Comparative Analysis of Coding and Non-Coding Features within Insect Tolerance Loci in Wheat with Their Homologs in Cereal Genomes.

Food insecurity and malnutrition have reached critical levels with increased human population, climate fluctuations, water shortage; therefore, higher-yielding crops are in the spotlight of numerous studies. Abiotic factors affect the yield of staple food crops; among all, wheat stem sawfly (Cephus cinctus Norton) and orange wheat blossom midge (Sitodiplosis mosellana) are two of the most economically and agronomically harmful insect pests which cause yield loss in cereals, especially in wheat in North America. There is no effective strategy for suppressing this pest damage yet, and only the plants with intrinsic tolerance mechanisms such as solid stem phenotypes for WSS and antixenosis and/or antibiosis mechanisms for OWBM can limit damage. A major QTL and a causal gene for WSS resistance were previously identified in wheat, and 3 major QTLs and a causal gene for OWBM resistance. Here, we present a comparative analysis of coding and non-coding features of these loci of wheat across important cereal crops, barley, rye, oat, and rice. This research paves the way for our cloning and editing of additional WSS and OWBM tolerance gene(s), proteins, and metabolites.

Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential.

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool. To further enhance the agronomic potential of rye, we produced a chromosome-scale annotated assembly of the 7.9-gigabase rye genome and extensively validated its quality by using a suite of molecular genetic resources. We demonstrate applications of this resource with a broad range of investigations. We present findings on cultivated rye's incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution, pathogen resistance, low-temperature tolerance, fertility control systems for hybrid breeding and the yield benefits of rye-wheat introgressions.

Characterization of a new gene for resistance to wheat powdery mildew on chromosome 1RL of wild rye Secale sylvestre.

KEY MESSAGE: PmSESY, a new wheat powdery mildew resistance gene was characterized and genetically mapped to the terminal region of chromosome 1RL of wild rye Secale sylvestre. The genus Secale is an important resource for wheat improvement. The Secale species are usually considered as non-adapted hosts of Blumeria graminis f. sp. tritici (Bgt) that causes wheat powdery mildew. However, as a wild species of cultivated rye, S. sylvestre is rarely studied. Here, we reported that 25 S. sylvestre accessions were susceptible to isolate BgtYZ01, whereas the other five confer effective resistance to all the tested isolates of Bgt. A population was then constructed by crossing the resistant accession SESY-01 with the susceptible accession SESY-11. Genetic analysis showed that the resistance in SESY-01 was controlled by a single dominant gene, temporarily designated as PmSESY. Subsequently, combining bulked segregant RNA-Seq (BSR-Seq) analysis with molecular analysis, PmSESY was mapped into a 1.88 cM genetic interval in the terminus of the long arm of 1R, which was closely flanked by markers Xss06 and Xss09 with genetic distances of 0.87 cM and 1.01 cM, respectively. Comparative mapping demonstrated that the corresponding physical region of the PmSESY locus was about 3.81 Mb in rye cv. Lo7 genome, where 30 disease resistance-related genes were annotated, including five NLR-type disease resistance genes, three kinase family protein genes, three leucine-rich repeat receptor-like protein kinase genes and so on. This study gives a new insight into S. sylvestre that shows divergence in response to Bgt and reports a new powdery mildew resistance gene that has potential to be used for resistance improvement in wheat.

Discovery of a Novel Leaf Rust (Puccinia recondita) Resistance Gene in Rye (Secale cereale L.) Using Association Genomics.

Leaf rust constitutes one of the most important foliar diseases in rye (Secale cereale L.). To discover new sources of resistance, we phenotyped 180 lines belonging to a less well-characterized Gülzow germplasm at three field trial locations in Denmark and Northern Germany in 2018 and 2019. We observed lines with high leaf rust resistance efficacy at all locations in both years. A genome-wide association study using 261,406 informative single-nucleotide polymorphisms revealed two genomic regions associated with resistance on chromosome arms 1RS and 7RS, respectively. The most resistance-associated marker on chromosome arm 1RS physically co-localized with molecular markers delimiting Pr3. In the reference genomes Lo7 and Weining, the genomic region associated with resistance on chromosome arm 7RS contained a large number of nucleotide-binding leucine-rich repeat (NLR) genes. Residing in close proximity to the most resistance-associated marker, we identified a cluster of NLRs exhibiting close protein sequence similarity with the wheat leaf rust Lr1 gene situated on chromosome arm 5DL in wheat, which is syntenic to chromosome arm 7RS in rye. Due to the close proximity to the most resistance-associated marker, our findings suggest that the considered leaf rust R gene, provisionally denoted Pr6, could be a Lr1 ortholog in rye.

