Molecular mapping of resistance genes to tan spot [Pyrenophora tritici-repentis race 1] in synthetic wheat lines

Synthetic wheat lines (2n = 6x = 42, AABBDD), which are amphiploids developed from the hybrid between tetraploid wheat (Triticum turgidum L., 2n = 4x = 28, AABB) and Aegilops tauschii Coss. (2n = 2x = 14, DD), are important sources of resistance against tan spot of wheat caused by Pyrenophora tritici-repentis. In the present study, inheritance, allelism and genetic linkage analysis in synthetic wheat lines have been carried out. Segregation analysis of the phenotypic and molecular data in F_2:3 populations of CS/XX41, CS/XX45, and CS/XX110 has revealed a 1:2:1 segregation ratio indicating that resistance of tan spot in these synthetic lines is controlled by a single gene. Allelism tests detected no segregation for susceptibility among F₁ and F₂ plants derived from intercrosses of the resistance lines XX41, XX45 and XX110 indicating that the genes are either allelic or tightly linked. Linkage analysis using SSR markers showed that all the three genes: tsn3a in XX41, Tsn3b in XX45 and tsn3c in XX110 are clustered in the region around Xgwm2a, located on the short arm of chromosome 3D. The linked markers and genetic relationship of these genes will greatly facilitate their use in wheat breeding and deployment of cultivars resistant to tan spot.     

RFLP mapping of resistance to chlorosis induction by Pyrenophora tritici-repentis in wheat

Tan spot, caused by Pyrenophora tritici-repentis, is an economically important disease in major wheat production areas. The fungus can produce two genetically distinct symptoms on leaves of susceptible wheat genotypes: tan necrosis (nec) and extensive chlorosis (chl). Our objectives were to determine the number of genes conditioning resistance to tan spot in a population of wheat recombinant inbred lines, and map the chromosomal location of the resistance genes using RFLPs. Conidia produced by the P. tritici-repentis isolate Pti2 (nec+chl+) were used to inoculate seedlings of 135 recombinant inbred lines derived from the cross of the synthetic hexaploid wheat W-7984 with Opata 85. A subset of the population was inoculated with conidia produced by the isolates D308 (nec−chl+) and 86-124 (nec+chl−). Inoculated seedlings were rated on a scale of 1 to 5 based on lesion type. Necrosis-inducing culture filtrate produced by the isolate 86-124 was also used to screen the entire population. A map consisting of 532 markers was employed to identify significant associations between marker loci and tan spot resistance. The entire population was insensitive to culture filtrate produced by the isolate 86-124, and the entire subset was resistant to conidial inoculation of the same isolate. The population segregated for reaction to isolates D308 and Pti2, indicating that this population segregates for resistance to extensive chlorosis only, and not to tan necrosis. RFLP analysis indicated the presence of a gene with a major effect in 1AS, a gene with a minor effect in 4AL, and an interaction between the 1AS gene and a gene in 2DL. Together, these loci explained 49.0% of the variation in this population for resistance to tan spot produced by the isolate Pti2. Two regions one in 1BL and one in 3BL, were significantly associated with resistance to extensive chlorosis, but were not significant in the multiple regression model. It should be feasible to introgress these resistance loci into adapted genetic backgrounds by using a marker-assisted selection scheme. Received: 30 March 1996 / Accepted: 31 May 1996     

Genome-wide association mapping for resistance to leaf rust,stripe rust and tan spot in wheat reveals potential candidate genes

Key message

Genome-wide association mapping in conjunction with population sequencing map and Ensembl plants was used to identify markers/candidate genes linked to leaf rust, stripe rust and tan spot resistance in wheat.

Abstract

Leaf rust (LR), stripe rust (YR) and tan spot (TS) are some of the important foliar diseases in wheat (Triticum aestivum L.). To identify candidate resistance genes for these diseases in CIMMYT’s (International Maize and Wheat Improvement Center) International bread wheat screening nurseries, we used genome-wide association studies (GWAS) in conjunction with information from the population sequencing map and Ensembl plants. Wheat entries were genotyped using genotyping-by-sequencing and phenotyped in replicated trials. Using a mixed linear model, we observed that seedling resistance to LR was associated with 12 markers on chromosomes 1DS, 2AS, 2BL, 3B, 4AL, 6AS and 6AL, and seedling resistance to TS was associated with 14 markers on chromosomes 1AS, 2AL, 2BL, 3AS, 3AL, 3B, 6AS and 6AL. Seedling and adult plant resistance (APR) to YR were associated with several markers at the distal end of chromosome 2AS. In addition, YR APR was also associated with markers on chromosomes 2DL, 3B and 7DS. The potential candidate genes for these diseases included several resistance genes, receptor-like serine/threonine-protein kinases and defense-related enzymes. However, extensive LD in wheat that decays at about 5?×?10⁷ bps, poses a huge challenge for delineating candidate gene intervals and candidates should be further mapped, functionally characterized and validated. We also explored a segment on chromosome 2AS associated with multiple disease resistance and identified seventeen disease resistance linked genes. We conclude that identifying candidate genes linked to significant markers in GWAS is feasible in wheat, thus creating opportunities for accelerating molecular breeding.

