Wheat Powdery Mildew Resistance Gene Research Articles

Pm57 from Aegilops searsii encodes a tandem kinase protein and confers wheat powdery mildew resistance

Powdery mildew is a devastating disease that affects wheat yield and quality. Wheat wild relatives represent valuable sources of disease resistance genes. Cloning and characterization of these genes will facilitate their incorporation into wheat breeding programs. Here, we report the cloning of Pm57, a wheat powdery mildew resistance gene from Aegilops searsii. It encodes a tandem kinase protein with putative kinase-pseudokinase domains followed by a von Willebrand factor A domain (WTK-vWA), being ortholog of Lr9 that mediates wheat leaf rust resistance. The resistance function of Pm57 is validated via independent mutants, gene silencing, and transgenic assays. Stable Pm57 transgenic wheat lines and introgression lines exhibit high levels of all-stage resistance to diverse isolates of the Bgt fungus, and no negative impacts on agronomic parameters are observed in our experimental set-up. Our findings highlight the emerging role of kinase fusion proteins in plant disease resistance and provide a valuable gene for wheat breeding.

Wheat powdery mildew resistance gene Pm13 encodes a mixed lineage kinase domain-like protein

Wheat powdery mildew is one of the most destructive diseases threatening global wheat production. The wild relatives of wheat constitute rich sources of diversity for powdery mildew resistance. Here, we report the map-based cloning of the powdery mildew resistance gene Pm13 from the wild wheat species Aegilops longissima. Pm13 encodes a mixed lineage kinase domain-like (MLKL) protein that contains an N-terminal-domain of MLKL (MLKL_NTD) domain in its N-terminus and a C-terminal serine/threonine kinase (STK) domain. The resistance function of Pm13 is validated by mutagenesis, gene silencing, transgenic assay, and allelic association analyses. The development of introgression lines with significantly reduced chromosome segments of Ae. longissima encompassing Pm13 enables widespread deployment of this gene into wheat cultivars. The cloning of Pm13 may provide valuable insights into the molecular mechanisms underlying Pm13-mediated powdery mildew resistance and highlight the important roles of kinase fusion proteins (KFPs) in wheat immunity.

Large-scale mutational analysis of wheat powdery mildew resistance gene Pm21.

Wheat powdery mildew is a devastating disease leading to severe yield loss. The powdery mildew resistance gene Pm21, encoding a nucleotide-binding leucine-rich repeat receptor (NLR) protein, confers broad-spectrum resistance to powdery mildew and has great potential for controlling this disease. In this study, a large-scale mutagenesis was conducted on wheat cultivar (cv.) Yangmai 18 carrying Pm21. As a result, a total of 113 independent mutant lines susceptible to powdery mildew were obtained, among which, only one lost the whole Pm21 locus and the other 112 harbored one- (107) or two-base (5) mutations in the encoding region of Pm21. From the 107 susceptible mutants containing one-base change, we found that 25 resulted in premature stop codons leading to truncated proteins and 82 led to amino acid changes involving in 59 functional sites. We determined the mutations per one hundred amino acids (MPHA) indexes of different domains, motifs, and non-domain and non-motif regions of PM21 protein and found that the loss-of-function mutations occurred in a tendentious means. We also observed a new mutation hotspot that was closely linked to RNBS-D motif of the NB-ARC domain and relatively conserved in different NLRs of wheat crops. In addition, we crossed all the susceptible mutants with Yangmai 18 carrying wild-type Pm21, subsequently phenotyped their F1 plants and revealed that the variant E44K in the coiled-coil (CC) domain could lead to dominant-negative effect. This study revealed key functional sites of PM21 and their distribution characteristics, which would contribute to understanding the relationship of resistance and structure of Pm21-encoded NLR.

