High-Resolution Melting-Based Marker Development for Wheat Leaf Rust Resistance Gene Lr34.

Deploying adult plant resistance (APR) against rust diseases is an important breeding objective of most wheat-breeding programs. The gene Lr34 is an effective and widely deployed broad-spectrum APR gene in wheat against leaf rust fungus Puccinia triticina. Various molecular markers have been developed for Lr34, but they either require post-PCR handling processes or are not economical. Herein, we developed a high-resolution melting (HRM)-based diagnostic assay for Lr34 based on a 3-bp 'TTC' deletion in exon 11 of the resistant allele. The susceptible cultivar Thatcher (Tc) and the near-isogenic Thatcher line (RL6058) with Lr34 yielded distinct melting profiles and were differentiated with high reproducibility. For further validation, all three copies of Lr34 were cloned in plasmid vectors, and HRM analysis using individual and combination (equimolar mixture of three copies) homoeologs yielded distinct melting profiles. An additional layer of genotyping was provided by a LunaProbe assay. The allele-specific probes successfully distinguished the homoeologs but not Tc and RL6058. Furthermore, the practical deployment of the HRM assay was tested by running the marker on a set of breeding lines. When compared with a kompetitive allele-specific PCR (KASP) Lr34 assay, the HRM assay had similar genotyping results and was able to accurately differentiate the resistant and susceptible breeding lines. However, our HRM assay was unable to detect the heterozygote. To our knowledge, this is the first report of an HRM assay for genotyping a wheat rust resistance gene.

Identification and characterization of the novel leaf rust resistance gene Lr81 in wheat.

KEY MESSAGE: The novel, leaf rust seedling resistance gene, Lr81, was identified in a Croatian breeding line and mapped to a genomic region of less than 100 Kb on chromosome 2AS. Leaf rust, caused by Puccinia triticina, is the most common and widespread rust disease in wheat. Races of Puccinia triticina evolve rapidly in the southern Great Plains of the USA, and leaf rust resistance genes often lose effectiveness shortly after deployment in wheat production. PI 470121, a wheat breeding line developed by the University of Zagreb in Croatia, showed high resistance to Puccinia triticina races collected from Oklahoma, suggesting that PI 470121 could be a leaf rust resistance source for the southern Great Plains of the USA. Genetic analysis based on an F2 population and F2:3 families derived from the cross PI 470121 × Stardust indicated that PI 470121 carries a dominant seedling resistance gene, designated as Lr81. Linkage mapping delimited Lr81 to a genomic region of 96,148 bp flanked by newly developed KASP markers Xstars-KASP320 and Xstars-KASP323 on the short arm of chromosome 2A, spanning 67,030,206-67,132,354 bp in the Chinese Spring reference assembly (IWGSC RefSeq v1.0). Deletion bin mapping assigned Lr81 to the terminal bin 2AS-0.78-1.00. Allelism tests indicated that Lr81 is a distinctive leaf rust resistance locus with the physical order Lr65-Lr17-Lr81. Marker-assisted selection based on a set of markers closely linked to leaf rust resistance genes in PI 470121 and Stardust enabled identification of a recombinant inbred line RIL92 carrying Lr81 only. Lr81 is a valuable leaf rust resistance source that can be rapidly introgressed into locally adapted cultivars using KASP markers Xstars-KASP320 and Xstars-KASP323.

Virulence Phenotypes of the Wheat Leaf Rust Pathogen, Puccinia triticina, in the United States from 2018 to 2020.

Collections of wheat leaves infected with the leaf rust fungus, Puccinia triticina, were obtained from the southeastern states, the Ohio Valley, the Great Plains, and Washington in 2018, 2019, and 2020 to determine the prevalent virulence phenotypes in the wheat-growing regions of the United States. In the hard red winter wheat region of the southern and mid Great Plains, MNPSD and MPPSD were the two most common phenotypes in 2018 and 2019. In 2020, BBBQD with high virulence to durum wheat was the most common phenotype in the southern Great Plains. In the hard red spring wheat region of the northern Great Plains, MNPSD, MPPSD, MBDSD, and TBBGS were the predominant phenotypes. In the soft red winter wheat region of the southeastern states and Ohio Valley region, MBTNB, MCTNB, and MNPSD were the three most common phenotypes. Collections in Washington had phenotypes LBDSG, LCDSG, LCDJG, and MBDSB that were not found in any other region. Isolates with virulence to the leaf rust resistance (Lr) gene Lr11 were most frequent in the southeastern states and Ohio Valley regions. The frequency of isolates with virulence to the Lr39 gene was highest in the Great Plains region and frequency of isolates with virulence to the Lr21 gene was highest in the northern Great Plains region. Selection of virulence phenotypes by Lr genes in the different market classes of wheat, combined with the effects of clonal reproduction, overwintering in southern regions, and low migration between the Great Plains region and eastern wheat-producing regions, has maintained the different P. triticina populations in the United States.

Genotyping-by-Sequencing for the Study of Genetic Diversity in Puccinia triticina.