     

Genetics of tan spot resistance in wheat

Tan spot is a devastating foliar disease of wheat caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis. Much has been learned during the past two decades about the genetics of wheat–P. tritici-repentis interactions. Research has shown that the fungus produces at least three host-selective toxins (HSTs), known as Ptr ToxA, Ptr ToxB, and Ptr ToxC, that interact directly or indirectly with the products of the dominant host genes Tsn1, Tsc2, and Tsc1, respectively. The recent cloning and characterization of Tsn1 provided strong evidence that the pathogen utilizes HSTs to subvert host resistance mechanisms to cause disease. However, in addition to host–HST interactions, broad-spectrum, race non-specific resistance QTLs and recessively inherited qualitative ‘resistance’ genes have been identified. Molecular markers suitable for marker-assisted selection against HST sensitivity genes and for race non-specific resistance QTLs have been developed and used to generate adapted germplasm with good levels of tan spot resistance. Future research is needed to identify novel HSTs and corresponding host sensitivity genes, determine if the recessively inherited resistance genes are HST insensitivities, extend the current race classification system to account for new HSTs, and determine the molecular basis of race non-specific resistance QTLs and their relationships with host–HST interactions at the molecular level. Necrotrophic pathogens such as P. tritici-repentis are likely to become increasingly significant under a changing global climate making it imperative to further characterize the wheat–P. tritici-repentis pathosystem and develop tan spot resistant wheat varieties.     

Chromosomal location and molecular mapping of a tan spot resistance gene in the winter wheat cultivar Red Chief

The winter wheat cultivar Red Chief has been identified as the wheat cultivar most resistant toPyrenophora tritici-repentis (Ptr). This study was undertaken to determine the inheritance, chromosomal location and molecular mapping of a tan spot resistance gene in Red Chief. χ² analysis of the F₂ segregation data of the hybrids between 21 monosomic lines of the susceptible wheat cultivar Chinese Spring and the resistant cultivar Red Chief revealed that tan spot resistance in cv. Red Chief is controlled by a single recessive gene located on chromosome 3A. Linkage analysis using SSR markers in the Red Chief/Chinese Spring F2 population showed that thetsr4 gene is clustered in the region aroundXgwm2a, on the short arm of chromosome 3A. This marker has also been identified as the closest marker to thetsr3 locus on chromosome 3D in synthetic wheat lines. Validation analysis of this marker for thetsr3 andtsr4 genes using 28 resistant and 6 susceptible genotypes indicated that the 120 bp allele (thetsr3 gene) specific fragment was observed in 11 resistant genotypes, including the three synthetic lines XX41, XX45 and XX110, while the 130 bp allele was amplified only in cv. Red Chief and Dashen.Xgwm2a can be used to trace the presence of the target gene in successive backcross generations and pyramiding of thetsr3 &tsr4 genes into a commonly grown and adaptable cultivar.     

The effect of Trichoderma harzianum and T. koningii on the control of tan spot (Pyrenophora tritici-repentis) and leaf blotch (Mycosphaerella graminicola) of wheat under field conditions in Argentina

The effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot (Pyrenophora tritici-repentis) and leaf blotch of wheat (Mycosphaerella graminicola) was evaluated under field conditions. Significant differences between wheat cultivars, inoculum types and growth stages were found. Three of the isolates tested (T2 for M. graminicola, T7 for P. tritici-repentis and T5 for both of them) showed the best performance in controlling leaf blotch and tan spot when coated onto seed or sprayed onto wheat leaves at different growth stages, with significant severity reduction up to 56%. At tillering, six of the isolates reduced the severity of P. tritici-repentis and M. graminicola compared to the control by up to 39% and 12-53%, respectively. In some experiments, the biocontrol preparation (T2 and T5) gave a level of disease control similar to that obtained with Tebuconazole (70 and 48%, respectively). The effect of Trichoderma against P. tritici-repentis was also observed at the heading stage, when six of the treatments reduced disease severity by 16-35%. This is the first report on the efficacy of Trichoderma spp. against wheat necrotrophic pathogens under field conditions in Argentina.     

Genome-wide association mapping for adult resistance to powdery mildew in common wheat

Molecular Biology Reports - Blumeria graminis f. sp. tritici, the causal agent of wheat powdery mildew disease, can occur at all stages of the crop and constantly threatens wheat production. To...     