Characterization of PmBN418, a wheat powdery mildew resistance gene on the rye 1RS chromosome arm

AbstractPowdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a disease of global importance, and new powdery mildew resistance genes that can be directly used in cultivar development are urgently needed. A recombinant inbred line population from ‘CI 17884’ × ‘Bainong 418’ was genotyped using single nucleotide polymorphism markers generated from genotyping‐by‐sequencing, and evaluated for responses to Great Plains Bgt isolates OKS(14)‐B‐3‐1 and NEI‐1‐3. Genetic mapping identified a powdery mildew resistance gene, designated PmBN418, on a 1BL.1RS translocation segment in Bainong 418. Given that PmBN418 conferred resistance to Bgt isolates virulent to Pm8 that resides on the chromosome arm 1RS translocated from rye (Secale cereale L.) cv. ‘Petkus’, PmBN418 is either a new gene or a new allele of Pm8. Phenotypic and genomic data analyses indicated segregation distortion of the powdery mildew resistance gene due to reduced transmission of the wheat (Triticum aestivum L.)–rye 1BL.1RS translocation segment from Bainong 418, while the wheat–Triticum speltoides translocation segment 7S#1 in CI 17884 was preferentially transmitted. Bainong 418, a high‐yielding cultivar with good resistance to several U.S. Bgt isolates, is a useful resistance source for wheat breeding, and the availability of the Kompetitive Allele Specific PCR (KASP) marker KASP‐1RS‐1 makes marker‐assisted introgression of PmBN418 into locally adapted cultivars feasible.

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

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 0.87cM and 1.01cM, 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.

Pm21 CC domain activity modulated by intramolecular interactions is implicated in cell death and disease resistance

Nucleotide‐binding (NB) leucine‐rich repeat (LRR) receptors (NLRs) provide resistance against several plant pathogens. We previously cloned the wheat powdery mildew resistance gene Pm21, which encodes a coiled‐coil (CC) NLR that confers broad‐spectrum resistance against Blumeria graminis f. sp. tritici. Here, we report comprehensive biochemical and functional analyses of Pm21 CC domain in Nicotiana benthamiana. Transient overexpression assay suggested that only the extended CC (eCC, amino acid residues 1–159) domain has cell‐death‐inducing activity, whereas the CC‐containing truncations, including CC‐NB and CC‐NB‐LRR, do not induce cell‐death responses. Coimmunoprecipitation (Co‐IP) assay showed that the eCC domain self‐associates and interacts with the NB and LRR domains in planta. These results imply that the activity of the eCC domain is inhibited by the intramolecular interactions of different domains in the absence of pathogens. We found that the LRR domain plays a crucial role in D491V‐mediated full‐length (FL) Pm21 autoactivation. Some mutations in the CC domain leading to the loss of Pm21 resistance to powdery mildew impaired the CC activity of cell‐death induction. Two mutations (R73Q and E80K) interfered with D491V‐mediated Pm21 autoactivation without affecting the cell‐death‐inducing activity of the eCC domain. Notably, some susceptible mutants harbouring mutations in the CC domain still exhibited cell‐death‐inducing activity. Taken together, these results implicate the CC domain of Pm21 in cell‐death signalling and disease‐resistance signalling, which are potentially independent of each other.

Fine mapping of wheat powdery mildew resistance gene Pm6 using 2B/2G homoeologous recombinants induced by the ph1b mutant

