Resistant Barley Research Articles

Genome-wide analysis of FRF gene family and functional identification of HvFRF9 under drought stress in barley.

FHY3 and its homologous protein FAR1 are the founding members of FRS family. They exhibited diverse and powerful physiological functions during evolution, and participated in the response to multiple abiotic stresses. FRF genes are considered to be truncated FRS family proteins. They competed with FRS for DNA binding sites to regulate gene expression. However, only few studies are available on FRF genes in plants participating in the regulation of abiotic stress. With wide adaptability and high stress-resistance, barley is an excellent candidate for the identification of stress-resistance-related genes. In this study, 22 HvFRFs were detected in barley using bioinformatic analysis from whole genome. According to evolution and conserved motif analysis, the 22 HvFRFs could be divided into subfamilies I and II. Most promoters of subfamily I members contained abscisic acid and methyl jasmonate response elements; however, a large number promoters of subfamily II contained gibberellin and salicylic acid response elements. HvFRF9, one of the members of subfamily II, exhibited a expression advantage in different tissues, and it was most significantly upregulated under drought stress. In-situ PCR revealed that HvFRF9 is mainly expressed in the root epidermal cells, as well as xylem and phloem of roots and leaves, indicating that HvFRF9 may be related to absorption and transportation of water and nutrients. The results of subcellular localization indicated that HvFRF9 was mainly expressed in the nuclei of tobacco epidermal cells and protoplast of arabidopsis. Further, transgenic arabidopsis plants with HvFRF9 overexpression were generated to verify the role of HvFRF9 in drought resistance. Under drought stress, leaf chlorosis and wilting, MDA and O2 - contents were significantly lower, meanwhile, fresh weight, root length, PRO content, and SOD, CAT and POD activities were significantly higher in HvFRF9-overexpressing arabidopsis plants than in wild-type plants. Therefore, overexpression of HvFRF9 could significantly enhance the drought resistance in arabidopsis. These results suggested that HvFRF9 may play a key role in drought resistance in barley by increasing the absorption and transportation of water and the activity of antioxidant enzymes. This study provided a theoretical basis for drought resistance in barley and provided new genes for drought resistance breeding.

A comprehensive review on mutation breeding milestones in cereals: Conventional to advanced molecular approaches to achieve sustainable goals in trait improvement

The grass family includes the annual common grasses known as cereals. The 5 main types of cereals are rice, wheat, barley, rye and oats. As a result of their high vitamin (20-80%) and fiber content (90%), these grains can help treat conditions like type II diabetes, complex metabolic syndrome, obesity, gastrointestinal disorders, and high cholesterol. Cereals have inspired a long history of research into their genetics, development, and evolution due to their significance in both economic and scientific terms. They need to be improved because they play a big part in global food production and crop quality is declining. The crop improvement can be done by incorporating desired features from non-adapted landraces/wild resources. That induced mutagenesis has made a significant contribution to crop improvement initiatives by producing mutant varieties with better and desired genetic modifications in the crop plants mainly in agronomically significant features. The mutants produced by the induction of physical and chemical mutagens were highly useful. The notable traits obtained by the induced mutations are semidwarfness and early maturity in rice varieties, disease resistance in barley, salinity, and drought tolerance in wheat, and lodging and disease resistance in oat and rye. In this review, we focused on identifying and developing elite mutants, their genetic function, and their utilization in future breeding programs for various traits in cereal crops.

Breeding for durable resistance against biotrophic fungal pathogens using transgenes from wheat

Breeding for resistant crops is a sustainable way to control disease and relies on the introduction of novel resistance genes. Here, we tested three strategies on how to use transgenes from wheat to achieve durable resistance against fungal pathogens in the field. First, we tested the highly effective, overexpressed single transgene Pm3e in the background of spring wheat cultivar Bobwhite in a long-term field trial over many years. Together with previous results, this revealed that transgenic wheat line Pm3e#2 conferred complete powdery mildew resistance during a total of nine field seasons without a negative impact on yield. Furthermore, overexpressed Pm3e provided resistance to powdery mildew isolates from our worldwide collection when crossed into the elite wheat cultivar Fiorina. Second, we pyramided the four overexpressed transgenes Pm3a, Pm3b, Pm3d, and Pm3f in the background of cultivar Bobwhite and showed that the pyramided line Pm3a,b,d,f was completely resistant to powdery mildew in five field seasons. Third, we performed field trials with three barley lines expressing adult plant resistance gene Lr34 from wheat during three field seasons. Line GLP8 expressed Lr34 under control of the pathogen-inducible Hv-Ger4c promoter and provided partial barley powdery mildew and leaf rust resistance in the field with small, negative effects on yield components which might need compensatory breeding. Overall, our study demonstrates and discusses three successful strategies for achieving fungal disease resistance of wheat and barley in the field using transgenes from wheat. These strategies might confer long-term resistance if applied in a sustainable way.