A novel approach to produce phage single domain antibody fragments for the detection of gluten in foods.

In this study, we demonstrated the feasibility of isolating recombinant phage-antibodies against gluten from a non-immunized library of human single-domain antibodies (dAbs). Phage display technology enabled the selection of affinity probes by successive rounds of biopanning against a biotinylated synthetic peptide comprising repetitive immunogenic gluten motifs. The analysis of a wide representation of heterologous plant species corroborated that two of the isolated clones were specific to wheat, barley and rye proteins. The phage antibody selected as the most appropriate clone for the detection of gluten in foods (dAb8E-phage) was further applied in an indirect ELISA to the analysis of 50 commercial food samples. Although the limit of detection achieved did not improve those of current immunoassays, the proposed methodology could provide promising new pathways for the generation of recombinant antibodies that allow a comprehensive determination of gluten in foods, whilst replacing the need for animal immunization.

Assessing the genetic diversity and characterizing genomic regions conferring Tan Spot resistance in cultivated rye.

Rye (Secale cereale L.) is known for its wide adaptation due to its ability to tolerate harsh environments in semiarid areas. To assess the diversity in rye we genotyped a panel of 178 geographically diverse accessions of four Secale sp. from U.S. National Small Grains Collection using 4,037 high-quality SNPs (single nucleotide polymorphisms) developed by genotyping-by-sequencing (GBS). PCA and STRUCTURE analysis revealed three major clusters that separate S. cereale L. from S. strictum and S. sylvestre, however, genetic clusters did not correlate with geographic origins and growth habit (spring/winter). The panel was evaluated for response to Pyrenophora tritici-repentis race 5 (PTR race 5) and nearly 59% accessions showed resistance or moderate resistance. Genome-wide association study (GWAS) was performed on S. cereale subsp. cereale using the 4,037 high-quality SNPs. Two QTLs (QTs.sdsu-5R and QTs.sdsu-2R) on chromosomes 5R and 2R were identified conferring resistance to PTR race 5 (p < 0.001) that explained 13.1% and 11.6% of the phenotypic variation, respectively. Comparative analysis showed a high degree of synteny between rye and wheat with known rearrangements as expected. QTs.sdsu-2R was mapped in the genomic region corresponding to wheat chromosome group 2 and QTs.sdsu-5R was mapped to a small terminal region on chromosome 4BL. Based on the genetic diversity, a set of 32 accessions was identified to represents more than 99% of the allelic diversity with polymorphic information content (PIC) of 0.25. This set can be utilized for genetic characterization of useful traits and genetic improvement of rye, triticale, and wheat.

Baker's Asthma: Is the Ratio of Rye Flour-Specific IgE to Total IgE More Suitable to Predict the Outcome of Challenge Test Than Specific IgE Alone.

Usually the diagnosis of baker's asthma is based on specific inhalation challenge with flours. To a certain extent the concentration of specific IgE to flour predicts the outcome of challenge test in bakers. The aim of this study was to evaluate whether the ratio of specific IgE (sIgE) to total IgE (tIgE) improves challenge test prediction in comparison to sIgE alone. Ninety-five bakers with work-related respiratory symptoms were challenged with rye flour. Total IgE, sIgE, and the sIgE/tIgE ratio were determined. Receiver operator characteristic (ROC) plots including the area under the curve (AUC) were calculated using the challenge test as gold-standard. Total IgE and sIgE concentrations, and their ratio were significantly higher in bakers with a positive challenge test than in those with a negative one (p < 0.0001, p < 0.0001, and p = 0.023, respectively). In ROC analysis, AUC was 0.83 for sIgE alone, 0.79 for tIgE, and 0.64 for the ratio. At optimal cut-offs, tIgE, sIgE, and the ratio reached a positive predicted value (PPV) of 95%, 84% and 77%, respectively. In conclusion, calculating the ratio of rye flour-sIgE to tIgE failed to improve the challenge test prediction in our study group.