Using the ph1b mutant, the recombination frequency between the homoeologous region of 2B and 2G was significantly increased. By this, we narrowed Pm6 to a 0.9Mb physical region. The powdery mildew (Pm) resistance gene Pm6 from Triticum timopheevii (2n = 48, AAGG) was mapped to the long arm of chromosome 2G and introduced into common wheat in the form of 2B-2G introgressions. The introgression line IGV1-465 has the shortest 2G segment, which is estimated 37Mb in size when referring to 2BL genome reference of Chinese Spring (CS). The further fine mapping of Pm6 was impeded by the inhibition of allogeneic chromosome recombination between 2B and 2G in the Pm6 region. In the present study, to overcome 2B/2G recombination suppression, a ph1b-based strategy was employed to produce introgressions with reduced 2G fragments for the fine mapping of Pm6. IGV1-465 was crossed and backcrossed to the CSph1b mutant to produce plants with increased 2B/2G chromosome pairing frequency at the Pm6 region. A total of 182 allogeneic recombinants were obtained through two-round screening, i.e., first round of screening of 820 BC1F2:3 progenies using the flanking markers CIT02g-14/CIT02g-19 and second round of screening of 642 BC1F2:4 progenies using the flanking markers CIT02g-13/CIT02g-18, respectively. Through marker analysis using 30 chromosome 2G-specific markers located in the Pm6 region, the identified recombinants were divided into 14 haplotypes. Pm resistance evaluation of these haplotypes enabled us to narrow Pm6 to a 0.9Mb physical region of 2BL, flanked by markers CIT02g-20 and CIT02g-18. Six wheat varieties containing Pm6 were identified from a natural population, and they showed increased Pm resistance. This implied Pm6 is still effective, especially when used in combination with other Pm resistance genes.

Wheat powdery mildew resistance gene Pm64 derived from wild emmer (Triticum turgidum var. dicoccoides) is tightly linked in repulsion with stripe rust resistance gene Yr5

Stripe rust and powdery mildew are both devastating diseases for durum and common wheat. Pyramiding of genes conferring resistance to one or more diseases in a single cultivar is an important breeding approach to provide broader spectra of resistances in wheat improvement. A new powdery mildew resistance gene originating from wild emmer (Triticum turgidum var. dicoccoides) backcrossed into common wheat (T. aestivum) line WE35 was identified. It conferred an intermediate level of resistance to Blumeria graminis f. sp. tritici isolate E09 at the seedling stage and a high level of resistance at the adult plant stage. Genetic analysis showed that the powdery mildew resistance in WE35 was controlled by a dominant gene designated Pm64. Bulked segregant analysis (BSA) and molecular mapping indicated that Pm64 was located in chromosome bin 2BL4-0.50–0.89. Polymorphic markers were developed from the corresponding genomic regions of Chinese Spring wheat and wild emmer accession Zavitan to delimit Pm64 to a 0.55 cM genetic interval between markers WGGBH1364 and WGGBH612, corresponding to a 15 Mb genomic region on Chinese Spring and Zavitan 2BL, respectively. The genetic linkage map of Pm64 is critical for fine mapping and cloning. Pm64 was completely linked in repulsion with stripe rust resistance gene Yr5. Analysis of a larger segregating population might identify a recombinant line with both genes as a valuable resource in breeding for resistance to powdery mildew and stripe rust.

Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene Pm61.

The gene Pm61 that confers powdery mildew resistance has been previously identified on chromosome arm 4AL in Chinese wheat landrace Xuxusanyuehuang (XXSYH). To facilitate the use of Pm61 in breeding practices, the bulked segregant analysis-RNA-Seq (BSR-Seq) analysis, in combination with the information on the Chinese Spring reference genome sequence, was performed in the F2:3 mapping population of XXSYH × Zhongzuo 9504. Two single nucleotide polymorphism (SNP), two Kompetitive Allele Specific PCR (KASP), and six simple sequence repeat (SSR) markers, together with previously identified polymorphic markers, saturated the genetic linkage map for Pm61, especially in the proximal side of the target gene that was short of gene-linked markers. In the newly established genetic linkage map, Pm61 was located in a 0.71 cM genetic interval and can be detected in a high throughput scale by the KASP markers Xicsk8 and Xicsk13 or by the standard PCR-based markers Xicscx497 and Xicsx538. The newly saturated genetic linkage map will be useful in molecular marker assisted-selection of Pm61 in breeding for disease resistant cultivar and in its map-based cloning.

Fine mapping of the wheat powdery mildew resistance gene Pm52 using comparative genomics analysis and the Chinese Spring reference genomic sequence.

