e2\u003c/sub\u003e individuals were advanced and evaluated as F\u003csub\u003e2:3\u003c/sub\u003e families. Among 133 AvSp F\u003csub\u003e2:3\u003c/sub\u003e families, 44 were homozygous resistant (Hr), 46 segregating (Seg), and 43 homozygous susceptible (Hs). This distribution deviated significantly from ratios expected for one (1Hr:2Seg:1Hs; χ\u0026sup2; = 12.7, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) or two genes (1Hr:14Seg:1Hs; χ\u0026sup2; = 340.5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A similar pattern was observed for the AvSi F\u003csub\u003e2:3\u003c/sub\u003e families, with 19 Hr, 38 Seg, and 71 Hs families, also deviating from Mendelian expectations (1 gene: χ\u0026sup2; = 63.4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; 2 genes: χ\u0026sup2; = 560.1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChi-square goodness-of-fit test for expected ratio for one or two resistance gene conferring resistance for F\u003csub\u003e2:3\u003c/sub\u003e populations. All populations were phenotyped under natural infection in stripe rust field nursery during the 2022 (AvSi, AvSp) or the 2023 (SiAv, SpAv) growing season. AvSi: Avocet/CDC Silex; AvSp: Avocet/10Spelt17; SiAv: CDC Silex/Avocet; SpAv:10Spelt17/Avocet.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExpected Ratio (Hr:Seg:Hs)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExpected\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObserved\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChi-Square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvSi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32:64:32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19:38:71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;128)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:14:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8:112:8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19:38:71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e560.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvSp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33:67:33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44:46:43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;133)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:14:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8:117:8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44:46:43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e340.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSiAv\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39:78:39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24:86:46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;156)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:14:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11:136:11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24:86:46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e174.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpAv\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:2:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20:41:20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27:43:11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:14:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5:71:5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27:43:11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e113.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePhenotypes in the F\u003csub\u003e2:3\u003c/sub\u003e populations were not strongly predictive/correlated of their respective F\u003csub\u003e2\u003c/sub\u003e parent ratings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Mean F\u003csub\u003e2\u003c/sub\u003e ratings differed significantly between AvSi and AvSp (F\u0026thinsp;=\u0026thinsp;14.4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), averaging 5.88 (SD\u0026thinsp;=\u0026thinsp;1.36) and 5.25 (SD\u0026thinsp;=\u0026thinsp;1.38), respectively. Within populations, F\u003csub\u003e2\u003c/sub\u003e plants classified as Hr or Seg produced significantly lower mean ITs than Hs individuals, though Hr and Seg did not differ significantly. For AvSp, no significant difference in F\u003csub\u003e2\u003c/sub\u003e ITs was observed between categories, while in AvSi, Hs F\u003csub\u003e2:3\u003c/sub\u003e families were derived from significantly more susceptible F\u003csub\u003e2\u003c/sub\u003e plants. Only 31.8% (14/44) of AvSp Hr families and 31.6% (6/19) of AvSi Hr families originated from resistant F\u003csub\u003e2\u003c/sub\u003e plants. The reciprocal F\u003csub\u003e2:3\u003c/sub\u003e populations, SiAv and SpAv, consisted of 24 Hr, 86 Seg, and 46 Hs and 27 Hr, 43 Seg, and 11 Hs families, respectively. None of these fit expected ratios for single or two-gene models.\u003c/p\u003e \u003cp\u003eFinally, 112 AvSp and 85 AvSi F\u003csub\u003e2:5\u003c/sub\u003e RILs were phenotyped in field nurseries in 2023. However, even the F\u003csub\u003e5\u003c/sub\u003e generation did not fit to a single-gene model. The consistent partial suppression of \u003cem\u003eQYr.cbl-5D\u003c/em\u003e in the bread wheat background observed across different bread wheat genotypes, reciprocal crosses and population stages, from segregating F\u003csub\u003e2\u003c/sub\u003e to inbred F\u003csub\u003e5\u003c/sub\u003e, indicates the presence of a suppressor factor or gene in the bread wheat genome.