Levan differentially primes barley defence against infections by Fusarium graminearum, Rhizoctonia solani and Pyricularia oryzae

AbstractThe widespread use of pesticides poses significant challenges to the safety of agricultural products and the ecological environment. Levan‐type fructans have the ability to act as an induced resistance stimulus, enhancing plant disease resistance, which aligns with the principles of green development. This study showed that levan polysaccharide, produced by Halomonas smyrnnensis levansucrase, could induce the resistance of barley to Fusarium graminearum much better than inulin‐type fructans or low‐molecular‐weight chitosan oligosaccharide. Three different application methods of levan, namely seed soaking, leaf spraying or their combination, all decreased the necrotic areas caused by F. graminearum and Rhizoctonia solani. When inoculated with F. graminearum, the content of hydrogen peroxide, proline or jasmonic acid and the activities of peroxidase and ascorbate peroxidase in levan‐treated barley were higher than those in the control. In contrast, when inoculated with R. solani, the hydrogen peroxide content and peroxidase activity in levan‐treated leaves showed a faster induction than the control at the early priming and the content of proline and methyl salicylate was higher than that of the control. Thus, levan induces distinct pathways in response to different pathogens.

Analysis of genetic variability and correlations between yield and its contributing traits in barley (Hordeum vulgare L.) for drought resistance

Analysis of genetic variability and correlations between yield and its contributing traits in barley (Hordeum vulgare L.) for drought resistance

New genetic diversity of winter two-row barley lines resistant to Loose smut Ustilago nuda (Jensen) Rostrup

The creation of resistant barley cultivars leads to reduction in direct yield losses and limits the accumulation of infection in crops. Studies of new genetic plasma make it possible to find forms that can serve as starting material for the creation of a future disease-resistant cultivar. In the period 2012/2014, in competitive variety experiments, perspective lines of winter two-row barley resistant to loose smut (Ustilago nuda (Jensen) Rostrup) were studied. The experiment was based on the method of the Latin rectangle, with a plot of 10 sq m, in four repetitions, with standards Obzor, Emon and Kaskadyor 3. The yield was reported and the results were statistically processed. Grain quality was determined on the basis of the following indicators: 1000-grain weight (g) by weight method, uniformity 1st class (%), expressed as the ratio of the weight of grains remaining on sorting sieves 2.8 mm wide and 2.5 mm to the total weight in a sample of 100 g, protein content (%) (Kjeldahl method), extract content (%) according to EBC (1998). The relationship between grain yield and quality in perspective lines resistant to loose smut (Ustilago nuda (Jensen) Rostrup) was studied by correlation and principle component analysis using software SPSS 19.0 and Statgraf 2.1. As a result, winter barley lines resistant to loose smut (Ustilago nuda (Jensen) Rostrup) were created. Perspective lines PG 4480, PG 4447 and PG 4628 combine resistance to loose smut (Ustilago nuda (Jensen) Rostrup), high productivity and grain quality. They can be successfully used in the barley breeding program as parent components for creating new cultivars.

Expression analysis and characterization of morpho-physiological traits for reduced height and increased lodging resistance in barley (Hordeum vulgare L.) hybrids under different water regimes

Expression analysis and characterization of morpho-physiological traits for reduced height and increased lodging resistance in barley (Hordeum vulgare L.) hybrids under different water regimes

Bacterial Community Structure in Rhizosphere of Barley at Maturity Stage

The crop rhizosphere is the main site of soil microbial activities. Understanding the structure and diversity of microbial communities in the crop rhizosphere will help us reveal interactions between rhizosphere microorganisms and plant growth. In this study, the rhizosphere soil was collected from 35 cultivated barley varieties at the mature stage. To investigate the structure and diversity of bacterial communities in the rhizosphere of different barley varieties, the 16S rDNA gene of microorganisms from the soil was sequenced using Illumina MiSeq next-generation high-throughput sequencing technology. The results showed that 13, 25, 49, and 59 bacterial flora with relative abundance >1% were detected from 35 barley rhizosphere samples at the phylum, class, order, and family levels, respectively. The abundance of bacteria among varieties differed relatively little, but the abundance of the same bacteria in rhizospheres of different varieties was different. In addition, both the cluster analysis and principal component analysis (PCA) divided the 35 samples into three clusters at the phylum level. Groups III and IV showed significantly higher abundance than group II in Proteobacteria, while group II exhibited significantly higher abundance of Chloroflexi than groups III and IV. This finding provides a realistic basis for further using the relationship between barley rhizosphere microorganisms and barley growth to improve the resistance and quality of barley.