A high-resolution genetic linkage map was constructed using the comparative genomics analysis approach and the wheat reference genome, which placed wheat powdery mildew resistance gene Pm52 in a 0.21-cM genetic interval on chromosome arm 2BL. The gene Pm52 confers resistance to powdery mildew and has been previously mapped on chromosome arm 2BL in winter wheat cultivar Liangxing 99. Because of its effectiveness against the disease, this study was initiated to finely map Pm52 using the comparative genomics analysis approach and the wheat reference genomic sequence. Based on the EST sequences that were located in the chromosome region flanking Pm52, four EST-SSR markers were developed, and another nine SSR markers were developed using the comparative genomics technology. These thirteen markers were integrated into a genetic linkage map using an F2:3 subpopulation of the Liangxing 99 × Zhongzuo 9504 cross. Pm52 was mapped within a 3.2-cM genetic interval in the subpopulation that corresponded to a ~40-Mb genomic interval on chromosome arm 2BL of the Chinese Spring reference genome. The Pm52-flanking markers Xicsl163 and Xicsl62 identified 344 recombinant individuals from 8820 F2 plants. Nine SSR markers generated from the Chinese Spring genomic interval were incorporated into a high-resolution genetic linkage map, which placed Pm52 in a 0.21-cM genetic interval corresponding to 5.6-Mb genomic region. The constructed high-resolution genetic linkage map will facilitate the map-based cloning of Pm52 and its marker-assisted selection.

Fine mapping of powdery mildew resistance gene Pm4e in bread wheat (Triticum aestivum L.).

Fine mapping of wheat powdery mildew-resistance gene Pm4e to a 0.19cM interval with sequence-based markers provides the foundation for map-based cloning and marker-assisted selection with breeder-friendly markers. Powdery mildew caused by Blumeria graminis f. sp. tritici is a wheat foliar disease that poses a serious threat to global wheat production. Pm4 is a resistance gene locus that has played a key role in controlling this disease in wheat production and a few resistance alleles of this locus have been identified. We have previously mapped the Pm4e allele to a 6.7cM interval on chromosome 2AL. In this study, Pm4e was delimited to a 0.19cM interval flanked by Xwgrc763 and Xwgrc865, through employment of a larger segregating population, derived from the cross of resistant parent D29 with susceptible parent Yangmai 158 (Y158), and enrichment of the genetic interval with markers developed on Chinese Spring (C.S.) survey sequence. In this interval, Pm4e co-segregated with a few markers, some of which were either D29-dominant or Y158-dominant, implying great sequence variation in the interval between D29 and Y158. Most of these co-segregation markers could not differentiate the Pm4 alleles from each other. Survey of 55 wheat cultivars with four co-dominant markers showed that the Pm4e-co-segregating loci always co-exist. Annotation of the Pm4e interval-corresponding C.S. sequence revealed more than a dozen resistance gene analogs clustered in a 2.4Mb region, although C.S. is susceptible to the Pm4e-avirulent isolate Bgt2. This study has established the foundation for map-based cloning of Pm4e. Moreover, some of the co-dominant markers developed in this study could help in marker-assisted transfer of Pm4e into elite cultivars.

Molecular mapping of a novel wheat powdery mildew resistance gene Ml92145E8-9 and its application in wheat breeding by marker-assisted selection