Coeliac disease: immunogenicity studies of barley hordein and rye secalin-derived peptides.

Coeliac disease (CD) is an inflammatory disorder of the small intestine. It includes aberrant adaptive immunity with presentation of CD toxic gluten peptides by HLA-DQ2 or DQ8 molecules to gluten-sensitive T cells. A ω-gliadin/C-hordein peptide (QPFPQPEQPFPW) and a rye-derived secalin peptide (QPFPQPQQPIPQ) were proposed to be toxic in CD, as they yielded positive responses when assessed with peripheral blood T-cell clones derived from individuals with CD. We sought to assess the immunogenicity of the candidate peptides using gluten-sensitive T-cell lines obtained from CD small intestinal biopsies. We also sought to investigate the potential cross-reactivity of wheat gluten-sensitive T-cell lines with peptic-tryptic digested barley hordein (PTH) and rye secalin (PTS). Synthesised candidate peptides were deamidated with tissue transglutaminase (tTG). Gluten-sensitive T-cell lines were generated by culturing small intestinal biopsies from CD patients with peptic-tryptic gluten (PTG), PTH or PTS, along with autologous PBMCs for antigen presentation. The stimulation indices were determined by measuring the relative cellular proliferation via incorporation of 3 H-thymidine. The majority of T-cell lines reacted to the peptides studied. There was also cross-reactivity between wheat gluten-sensitive T-cell lines and the hordein, gliadin and secalin peptides. PTH, PTS, barley hordein and rye secalin-derived CD antigen-sensitive T-cell lines showed positive stimulation with PTG. ω-gliadin/C-hordein peptide and rye-derived peptide are immunogenic to gluten-sensitive T-cell lines and potentially present in wheat, rye and barley. Additional CD toxic peptides may be shared.

Analyzing Genetic Diversity for Virulence and Resistance Phenotypes in Populations of Stem Rust (Puccinia graminis f. sp. secalis) and Winter Rye (Secale cereale).

Stem rust (Puccinia graminis f. sp. secalis) leads to considerable yield losses in rye-growing areas with continental climate, from Eastern Germany to Siberia. For implementing resistance breeding, it is of utmost importance to (i) analyze the diversity of stem rust populations in terms of pathotypes (= virulence combinations) and (ii) identify resistance sources in winter rye populations. We analyzed 323 single-uredinial isolates mainly collected from German rye-growing areas across 3 years for their avirulence/virulence on 15 rye inbred differentials. Out of these, 226 pathotypes were detected and only 56 pathotypes occurred more than once. This high diversity was confirmed by a Simpson index of 1.0, a high Shannon index (5.27), and an evenness index of 0.97. In parallel, we investigated stem rust resistance among and within 121 heterogeneous rye populations originating mainly from Russia, Poland, Austria, and the United States across 3 to 15 environments (location-year combinations). While German rye populations had an average stem rust severity of 49.7%, 23 nonadapted populations were significantly (P < 0.01) more resistant with a stem rust severity ranging from 3 to 40%. Out of these, two modern Russian breeding populations and two old Austrian landraces were the best harboring 32 to 70% fully resistant plants across 8 to 10 environments. These populations with the lowest disease severity in adult-plant stage in the field also displayed resistance in leaf segment tests. In conclusion, stem rust populations are highly diverse and the majority of resistances in rye populations seems to be race specific.

Should wheat, barley, rye, and/or gluten be avoided in a 6-food elimination diet?

Eosinophilic esophagitis (EoE), a food antigen-mediated disease, is effectively treated with the dietary elimination of 6 foods commonly associated with food allergies (milk, wheat, egg, soy, tree nuts/peanuts, and fish/shellfish). Because wheat shares homologous proteins (including gluten) with barley and rye and can also be processed with these grains, some clinicians have suggested that barley and rye might also trigger EoE as a result of cross-reaction and/or cross-contamination with wheat. In this article, we discuss the theoretical risks of cross-reactivity and cross-contamination among wheat, barley, and rye proteins (including gluten); assess common practices at EoE treatment centers; and provide recommendations for dietary treatment and future studies of EoE.