Genetic diversity of barley accessions from East Asian countries in terms of resistance to powdery mildew

Background. Barley is an ancient crop currently cultivated in more than 100 countries. High adaptability makes it possible to cultivate it in environments unfavorable for many other crops. At the same time, fungal diseases can have a negative impact on its grain yield and quality. One of the most harmful is the fungus Blumeria graminis (DC.) Golovin ex Speer f. sp. hordei Marchal. The pathogen can overcome a cultivar’s resistance, resulting in significant harvest losses. There is a need for a constant search for new effective sources of resistance to powdery mildew for barley breeding.Materials and methods. A set of 950 barley accessions from the East Asian center of the crop’s morphogenesis were studied at the adult stage of plant development. They were screened and crossed in the fields of Pushkin and Pavlovsk Laboratories of VIR (St. Petersburg, Russia). Genetic control of barley resistance to B. graminis was analyzed in a climate chamber under artificial infection pressure with the northwestern population of the fungus. Scoring scales were used for screening.Results and conclusions. Weak development of B. graminis during the heading phase was observed on plants of 38 barley accessions from the East Asian center. High resistance at all stages of development was manifested by 20 genotypes that can be used in breeding programs. Resistance genes in 18 selected forms differed in their effectiveness at the seedling stage of development and during the heading phase. Accessions k-3433, k-10931, k-10934, k-11608, k-17545, k-20272, k-20279, k-20354 and k-27867 had one dominant resistance gene each. Accessions k-11608, k-12278, and k-17545 are protected by the identical gene, while the resistance of k-3433 is induced by a gene that differs from the genes present in accessions k-10931 and k-20279.

Comparative transcriptome analysis of major lodging resistant factors in hulless barley.

Hulless barley (Hordeum vulgare L. var. nudum Hook. f.), belonging to the genus Gramineae, has high and steady output and thus considered as a principal food crop by Tibetan people. Hulless barley grain can be used for food, brewing, and functional health product development, while its straw serves as an essential supplementary forage and is a crucial cereal crop. Lodging can reduce the yield and quality of barley grain and straw, and it hinders mechanical harvesting. It is a significant factor affecting high and stable yields of barley. Unlike other Poaceae plants (such as rice, wheat), hulless barley is mainly grown in high-altitude regions, where it is susceptible to low temperatures, strong winds, and heavy rainfall. As a result, its stem lodging resistance is relatively weak, making it prone to lodging during the growth period. In this study, we observed that the lignin concentration and the contents of lignin monomers (H, S, and G), and neutral detergent fibre of the lodging-resistant variety Kunlun14 were substantially greater than those of the lodging-sensitive variety Menyuanlianglan. We performed the weighted gene co-expression network analysis (WGCNA) and Short Time-series Expression Miner (STEM) analysis of both the lodging-resistant and lodging-sensitive varieties. Through transcriptome sequencing analysis at different developmental stages, combined with the previously annotated genes related to lodging resistance, a total of 72 DEGs were identified. Among these DEGs, 17 genes were related to lignin, cellulose, and hemicellulose synthesis or regulation, including five transcription factors about NAC, MYB and WRKY. Our results provide a basis for further exploring the molecular mechanism of stem lodging resistance in hulless barley and provide valuable gene resources for stem lodging resistance molecular breeding.