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most devastating diseases of common wheat (Triticum aestivum L.). The wheat line 92145E8-9 is immune to Bgt isolate E09. Genetic analysis reveals that the powdery mildew resistance in 92145E8-9 is controlled by a single dominant gene, temporarily designated Ml92145E8-9. Bulked-segregant analysis (BSA) with simple sequence repeat (SSR) markers indicates that Ml92145E8-9 is located on chromosome 2AL. According to the reactions of 92145E8-9, VPM1 (Pm4b carrier), and Lankao 906 (PmLK906 carrier) to 14 Bgt isolates, the resistance spectrum of 92145E8-9 differs from those of Pm4b and PmLK906, both of which were previously localized to 2AL. To test the allelism among Ml92145E8-9, Pm4b and PmLK906, two F2 populations of 92145E8-9 × VPM1 (Pm4b) and 92145E8-9 × Lankao 906 (PmLK906) were developed in this study. Screening of 784 F2 progeny of 92145E8-9 × VPM1 and 973 F2 progeny of 92145E8-9 × Lankao 906 for Bgt isolate E09 identified 37 and 19 susceptible plants, respectively. These findings indicated that Ml92145E8-9 is non-allelic to either Pm4b or PmLK906. Thus, Ml92145E8-9 is likely to be a new powdery mildew resistance gene on 2AL. New polymorphic markers were developed based on the collinearity of genomic regions of Ml92145E8-9 with the reference sequences of the International Wheat Genome Sequencing Consortium (IWGSC). Ml92145E8-9 was mapped to a 3.6 cM interval flanked by molecular markers Xsdauk13 and Xsdauk682. This study also developed five powdery mildew-resistant wheat lines (SDAU3561, SDAU3562, SDAU4173, SDAU4174, and SDAU4175) using flanking marker-aided selection. The markers closely linked to Ml92145E8-9 would be useful in marker-assisted selection for wheat powdery mildew resistance breeding.

The NB-LRR gene Pm60 confers powdery mildew resistance in wheat.

Powdery mildew is one of the most devastating diseases of wheat. To date, few powdery mildew resistance genes have been cloned from wheat due to the size and complexity of the wheat genome. Triticum urartu is the progenitor of the A genome of wheat and is an important source for powdery mildew resistance genes. Using molecular markers designed from scaffolds of the sequenced T.urartu accession and standard map-based cloning, a powdery mildew resistance locus was mapped to a 356-kb region, which contains two nucleotide-binding and leucine-rich repeat domain (NB-LRR) protein-encoding genes. Virus-induced gene silencing, single-cell transient expression, and stable transformation assays demonstrated that one of these two genes, designated Pm60, confers resistance to powdery mildew. Overexpression of full-length Pm60 and two allelic variants in Nicotiana benthamiana leaves induced hypersensitive cell death response, but expression of the coiled-coil domain alone was insufficient to induce hypersensitive response. Yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation imaging assays showed that Pm60 protein interacts with its neighboring NB-containing protein, suggesting that they might be functionally related. The identification and cloning of this novel wheat powdery mildew resistance gene will facilitate breeding for disease resistance in wheat.

Development of Markers Closely Linked with Wheat Powdery Mildew Re-sistance Gene Pm48

Development of Markers Closely Linked with Wheat Powdery Mildew Re-sistance Gene <i>Pm48</i>

Collinearity Analysis and High-Density Genetic Mapping of the Wheat Powdery Mildew Resistance Gene Pm40 in PI 672538.

The wheat powdery mildew resistance gene Pm40, which is located on chromosomal arm 7BS, is effective against nearly all prevalent races of Blumeria graminis f. sp tritici (Bgt) in China and is carried by the common wheat germplasm PI 672538. A set of the F1, F2 and F2:3 populations from the cross of the resistant PI 672538 with the susceptible line L1034 were used to conduct genetic analysis of powdery mildew resistance and construct a high-density linkage map of the Pm40 gene. We constructed a high-density linkage genetic map with a total length of 6.18 cM and average spacing between markers of 0.48 cM.Pm40 is flanked by Xwmc335 and BF291338 at genetic distances of 0.58 cM and 0.26 cM, respectively, in deletion bin C-7BS-1-0.27. Comparative genomic analysis based on EST-STS markers established a high level of collinearity of the Pm40 genomic region with a 1.09-Mbp genomic region on Brachypodium chromosome 3, a 1.16-Mbp genomic region on rice chromosome 8, and a 1.62-Mbp genomic region on sorghum chromosome 7. We further anchored the Pm40 target intervals to the wheat genome sequence. A putative linear index of 85 wheat contigs containing 97 genes on 7BS was constructed. In total, 9 genes could be considered as candidates for the resistances to powdery mildew in the target genomic regions, which encoded proteins that were involved in the plant defense and response to pathogen attack. These results will facilitate the development of new markers for map-based cloning and marker-assisted selection of Pm40 in wheat breeding programs.