Serum autoantibodies directed against transglutaminase-2 have a low avidity compared with alloantibodies against gliadin in coeliac disease.

Coeliac disease is characterized by intolerance to gliadin and related gluten components present in wheat, barley and rye. Coeliac disease patients harbour antibodies directed against alloantigens such as gliadin, but also against the autoantigen transglutaminase-2 (TG2). The type and quality of antibody responses provides insight into the underlying immune activation processes. Therefore, in this study we have analysed the avidity of the antibody response directed against the autoantigen TG2 and compared this with antibody responses against the alloantigens gliadin and Escherichia coli. We observed that the immunoglobulin (Ig)A autoantibody response directed against TG2 is of low avidity compared with the IgA response against the alloantigens gliadin and E. coli in the same patients; the same was true for IgG, both in IgA-deficient and in -sufficient coeliac patients. The observed avidities appear not to be related to disease stage, antibody levels, age or duration of exposure to gluten. In conclusion, in coeliac disease there is a clear difference in avidity of the antibody responses directed against the auto- and alloantigens, indicating different regulation or site of initiation of these responses.

Testing safety of germinated rye sourdough in a celiac disease model based on the adoptive transfer of prolamin-primed memory T cells into lymphopenic mice.

UNLABELLED: The current treatment for celiac disease is strict gluten-free diet. Technical processing may render gluten-containing foods safe for consumption by celiac patients, but so far in vivo safety testing can only be performed on patients. We modified a celiac disease mouse model to test antigenicity and inflammatory effects of germinated rye sourdough, a food product characterized by extensive prolamin hydrolysis. Lymphopenic Rag1-/- or nude mice were injected with splenic CD4+CD62L-CD44high-memory T cells from gliadin- or secalin-immunized wild-type donor mice. We found that: 1) Rag1-/- recipients challenged with wheat or rye gluten lost more body weight and developed more severe histological duodenitis than mice on gluten-free diet. This correlated with increased secretion of IFNγ, IL-2, and IL-17 by secalin-restimulated splenocytes. 2) In vitro gluten testing using competitive R5 ELISA demonstrated extensive degradation of the gluten R5 epitope in germinated rye sourdough. 3) However, in nude recipients challenged with germinated rye sourdough (vs. native rye sourdough), serum anti-secalin IgG/CD4+ T helper 1-associated IgG2c titers were only reduced, but not eliminated. In addition, there were no reductions in body weight loss, histological duodenitis, or T cell cytokine secretion in Rag1-/- recipients challenged accordingly. IN CONCLUSION: 1) prolamin-primed CD4+CD62L-CD44high-memory T cells induce gluten-sensitive enteropathy in Rag1-/- mice. 2) Hydrolysis of secalins in germinated rye sourdough remains incomplete. Secalin peptides retain B and T cell stimulatory capacity and remain harmful to the intestinal mucosa in this celiac disease model. 3) Current antibody-based prolamin detection methods may fail to detect antigenic gluten fragments in processed cereal food products.

[Celiac disease--the chameleon among the food intolerances]. / Zöliakie. Das Chamäleon unter den Nahrungsmittelintoleranzen.

Celiac disease is an autoimmune disorder resulting from gluten intolerance and is based on a genetically predisposition. Symptoms occur upon exposure to prolamin from wheat, rye, barley and related grain. The pathogenesis of celiac disease has not yet been sufficiently elucidated but is being considered as an autoimmune process. At its core are the deamidation of prolamin fragments, the building of specific antibodies and the activation of cytotoxic T-cells. The immunological inflammatory process is accompanied by structural damages of the enterocytes (villous atrophy, colonization and crypt hyperplasia). The symptoms and their extent depend on the type of the celiac disease; classic and non-classic forms are being distinguished (atypical, oligosymptomatic, latent and silent celiac disease). Characteristics of the classic presentation are malabsorption syndrome and intestinal symptoms such as mushy diarrhea and abdominal distension. The diagnosis of celiac disease is based on four pillars: Anamnesis and clinical presentation, serological evidence of coeliac specific antibodies (IgA-t-TG; IgA-EmA), small intestine biopsy and improvement of symptoms after institution of a gluten-free diet. The basis of the therapy is a lifelong gluten-free diet, i. e. wheat, rye, barley, spelt, green-core, faro-wheat, kamuth and conventional oats as well as food items obtained therefrom. Small amounts of up to 50 mg gluten per day are usually tolerated by most patients; amounts of > or = 100 mg/day lead mostly to symptoms. Gluten-free foods contain < or = 20 ppm or 20 mg/kg (Sign: symbol of the 'crossed ear' or label 'gluten-free'). At the beginning of the therapy the fat and lactose intake may need to be reduced; also the supplementation of single micronutrients (fat-soluble vitamins, folic acid, B12, iron, and calcium) may be required. Alternative therapies are being developed but have not yet been clinically tested.

Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew.

The improvement of wheat through breeding has relied strongly on the use of genetic material from related wild and domesticated grass species. The 1RS chromosome arm from rye was introgressed into wheat and crossed into many wheat lines, as it improves yield and fungal disease resistance. Pm8 is a powdery mildew resistance gene on 1RS which, after widespread agricultural cultivation, is now widely overcome by adapted mildew races. Here we show by homology-based cloning and subsequent physical and genetic mapping that Pm8 is the rye orthologue of the Pm3 allelic series of mildew resistance genes in wheat. The cloned gene was functionally validated as Pm8 by transient, single-cell expression analysis and stable transformation. Sequence analysis revealed a complex mosaic of ancient haplotypes among Pm3- and Pm8-like genes from different members of the Triticeae. These results show that the two genes have evolved independently after the divergence of the species 7.5 million years ago and kept their function in mildew resistance. During this long time span the co-evolving pathogens have not overcome these genes, which is in strong contrast to the breakdown of Pm8 resistance since its introduction into commercial wheat 70 years ago. Sequence comparison revealed that evolutionary pressure acted on the same subdomains and sequence features of the two orthologous genes. This suggests that they recognize directly or indirectly the same pathogen effectors that have been conserved in the powdery mildews of wheat and rye.

Characterization of wheat: Secale africanum introgression lines reveals evolutionary aspects of chromosome 1R in rye.

Wild Secale species, Secale africanum Stapf., serve as a valuable source for increasing the diversity of cultivated rye (Secale cereale L.) and provide novel genes for wheat improvement. New wheat - S. africanum chromosome 1R(afr) addition, 1R(afr)(1D) substitution, 1BL.1R(afr)S and 1DS.1R(afr)L translocation, and 1R(afr)L monotelocentric addition lines were identified by chromosome banding and in situ hybridization. Disease resistance screening revealed that chromosome 1R(afr)S carries resistance gene(s) to new stripe rust races. Twenty-nine molecular markers were localized on S. africanum chromosome 1R(afr) by the wheat - S. africanum introgression lines. Twenty markers can also identically amplify other reported wheat - S. cereale chromosome 1R derivative lines, indicating that there is high conservation between the wild and cultivated Secale chromosome 1R. Nine markers displayed polymorphic amplification between S. africanum and S. cereale chromosome 1R(afr) derivatives. The comparison of the nucleotide sequences of these polymorphic markers suggested that gene duplication and sequence divergence may have occurred among Secale species during its evolution and domestication.