Root Silicon Amendment Enhances Barley’s Resistance to Fusarium Head Blight in the Field

Background: Silicon (Si) amendment plays an important role in enhancing the resistance of several plant species to diverse pathogens. To date, a few studies have focused on how Si application helps barley, a higher Si absorber and accumulator monocot, to resist fungal diseases, including Fusarium head blight (FHB), which reduces the quality and safety of harvested products worldwide. However, no study has ever been conducted to demonstrate Si's ability to suppress FHB development in barley heads under uncontrolled climatic conditions. Materials and Methods: This 2-year field study elucidated the effect of multiple Si applications at 1.7 mM via roots in two barley cultivars, Arabi Aswad (AS moderately resistant) and Arabi Abiad (AB moderately susceptible), to control four Fusarium species with diverse pathogenicity. The incidence of FHB (DI type I resistance), severity of FHB (DS type II), and Fusarium-damaged kernels (FDK type III) were also tested to describe the nature of the Si-enhanced barley resistance. Results: Si treatment at 1.7 mM under soil culture decreased FHB development by enhancing all resistance types measured in the present research. DI, DS, and FDK were reduced by 18.7%, 20.3%, and 20.2%, respectively, in Si-Fusarium-inoculated treatments relative to fungal-inoculated controls. Si absorption in barley strengthened the defense system measured by type I and type II on AB to a level comparable to AS not amended with Si. Irrespective of the barley cultivar, however, Si resulted in a quasi-similar reduction of FDK. Importantly, Si treatment at 1.7 mM decreased the damage of FHB in previous analyses conducted on AS and AB under in vitro and growth chamber environments, showing that Si enhanced the expression of resistance to FHB infection in seedlings and adult barley plants. Conclusion: All of these results are promising outcomes for the application of Si as a safe and effective method against Fusarium species. This study provides new insights into the potential of multiple Si applications at 1.7 mM via roots for boosting barley’s resistance to FHB with a bright prospect for Si use in barley cultivation under field conditions.

Deciphering the genetic diversity and population structure of wild barley germplasm against corn leaf aphid, Rhopalosiphum maidis (Fitch)

Corn-leaf aphid (CLA-Rhopalosiphum maidis) is a major insect pest of barley (Hordeum vulgare) causing yield loss upto 30% under severe infestation. Keeping in view of the availability of very few sources of CLA resistance in barley, the present investigation was framed to assess the genetic diversity and population structure of 43 wild barley (H. vulgare subsp. spontaneum) genotypes using eight microsatellite markers against R. maidis. Three statistical methods viz. multivariate-hierarchical clustering, Bayesian clustering and PCoA, unanimously grouped genotypes into three subpopulations (K = 3) with 25.58% (SubPop1-Red), 39.53% (SubPop2-Green) and 34.88% (SubPop3-Blue) genotypes including admixtures. Based on Q ≥ 66.66%, 37.20% genotypes formed a superficial “Mixed/Admixture” subpopulation. All polymorphic SSR markers generated 36 alleles, averaging to 4.5 alleles/locus (2–7 range). The PIC and H were highest in MS31 and lowest in MS28, with averages of 0.66 and 0.71. MAF and mean genetic diversity were 0.16 and 89.28%, respectively. All these parameters indicated the presence of predominant genetic diversity and population structure amongst the studied genotypes. Based on AII, only 6 genotypes were found to be R. maidis resistant. SubPop3 had 91.66% (11) of the resistant or moderately resistant genotypes. SubPop3 also had the most pure genotypes (11), the least aphid infestation (8.78), and the highest GS (0.88), indicating its suitability for future R. maidis resistance breeding initiatives.

Response of salt stress resistance in highland barley (Hordeum vulgare L. var. nudum) through phenylpropane metabolic pathway.

Highland barley (Hordeum vulgare L. var. nudum) is a grain crop that grows on the plateau under poor and high salt conditions. Therefore, to cultivate high-quality highland barley varieties, it is necessary to study the molecular mechanism of strong resistance in highland barley, which has not been clearly explained. In this study, a high concentration of NaCl (240 mmol/L), simulating the unfavorable environment, was used to spray the treated highland barley seeds. Transcriptomic analysis revealed that the expression of more than 8,000 genes in highland barley seed cells was significantly altered, suggesting that the metabolic landscape of the cells was deeply changed under salt stress. Through the KEGG analysis, the phenylpropane metabolic pathway was significantly up-regulated under salt stress, resulting in the accumulation of polyphenols, flavonoids, and lignin, the metabolites for improving the stress resistance of highland barley seed cells, being increased 2.71, 1.22, and 1.17 times, respectively. This study discovered that the phenylpropane metabolic pathway was a significant step forward in understanding the stress resistance of highland barley, and provided new insights into the roles of molecular mechanisms in plant defense.