Haynaldia villosa NAM-V1 is linked with the powdery mildew resistance gene Pm21 and contributes to increasing grain protein content in wheat.

BackgroundThe 6AL/6VS translocation lines, carrying the wheat powdery mildew resistance gene Pm21, are planted on more than 3.4 million hectares. The NAM-A1 gene, located on chromosome 6AS of hexaploid wheat, has been implicated with increased wheat grain protein content (GPC). However, the NAM-A1 gene was removed from the 6AL/6VS translocation lines after the original chromosome 6AS was replaced by chromosome 6VS of Haynaldia villosa. The present study aimed to clone the NAM homologous gene from chromosome 6VS, to analyze the changes of GPC in the 6AL/6VS translocation lines, and to develop related molecular markers for wheat molecular breeding.ResultsA new NAM family gene, NAM-V1, was cloned from 6VS of H. villosa (GenBank ACC. no. KR873101). NAM-V1 contained an intact open reading frame (ORF) and putatively encodes a protein of 407 amino acids. Phylogenetic analysis indicated that NAM-V1 was an orthologous gene of NAM-A1, B1, and D1. The determination of GPC in four Pm21 F2 segregation populations demonstrated that the replacement of NAM-A1 by NAM-V1 confers increased GPC in hexaploid wheat. Multiple sequence alignment of NAM-A1, B1, B2, D1, D2, and V1 showed the single nucleotide polymorphism (SNP) sites for each of the NAM genes, allowing us to develop a molecular marker, CauNAM-V1, for the specific detection of NAM-V1 gene. Our results indicate that CauNAM-V1 can be used as a novel DNA marker for NAM-V1, and can also be used for selecting Pm21 in wheat breeding programs. Further, we developed a marker, CauNAM-ABD, for the amplification and simultaneously distinguish among the NAM-A1, NAM-B1, NAM-B2, NAM-D1, and NAM-D2 genes in a single step. CauNAM-ABD enabled us to develop an efficient “one-marker-for-five-genes” procedure for identifying genes and its copy numbers related with grain protein content.ConclusionHere, we report the isolation of the NAM-V1 gene of H. villosa. This gene contributes to increasing GPC in 6AL/6VS translocation wheat lines. We developed a molecular marker for the specific detection of NAM-V1 and a molecular marker that can be used to simultaneously distinguished among the NAM-A1, NAM-B1, NAM-B2, NAM-D1, and NAM-D2 genes in a single step.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0391-4) contains supplementary material, which is available to authorized users.

Dynamic evolution of resistance gene analogs in the orthologous genomic regions of powdery mildew resistance gene MlIW170 in Triticum dicoccoides and Aegilops tauschii

Rapid evolution of powdery mildew resistance gene MlIW170 orthologous genomic regions in wheat subgenomes. Wheat is one of the most important staple grain crops in the world and also an excellent model for plant ploidy evolution research with different ploidy levels from diploid to hexaploid. Powdery mildew disease caused by Blumeria graminis f.sp. tritici can result in significant loss in both grain yield and quality in wheat. In this study, the wheat powdery mildew resistance gene MlIW170 locus located at the Triticum dicoccoides chromosome 2B short arm was further characterized by constructing and sequencing a BAC-based physical map contig covering a 0.3cM genetic distance region (880kb) and developing additional markers to delineate the resistance gene within a 0.16cM genetic interval (372kb). Comparative analyses of the T. dicoccoides 2BS region with the orthologous Aegilops tauschii 2DS region showed great gene colinearity, including the structure organization of both types of RGA1/2-like and RPS2-like resistance genes. Comparative analyses with the orthologous regions from Brachypodium and rice genomes revealed considerable dynamic evolutionary changes that have re-shaped this MlIW170 region in the wheat genome, resulting in a high number of non-syntenic genes including resistance-related genes. This result might reflect the rapid evolution in R-gene regions. Phylogenetic analysis on these resistance-related gene sequences indicated the duplication of these genes in the MlIW170 region, occurred before the separation of the wheat B and D genomes.