Association mapping for Fusarium head blight resistance in European soft winter wheat

Resistance to Fusarium head blight (FHB) is of great importance in wheat breeding programs in the northern hemisphere. In Europe, breeders prefer adapted germplasm as resistance donor because of high grain yield and quality demands. Our objective was to identify chromosomal regions affecting FHB resistance among 455 European soft winter wheat (Triticum aestivum L.) lines using a genome-wide association mapping approach and to analyze the importance of epistatic interactions. All entries were evaluated for FHB resistance by inoculation in two environments and several ratings. Wheat was genotyped by 115 simple sequence repeat markers randomly distributed across the genome and two allele-specific markers for Rht-B1 and Rht-D1 genes. The genome-wide scan revealed nine significant (P < 0.05) marker–phenotype associations on seven chromosomes including dwarfing gene Rht-D1. Using a Bonferroni–Holm correction, three significant associations remained on chromosomes 1B, 1D, and 2D. The proportion of the genotypic variance explained simultaneously by individual markers was 36% and increased to 50% when two digenic epistatic interactions were considered, one of them associated with Rht-B1. In conclusion, new genomic regions on chromosomes 1D and 3A could be found for FHB resistance in European wheat and the effect of epistatic interactions was substantial.     

Genome-wide association mapping reveals genetic architecture of durable spot blotch resistance in US barley breeding germplasm

Spot blotch, an economically important disease of both barley and wheat, is caused by Cochliobolus sativus (anamorph: Bipolaris sorokiniana). The disease has been reported in many regions of the world, but is particularly severe on barley in the Upper Midwest region of the USA and adjacent areas of Canada. For over 50 years, spot blotch has been effectively controlled through the deployment of durable resistance in six-rowed malting cultivars. To characterize loci conferring spot blotch resistance in US barley germplasm, we employed an association mapping approach using 3,840 breeding lines and cultivars. Three quantitative trait loci (QTL), Rcs-qtl-1H-11_10764, Rcs-qtl-3H-11_10565 and Rcs-qtl-7H-11_20162, were found to confer both seedling and adult plant resistance. Together, these three QTL comprise the Midwest Six-rowed Durable Resistant Haplotype (MSDRH), which is present in all Midwest six-rowed cultivars released since the 1960s. Each QTL alone only partially reduced disease levels, but combining all three together reduced the seedling infection response and adult plant disease severity by 47 and 83 %, respectively. The identified MSDRH will be valuable for marker-assisted selection of breeding lines to deploy spot blotch resistance and can also be incorporated into genomic selection as one of the disease resistance traits.     

Chromosome-based molecular characterization of pathogenic and non-pathogenic wheat isolates of Pyrenophora tritici-repentis

The ToxA gene of Pyrenophora tritici-repentis encodes a host-selective toxin (Ptr ToxA) that has been shown to confer pathogenicity when used to transform a non-pathogenic wheat isolate. Major karyotype polymorphisms between pathogenic and non-pathogenic strains, and to a lesser extent among pathogenic strains, and among non-pathogenic strains were identified. ToxA was localized to a 3.0 Mb chromosome. PCR-based subtraction was carried out with the ToxA chromosome as tester DNA and genomic DNA from a non-pathogenic isolate as driver DNA. Seven of 8 single-copy probes that originated from the 3.0 Mb chromosome could be assigned to a 2.75 Mb chromosome of a non-pathogenic isolate. Nine different repetitive DNA probes originated from the 3.0 Mb chromosome, including sequences that correspond to known fungal transposable elements. Two additional single-copy probes that originated from a 3.4 Mb chromosome were unique to the pathogens and they correspond to a peptide synthetase gene. Our findings suggest substantial differences between pathogenic and non-pathogenic isolates of P. tritici-repentis.     

Identification of quantitative trait loci for race-nonspecific resistance to tan spot in wheat

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is an economically important foliar disease in the major wheat growing areas throughout the world. Multiple races of the pathogen have been characterized based on their ability to cause necrosis and/or chlorosis on differential wheat lines. In this research, we evaluated a population of recombinant inbred lines derived from a cross between the common wheat varieties Grandin and BR34 for reaction to tan spot caused by Ptr races 1–3 and 5. Composite interval mapping revealed QTLs on the short arm of chromosome 1B and the long arm of chromosome 3B that were significantly associated with resistance to all four races. The effects of the two QTLs varied for the different races. The 1B QTL explained from 13% to 29% of the phenotypic variation, whereas the 3B QTL explained from 13% to 41% of the variation. Additional minor QTLs were detected but not associated with resistance to all races. The host-selective toxin Ptr ToxA, which is produced by races 1 and 2, was not a significant factor in the development of disease in this population. The race-nonspecific resistance derived from BR34 may take precedence over the gene-for-gene interaction known to be associated with the wheat–Ptr system.     