Immunologic effect and tolerability of intra-seasonal subcutaneous immunotherapy with an 8-day up-dosing schedule to 10,000 standardized quality-units: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: International guidelines recommend that allergen-specific immunotherapy for pollen-induced rhinoconjunctivitis is initiated preseasonally. However, because subjects often present to physicians with allergy symptoms during the pollen season, "within-season" initiation of specific immunotherapy is of special interest. OBJECTIVE: We evaluated the immunomodulatory effects and tolerability of subcutaneous immunotherapy (SCIT) with Standardized Quality (SQ) 6-grass mix and rye allergen extract, using an 8-day intra-seasonal up-dosing schedule to 10,000 SQ-units (SQ-U). METHODS: In a 9-week, multicenter, randomized, double-blind, placebo-controlled trial, adults (mean age, 34.6 years; 99.3% whites) with grass pollen-induced rhinoconjunctivitis (mean disease duration, 15.1 years) were randomized 3:1 to receive SCIT or placebo. Treatment was initiated during the 2008 pollen season, with an 8-day up-dosing from 100 to 10,000 SQ-U (6 daily injections) followed by 2 maintenance injections of 10,000 SQ-U at intervals of 2 and 4 weeks, respectively. The primary end point examined immunologic effects, assessing the difference in IgE-blocking factor (serum components competing with IgE for allergen binding) between SCIT and placebo at week 9. Secondary/explorative end points included the difference in IgE-blocking factor, specific IgG(4), and specific IgE at various times. Tolerability (adverse events, local and systemic allergic reactions) of the up-dosing schedule was also evaluated. RESULTS: Of the 148 treated subjects 144 (SCIT n = 109; placebo n = 35) were analyzed for the primary parameter. Immunologic response (significantly higher increase in IgE-blocking factor and IgG(4) levels) occurred with SCIT versus placebo at week 9 (IgE-blocking factor, P = 0.0017; IgG(4,)P = 0.0215). Significant differences were observed as early as week 3. AEs were reported in 60.7% of SCIT- and 30.6% of placebo-treated subjects, with no treatment-related serious AEs. Local allergic reactions occurred in 46.4% of SCIT and 8.3% of placebo subjects (χ(2) test, P < 0.0001). No significant difference was observed between groups in the incidence of systemic reactions (7.1% SCIT vs 5.6% placebo; χ(2) test, P = 0.7413). CONCLUSIONS: This trial provides the first description of short (8-day) intra-seasonal up-dosing with SCIT, which induced immunologic effects after only 3 weeks, and was generally well tolerated, although it induced a marked increase in the rate of local reactions compared with placebo. ClinicalTrials.gov identifier NCT00807547; ALK trial ID SHX0562.

Repertoire of gluten peptides active in celiac disease patients: perspectives for translational therapeutic applications.

Celiac disease is a common and lifelong food intolerance, affecting approximately 1% of the population. Because of a mechanism not completely understood, the ingestion of wheat gluten, and of homologue proteins of barley and rye, induces in genetically predisposed individuals pronounced inflammatory reactions mainly at the site of small intestine. Gluten, the triggering factor, is a complex protein mixture highly resistant to the gastrointestinal enzymatic proteolysis, and this results in the presence of large, and potentially immunogenic, peptides at the intestinal mucosa surface. During the last decade, several studies have defined gluten peptides able to stimulate adaptive T cells, of either CD4 or CD8 phenotype, and to activate innate (non T) immune cells. This review examines the complete repertoire of gluten peptides recognized by celiac T cells and discusses the several translational implications that the identification of these epitopes opens.

Aegilops-Secale amphiploids: chromosome categorisation, pollen viability and identification of fungal disease resistance genes.

The aim of this study was to assess the potential breeding value of goatgrass-rye amphiploids, which we are using as a "bridge" in a transfer of Aegilops chromatin (containing, e.g. leaf rust resistance genes) into triticale. We analysed the chromosomal constitution (by genomic in situ hybridisation, GISH), fertility (by pollen viability tests) and the presence of leaf rust and eyespot resistance genes (by molecular and endopeptidase assays) in a collection of 6× and 4× amphiploids originating from crosses between five Aegilops species and Secale cereale. In the five hexaploid amphiploids Aegilops kotschyi × Secale cereale (genome UUSSRR), Ae. variabilis × S. cereale (UUSSRR), Ae. biuncialis × S. cereale (UUMMRR; two lines) and Ae. ovata × S. cereale (UUMMRR), 28 Aegilops chromosomes were recognised, while in the Ae. tauschii × S. cereale amphiploid (4×; DDRR), only 14 such chromosomes were identified. In the materials, the number of rye chromosomes varied from 14 to 16. In one line of Ae. ovata × S. cereale, the U-R translocation was found. Pollen viability varied from 24.4 to 75.4%. The leaf rust resistance genes Lr22, Lr39 and Lr41 were identified in Ae. tauschii and the 4× amphiploid Ae. tauschii × S. cereale. For the first time, the leaf rust resistance gene Lr37 was found in Ae. kotschyi, Ae. ovata, Ae. biuncialis and amphiploids derived from those parental species. No eyespot resistance gene Pch1 was found in the amphiploids.