Enhancement of some barley (Hordeum vulgare L.) resistance for nematode (Heterdra avanae) using DNA fingerprinting analysis

Background Heterodera avenae, the cereal cyst nematode or European cyst nematode, is a plant pathogen and an obligate parasite of cereal crops including barley, oats, wheat, and rye. Cereal crops infected with this nematode are more susceptible to infection by fungal diseases such as Rhizoctonia root rot. Objective The main objective of the present study was to map Heterodera avenae resistance and to compare seven different species. This study also aimed to increase the efficiency and precision of standard procedures for testing the resistance of barley toward root-lesion nematodes. Materials and methods The genetic variability analysis of seven barley genotypes, of Egypt was achieved using 7 Inter simple sequence repeat (ISSR) primers. Phylogenetic relationships of seven accessions of (Hordeum vulgare L.) collected from different region of Egypt were assessed. The soil was sterilized in an autoclave of each pot was planted in the comparison experiment, as well as in the treatment experiment with nematode Heterdra avanae infected soil brought from an infected field, and this soil was added to the treated pots. Also (ISSR) molecular marker technique was used for DNA fingerprinting and assessing genetic diversity and phylogenetic relationships in barley germplasm. Results and conclusion A significant correlation was observed between the Jaccard’s dissimilarity matrices based on ISSR markers, as revealed by Mantel test using the Pearson correlation coefficient (r = 0.69; P < 0.05). The results showed that ISSR primers produced 37 bands their size ranged between 100-2000 bp with (87.5%). polymorphism percentage. Polymorphic information content PIC was 0.74 for ISSR. Unweighted Pair Group Method with Arithmetic (UPGMA), Dendrogram was divided into two clusters by morphological traits and ISSR analysis. Genetic similarity matrix was examined with Jaccard’s coefficient, maximum similarity was found between Giza126 and Giza127 (99%) with morphological analysis both and lowest similarity between Giza123 and Giza126 (6%) with ISSR analysis. Determination of genetic diversity between barley is of major importance for characterization of barley germplasm, breeding programs and conservation purposes. Morphological traits and ISSR analysis are effective tools for detecting genetic variations. The results showed that H. vulgare have high ratio of variation. This study may be considered as reference study for further studies on H. vulgare and may contribute to species concept and breeding programs.

English

English

Exploring Wild Hordeum spontaneum and Hordeum marinum Accessions as Genetic Resources for Fungal Resistance.

Crop Wild Relatives (CWRs), as potential sources of new genetic variants, are being extensively studied to identify genotypes that will be able to confer resistance to biotic stresses. In this study, a collection of barley wild relatives was assessed in the field, and their phenotypic variability was evaluated using a Barley Description List, reflecting the identified ecosites. Overall, the CWRs showed significant field resistance to various fungal diseases. To further investigate their resistance, greenhouse tests were performed, revealing that several CWRs exhibited resistance against Fusarium culmorum, Pyrenophora teres, and Puccinia hordei G.H. Otth. Additionally, to characterize the genetic diversity within the collection, DNA polymorphisms at 21 loci were examined. We successfully employed barley-specific SSR markers, confirming their suitability for identifying H. spontaneum and even H. marinum, i.e., perennial species. The SSR markers efficiently clustered the investigated collection according to species and ecotypes, similarly to the phenotypic assessment. Moreover, SSR markers associated with disease resistance revealed different alleles in comparison to those found in resistant barley cultivars. Overall, our findings highlight that this evaluated collection of CWRs represents a valuable reservoir of genetic variability and resistance genes that can be effectively utilized in breeding programs.

A pathogen-induced putative NAC transcription factor mediates leaf rust resistance in barley

Leaf rust, caused by Puccinia hordei, is one of the most widespread and damaging foliar diseases affecting barley. The barley leaf rust resistance locus Rph7 has been shown to have unusually high sequence and haplotype divergence. In this study, we isolate the Rph7 gene using a fine mapping and RNA-Seq approach that is confirmed by mutational analysis and transgenic complementation. Rph7 is a pathogen-induced, non-canonical resistance gene encoding a protein that is distinct from other known plant disease resistance proteins in the Triticeae. Structural analysis using an AlphaFold2 protein model suggests that Rph7 encodes a putative NAC transcription factor with a zinc-finger BED domain with structural similarity to the N-terminal DNA-binding domain of the NAC transcription factor (ANAC019) from Arabidopsis. A global gene expression analysis suggests Rph7 mediates the activation and strength of the basal defence response. The isolation of Rph7 highlights the diversification of resistance mechanisms available for engineering disease control in crops.