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQYr.cbl-5D\u003c/b\u003e \u003cb\u003eis a novel gene and does not overlap with any known loci\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eQYr.cbl-5D\u003c/em\u003e was mapped to the short arm of chromosome 5D and delimited to a physical interval centered at ~\u0026thinsp;139 Mb on the reference genome. Examination of this region revealed no overlap with any previously reported stripe rust resistance genes or quantitative trait loci on chromosome 5D. Among the known loci on this chromosome, \u003cem\u003eYr70\u003c/em\u003e, a high-temperature adult-plant resistance gene, is located at approximately 8.86 Mb on chromosome arm 5DS (Bansal et al. 2017), which is physically distant from the \u003cem\u003eQYr.cbl-5D\u003c/em\u003e interval (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). \u003cem\u003eYr40\u003c/em\u003e, an alien introgression from \u003cem\u003eAegilops umbellulata\u003c/em\u003e conferring race-specific all-stage resistance, has been genetically mapped to chromosome 5D (Kuraparthy et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e); however, its precise physical position on the wheat reference genome remains undefined due to the non-collinearity of the alien introgressed chromatin.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSpelt wheat is a proven reservoir of diverse rust resistance genes which can be used in bread wheat improvement, having contributed major loci such as \u003cem\u003eYr5\u003c/em\u003e and several leaf rust genes (Dyck and Sykes \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Law \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Mohler et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Continued mining of spelt germplasm is therefore well justified, particularly as stripe rust populations remain dynamic and widely distributed in major wheat-growing regions, including Canada (Brar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Holden et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003eQYr.cbl-5D\u003c/em\u003e confers a near-immune adult-plant response with non-race-specific, broad-spectrum effectiveness against stripe rust. If successfully expressed in bread wheat, this locus could complement APR genes already deployed in Canadian wheat breeding, such as \u003cem\u003eYr18/Lr34\u003c/em\u003e and \u003cem\u003eYr46/Lr67\u003c/em\u003e, to further strengthen durable stripe rust resistance.\u003c/p\u003e \u003cp\u003eA key finding of this study is that resistance identified in a donor background did not manifest predictably following introgression into elite germplasm. The reduced expression of the spelt-derived APR in bread wheat backgrounds is most consistent with epistasis, potentially involving a dominant suppressor present in common wheat. Suppressors of rust resistance have been documented previously and can mask otherwise effective loci, complicating both genetic analysis and breeding deployment (Hiebert et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jin et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Such interactions are particularly relevant in hexaploid wheat, where homeologous genes and regulatory networks can buffer or override allele effects. These observations highlight the importance of evaluating resistance alleles directly in target breeding backgrounds and of identifying genetic modifiers that constrain their expression (Poland et al. 2009; Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe candidate gene content in the \u003cem\u003eQYr.cbl-5D\u003c/em\u003e locus supports a resistance mechanism consistent with an emerging theme in adult-plant resistance: unusual or non-canonical genes providing strong, durable resistance through an unknown mechanism that is not traditional extracellular or intracellular recognition by receptor kinase or NLR proteins. No candidate gene in the region matches these expectations for R-gene predicted domain structure and function. A putative candidate encodes a fusion protein containing a DnaJ/HSP domain and a plant-specific C2H2 zinc-finger domain, suggesting roles in protein homeostasis and transcriptional regulation rather than classical immune receptor-mediated recognition. This interpretation is consistent with the observation that several cloned APR genes encode non-canonical proteins, including transporters such as \u003cem\u003eLr34/Yr18\u003c/em\u003e and \u003cem\u003eLr67/Yr46\u003c/em\u003e (Krattinger et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Moore et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Transcriptional regulators are increasingly recognized as key components of wheat-rust interactions, with both positive and negative effects on disease outcomes (Zheng et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, a C2H2 zinc-finger transcription factors, \u003cem\u003eTaZFP8-5B\u003c/em\u003e, was shown to negatively regulate stripe rust resistance as its overexpression led to increased fungal infection in inoculated plants (Huang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), demonstrating that members of this family can modulate wheat-rust interactions. Also, the presence of a conserved ortholog of the putative candidate gene in bread wheat and \u003cem\u003eAegilops tauschii\u003c/em\u003e suggests that functional differences may arise from allele-specific variation or regulatory context rather than gene presence alone.\u003c/p\u003e \u003cp\u003eFrom a breeding perspective, this study highlights both the opportunity and the complexity of exploiting non-elite germplasm for durable disease resistance. The \u003cem\u003eQYr.cbl-5D\u003c/em\u003e represents a potentially valuable addition to the stripe rust resistance portfolio, though its practical utility will depend on understanding and overcoming suppression in bread wheat backgrounds.