Substitutions of Two Amino Acids in the Nucleotide-Binding Site Domain of a Resistance Protein Enhance the Hypersensitive Response and Enlarge the PM3F Resistance Spectrum in Wheat

Proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains are major components of the plant immune system. They usually mediate resistance against a subgroup of races of a specific pathogen. For the allelic series of the wheat powdery mildew resistance gene Pm3, alleles with a broad and a narrow resistance spectrum have been described. Here, we show that a broad Pm3 spectrum range correlates with a fast and intense hypersensitive response (HR) in a Nicotiana transient-expression system and this activity can be attributed to two particular amino acids in the ARC2 subdomain of the NBS. The combined substitution of these amino acids in narrow-spectrum PM3 proteins enhances their capacity to induce an HR in Nicotiana benthamiana, and we demonstrate that these substitutions also enlarge the resistance spectrum of the Pm3f allele in wheat. Finally, using Bph14, we show that the region carrying the relevant amino acids also plays a role in the HR regulation of another coiled-coil NBS-LRR resistance protein. These results highlight the importance of an optimized NBS-'molecular switch' for the conversion of initial pathogen perception by the LRR into resistance-protein activation, and we describe a possible approach to extend the effectiveness of resistance genes via minimal targeted modifications in the NBS domain.

MlNCD1: A Novel Aegilops tauschii‐Derived Powdery Mildew Resistance Gene Identified in Common Wheat

ABSTRACTPowdery mildew is a major fungal disease in wheat, especially in cool maritime climates. A novel Aegilops tauschii Coss.‐derived wheat powdery mildew resistance gene present in the germplasm line NC96BGTD1 was genetically characterized as a monogenic trait in field trials using F2– and F4–derived lines from a NC96BGTD1 × ‘Saluda’ cross. Simple sequence repeat (SSR) markers were used to map and tag the resistance gene present in NC96BGTD1. Two dominant SSR markers flanking the resistance gene were identified. Xgwm635 mapped 5.5 and 8.3 cM distal to the resistance gene in the F2 and F4 generations, respectively. Xgpw328 mapped 16.2 cM proximal to the resistance gene in the F2 and 13.6 cM proximal to the resistance gene in the F4 generation. These SSR markers were previously mapped to the short arm of chromosome 7D and their positions were confirmed using Chinese Spring aneuploid and deletion stocks. Significant segregation distortion was observed in the F4 generation with markers distal to the resistance gene showing a preference for the A. tauschii alleles while markers proximal to the resistance gene showed less significant preference for Saluda alleles. Only the adult plant resistance gene Pm38 has been mapped to the short arm of chromosome 7D but to a more proximal position than the gene in NC96BGTD1. The resistance gene described herein should be temporarily designated MlNCD1.

Molecular Tagging Wheat Powdery Mildew Resistance Gene Pm21 by EST-SSR and STS Markers

In order to enrich the molecular markers linked to wheat powdery mildew resistance gene Pm21 and facilitate the molecular marker-assisted selection in multi-background breeding procedure, preferred small groups (PSG) analysis was conducted using F 2 segregating population cross between the wheat germplasm line CB033 with Pm21 and a common wheat variety Huixianhong (susceptible to wheat powdery mildew). One hundred and two markers revealed polymorphism between the parents when screening with 258 SSR or STS markers located on wheat homoeologous group VI, and two EST-SSR markers ( Xcfe164 and Xedm129 ) and one STS marker ( Xcinau188 ) showed good consistency between the genotype and the phenotype to mildew in PSG. Linkage relationship between Pm21 and molecular markers of Xcfe164 , Xedm129 and Xcinau188 were then established, with the distance 3.75 cM, 1.26 cM and 0.98 cM respectively. Authenticity verification of the linkage between Pm21 and molecular markers were tested via F 2: 3 families and results showed that the three markers were reliable new markers linked to Pm21 . The results above would facilitate molecular marker-assisted selection in breeding program.