Genome-wide association and targeted transcriptomic analyses reveal loci and candidate genes regulating preharvest sprouting in barley.

Genome-wide association study of diverse barley genotypes identified loci, single nucleotide polymorphisms and candidate genes that control seed dormancy and therefore enhance resistance to preharvest sprouting. Preharvest sprouting (PHS) causes significant yield and quality loss in barley and it is strongly associated with the level of seed dormancy. This study performed genome-wide association study using a collection of 255 diverse barley genotypes grown over four environments to identify loci controlling dormancy/PHS. Our phenotypic analysis revealed substantial variation in germination index/dormancy levels among the barley genotypes. Marker-trait association and linkage disequilibrium (LD) decay analyses identified 16 single nucleotide polymorphisms (SNPs) and two QTLs associated with dormancy/PHS, respectively, on chromosome 3H and 5H explaining 6.9% to 11.1% of the phenotypic variation. QTL.5H consist of 14 SNPs of which 12 SNPs satisfy the FDR threshold of α = 0.05, and it may represent the SD2 locus. The QTL on 3H consists of one SNP that doesn't satisfy FDR (α = 0.05). Genes harbouring the significant SNPs were analyzed for their expression pattern in the seeds of selected dormant and non-dormant genotypes. Of these genes, HvRCD1, HvPSRP1 and HvF3H exhibited differential expression between the dormant and non-dormant seed samples, suggesting their role in controlling seed dormancy/PHS. Three SNPs located within the differentially expressed genes residing in QTL.5H explained considerable phenotypic variation (≥ 8.6%), suggesting their importance in regulating PHS resistance. Analysis of the SNP marker data in QTL.5H identified a haplotype for PHS resistance. Overall, the study identified loci, SNPs and candidate genes that control dormancy and therefore play important roles in enhancing PHS resistance in barley through marker-assisted breeding.

Improved quantification of Fusarium pseudograminearum (Fusarium crown rot) using qPCR measurement of infection in multi-species winter cereal experiments.

Fusarium crown rot (FCR) causes significant grain yield loss in winter cereals around the world. Breeding for resistance and/or tolerance to FCR has been slow with relatively limited success. In this study, multi-species experiments were used to demonstrate an improved method to quantify FCR infection levels at plant maturity using quantitative PCR (qPCR), as well as the genotype yield retention using residual regression deviation. Using qPCR to measure FCR infection allowed a higher degree of resolution between genotypes than traditional visual stem basal browning assessments. The results were consistent across three environments with different levels of disease expression. The improved measure of FCR infection along with genotype yield retention allows for partitioning of both tolerance and partial resistance. Together these methods offer new insights into FCR partial resistance and its relative importance to tolerance in bread wheat and barley. This new approach offers a more robust, unbiased way to select for both FCR traits within breeding programs. Key message: Genetic gain for tolerance and partial resistance against Fusarium crown rot (FCR) in winter cereals has been impeded by laborious and variable visual measures of infection severity. This paper presents results of an improved method to quantify FCR infection that are strongly correlated to yield loss and reveal previously unrecognised partial resistance in barley and wheat varieties.

Highly effective mlo-based powdery mildew resistance in hexaploid wheat without pleiotropic effects

Application of the mlo-based resistance in barley against powdery mildew attacks is a major success in crop breeding, since it confers durable disease resistance. Resistance caused by mutations in the Mlo gene seems to be ubiquitous across a range of species. This work addresses the introduction of mlo-based resistance into hexaploid wheat, which is complicated by the occurrence of three homoeologous genes: Mlo-A1, Mlo-B1 and Mlo-D1. EMS-generated mutant plants were screened for mutations in the three homoeologues. We selected and combined 6, 8, and 4 mutations, respectively, to obtain triple homozygous mlo mutant lines. Twenty-four mutant lines showed highly effective resistance towards attack by the powdery mildew pathogen under field conditions. All 18 mutations appeared to contribute to resistance; however, they had different effects on the occurrence of symptoms such as chlorotic and necrotic spots, which are pleiotropic to the mlo-based powdery mildew resistance. We conclude that to obtain highly effective powdery mildew resistance in wheat and to avoid detrimental pleiotropic effects, all three Mlo homoeologues should be mutated; however, at least one of the mutations should be of the weaker type in order to alleviate strong pleiotropic effects from the other mutations.