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe funding for this research was provided by Saskatchewan Ministry of Agriculture (Project ID: ADF20200205), Saskatchewan Wheat Development Commission (Project ID: 173-201127), Alberta Grains (Project ID: 2021AWC100B), and Manitoba Crop Alliance (Project ID: MCA2184).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eGSB conceived the idea and supervised the project. GSB and VF developed the mapping populations. VF and JSG performed the stripe rust phenotyping. VF, ML extracted DNA and prepared the libraries. VF and JS performed bioinformatic and other data analyses. VF, JS, and GSB wrote the manuscript. VF and JS prepared figures and tables. JE, CJP generated the CDC Silex genome assembly. CJP and PJH made significant intellectual contributions, provided the spelt wheat lines, and the genome assembly of CDC Silex. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe first author (VF) of the paper acknowledges various scholarships and awards that he has received during his tenure of M.Sc. degree program. The authors acknowledge the support and help received from members of the Cereal Breeding Lab (CBL).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData are included in the article and its supplementary files with an exception of genome assembly of CDC Silex .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrar GS, Kutcher HR (2016) Race characterization of \u003cem\u003ePuccinia striiformis\u003c/em\u003e f. sp. \u003cem\u003etritici\u003c/em\u003e, the cause of wheat stripe rust, in Saskatchewan and southern Alberta, Canada, and virulence comparison with races from the United States. Plant Dis 100:1744\u0026ndash;1753.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrar GS, Fetch T, McCallum BD, Hucl PJ, Kutcher HR (2019) Virulence dynamics and breeding for resistance to stripe, stem, and leaf rust in Canada since 2000. 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Euphytica 220:107.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng P, Liu M, Pang L, Sun R, Yao M, Wang X, Kang Z, Liu J (2023) Stripe rust effector Pst21674 compromises wheat resistance by targeting transcription factor \u003cem\u003eTaASR3\u003c/em\u003e. Plant Physiol 193:2806\u0026ndash;2824.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSupplementary Figures and Tables\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Wheat, adult-plant resistance (APR), stripe rust, yellow rust, BSAseq.","lastPublishedDoi":"10.21203/rs.3.rs-8991239/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8991239/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdult-plant resistance (APR) genes provide durable, non-race-specific resistance against wheat rust diseases. We identified a novel APR locus in the spelt wheat cultivar CDC Silex that confers non-race-specific near-immune adult-plant resistance to stripe rust. The trait segregates as a single dominant gene and bulked segregant analysis sequencing (BSA-seq) mapped the locus, \u003cem\u003eQYr.cbl-5D\u003c/em\u003e, to a single 0.31 Mb interval on chromosome 5D, confirming monogenic inheritance. Interestingly, introgression into bread wheat suppresses this resistance, suggesting the presence of a dominant suppressor factor in the bread wheat genome. We generated a scaffold-scale assembly of CDC Silex to annotate the locus and its flanking region on 5D. Within a\u0026thinsp;\u0026plusmn;\u0026thinsp;1 Mb interval flanking the \u003cem\u003eQYr.cbl-5D\u003c/em\u003e peak, six genes were identified, of which five had no predicted function and one encodes a C2H2 zinc-finger transcription factor. Future work will aim to clone this APR and its suppressor in bread wheat. Flanking and linked SNPs from this region will enable marker-assisted selection to deploy this APR locus, providing a durable resistance resource to support sustainable wheat breeding and reduce reliance on fungicides.\u003c/p\u003e","manuscriptTitle":"Discovery of a novel adult-plant resistance locus conferring broad-spectrum stripe rust immunity in spelt wheat and its suppression in bread wheat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 17:57:20","doi":"10.21203/rs.3.rs-8991239/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"52632725216265400693848521679968422620","date":"2026-05-18T09:23:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-17T06:22:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192517973330137711771172859525278208811","date":"2026-03-09T23:43:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-09T07:14:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-08T00:03:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-28T11:44:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Theoretical and Applied Genetics","date":"2026-02-27T19:33:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"12e1ca35-c35d-418d-bf6e-e8d873f71d10","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"52632725216265400693848521679968422620","date":"2026-05-18T09:23:24+00:00","index":20,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-12T17:57:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 17:57:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8991239","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8991239","identity":"rs-8991239","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}