Resistance of winter wheat varieties to tan spot in the North Caucasus region of Russia

Tan spot caused by Pyrenophora tritici-repentis (Died.) Drechsler, in recent years, occupies an increasingly large area on the territory of Russia. Due to the wide distribution and economic significance of this disease, the search for resistant plants to the pathogen is relevant. This paper presents the results of a field assessment for 2017–2019 of 34 regionally distributed winter wheat varieties of Russian selection for resistance to P. tritici-repentis in the North Caucasus region of Russia. Field resistance - the development of the disease up to 30% against the background of artificial infection for three years was shown by 20.5% of the studied varieties. Wheat varieties were assessed for resistance to isolates of tan spot identified as races 1, 3, and 4 in the greenhouse at the seedling stage. The number of resistant accessions for each race was different and ranged from 12 to 20. The 12 varieties showed resistance to race 1, 14 varieties to race 3, 20 varieties to race 4. This research showed that the resistance to tan spot of studied varieties was race-specific. A functional allele of the susceptibility gene Tsn1 to P. tritici-repentis isolates, producing the toxin Ptr ToxA, was diagnosed by PCR method. Of the analyzed 34 varieties, 13 had a dominant allele of the Tsn1 (Tsn1⁺), and 21 had a recessive allele in the tsn1tsn1 homozygous state. All Tsn1⁺ varieties, and most varieties with recessive alleles tsn1tsn1, were susceptible to tan spot in the field. Varieties Dolya, Gurt, Lebed and Sila, which showed field resistance, had the tsn1tsn1 genotype. The expected reaction of varieties with different allelic composition of the Tsn1 gene to inoculation with the isolate of race 1, according to the generally accepted model of “gene-to-gene” interaction, did not coincide with that observed in reality, which confirms the results obtained by other authors. Research results demonstrate the effect of weather conditions on the susceptibility of wheat varieties to tan spot. In years with higher humidity and higher average air temperatures, the susceptibility response to the disease was observed in more varieties than in drier years. The studies show that the main part (79.5%) of winter wheat varieties of Russian selection widely zoned in the North Caucasus region of Russia are susceptible to P. tritici-repentis. Varieties that have been resistant to the pathogen in the adult phase in the field for three years and to the pathogen races in which the recessive allele of the tsn1 gene has been identified may be of interest as sources of resistance for developing new disease-resistant varieties.     

Genome-wide association mapping for five major pest resistances in wheat

Insect pests cause substantial damage to wheat production in many wheat-producing areas of the world. Amongst these, Hessian fly (HF), Russian wheat aphid (RWA), Sunn pest (SP), wheat stem saw fly (WSSF) and cereal leaf beetle (CLB) are the most damaging in the areas where they occur. Historically, the use of resistance genes in wheat has been the most effective, environmentally friendly, and cost-efficient approach to controlling pest infestations. In this study, we carried out a genome-wide association study with 2518 Diversity Arrays Technology markers which were polymorphic on 134 wheat genotypes with varying degrees of resistance to the five most destructive pests (HF, RWA, SP, WSSF and CLB) of wheat, using mixed linear model (MLM) analysis with population structure as a covariate. We identified 26 loci across the wheat genome linked to genes conferring resistance to these pests, of which 20 are potentially novel quantitative trait loci with significance values which ranged between 5 × 10^?3 and 10^?11. We used an in silico approach to identify probable candidate genes at some of the genomic regions and found that their functions varied from defense response with transferase activity to several genes of unknown function. Identification of potentially new loci associated with resistances to pests would contribute to more rapid marker-aided incorporation of new and diverse genes to develop new varieties with improved resistance against these pests.     

RNA-mediated gene silencing of ToxB in Pyrenophora tritici-repentis

The fungus Pyrenophora tritici-repentis causes tan spot, a wheat leaf disease of worldwide importance. The pathogen produces three host-selective toxins, including Ptr ToxB, which causes chlorophyll degradation and foliar chlorosis on toxin-sensitive wheat genotypes. The ToxB gene, which codes for Ptr ToxB, was silenced in a wild-type race 5 isolate of the fungus thorough a sense- and antisense-mediated silencing mechanism. Toxin production by the silenced strains was evaluated in culture filtrates of the fungus via Western blotting analysis, and plant bioassays were conducted to test the virulence of the transformants in planta. The chlorosis-inducing ability of the silenced strains was correlated with the quantity of Ptr ToxB, and transformants in which toxin production was strongly decreased also caused very little disease on toxin-sensitive wheat genotypes. Cytological analysis of the infection process revealed that, in addition to a reduced capacity to induce chlorosis, the silenced strains with the greatest decrease in the levels of Ptr ToxB produced significantly fewer appressoria than the wild-type isolate, 12 and 24 h after inoculation onto wheat leaves. The results provide strong support for the suggestion that the amount of Ptr ToxB protein produced by fungal isolates plays a significant role in the quantitative variation in the virulence of P. tritici-repentis.