Leaf rust, caused by Puccinia triticina Erikss., is globally the most widespread rust of wheat. Populations of P. triticina are highly diverse for virulence, with many different races found annually. The genetic diversity of P. triticina populations has been previously assessed using different types of DNA markers. Genotyping technologies that provide a higher density of markers distributed across the genome will be more powerful for analysis of genetic and phylogenetic relationships in P. triticina populations. In this study, we utilized restriction-associated DNA (RAD) genotyping-by-sequencing (GBS) adapted for the Ion Torrent sequencing platform for the study of population diversity in P. triticina. A collection of 102 isolates, collected mainly from tetraploid and hexaploid wheat, was used. The virulence phenotypes of the isolates were determined on 20 lines of Thatcher wheat near isogenic for leaf rust resistance genes. Seven races were found among 57 isolates collected from tetraploid wheat, and 21 races were observed among 40 hexaploid wheat type isolates. This is the first study to report durum wheat virulent races to Lr3bg in Tunisia, Lr14a in Morocco, and Lr3bg and Lr28 in Mexico. Ethiopian isolates with high virulence to durum wheat but avirulent on Thatcher (hexaploid wheat) were tested for virulence on a set of durum (tetraploid) differentials. A subset of 30 isolates representing most of the virulence phenotypes in the 102 isolates were genotyped using RAD-GBS. Phylogenetic analysis of 30 isolates using 2,125 single nucleotide polymorphism (SNP) markers showed nine distinct clusters. There was a general correlation between virulence phenotypes and SNP genotypes. The high bootstrap values between clusters of isolates in the phylogenetic tree indicated that RAD-GBS can be used as a new genotyping tool that is fast, simple, high throughput, cost effective, and provides a sufficient number of markers for the study of genetic diversity in P. triticina.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Identifying Loci Conferring Resistance to Leaf and Stripe Rusts in a Spring Wheat Population (Triticum aestivum) via Genome-Wide Association Mapping.

A previous genome-wide association study (GWAS) for leaf rust (caused by Puccinia triticina) resistance identified 46 resistance quantitative trait loci (QTL) in an elite spring wheat leaf rust resistance diversity panel. With the aim of characterizing the pleiotropic resistance sources to both leaf rust and stripe rust (caused by P. striiformis f. sp. tritici), stripe rust responses were tested in five U.S. environments at the adult-plant stage and to five U.S. races at the seedling stage. The data revealed balanced phenotypic distributions in this population except for the seedling response to P. striiformis f. sp. tritici race PSTv-37. GWAS for stripe rust resistance discovered a total of 21 QTL significantly associated with all-stage or field resistance on chromosomes 1B, 1D, 2B, 3B, 4A, 5A, 5B, 5D, 6A, 6B, 7A, and 7B. Previously documented pleiotropic resistance genes Yr18/Lr34 and Yr46/Lr67 and tightly linked genes Yr17-Lr37 and Yr30-Sr2-Lr27 were also detected in this population. In addition, stripe rust resistance QTL Yrswp-2B.1, Yrswp-3B, and Yrswp-7B colocated with leaf rust resistance loci 2B_3, 3B_t2, and 7B_4, respectively. Haplotype analysis uncovered that Yrswp-3B and 3B_t2 were either tightly linked genes or the same gene for resistance to both stripe and leaf rusts. Single nucleotide polymorphism markers IWB35950, IWB74350, and IWB72134 for the 3B QTL conferring resistance to both rusts should be useful in incorporating the resistance allele(s) in new cultivars.

Mapping and characterization of the new adult plant leaf rust resistance gene Lr77 derived from Santa Fe winter wheat.

KEY MESSAGE: A new gene for adult plant leaf rust resistance in wheat was mapped to chromosome 3BL. This gene was designated as Lr77. 'Santa Fe' is a hard red winter cultivar that has had long-lasting resistance to the leaf rust fungus, Puccinia triticina. The objective of this study was to determine the chromosome location of the adult plant leaf rust resistance in Santa Fe wheat. A partial backcross line of 'Thatcher' (Tc) wheat with adult plant leaf rust resistance derived from Santa Fe was crossed with Thatcher to develop a Thatcher//Tc*2/Santa Fe F6 recombinant inbred line (RIL) population. The RIL population and parental lines were evaluated for segregation of leaf rust resistance in three field plot tests and in an adult plant greenhouse test. A genetic map of the RIL population was constructed using 90,000 single-nucleotide polymorphism (SNP) markers with the Illumina Infinium iSelect 90K wheat bead array. A significant quantitative trait locus for reduction of leaf rust severity in all four tests was found on chromosome 3BL that segregated as a single adult plant resistance gene. The RILs with the allele from the resistant parent for SNP marker IWB10344 had lower leaf rust severity and a moderately resistant to moderately susceptible response compared to the susceptible RILs and Thatcher. The gene derived from Santa Fe on chromosome 3BL was designated as Lr77. Kompetitive allele-specific polymerase chain reaction assay markers linked to Lr77 on 3BL should be useful for selection of wheat germplasm with this gene.

Association mapping of leaf rust resistance loci in a spring wheat core collection.

KEY MESSAGE: We identified 15 potentially novel loci in addition to previously characterized leaf rust resistance genes from 1032 spring wheat accessions. Targeted AM subset panels were instrumental in revealing interesting loci. Leaf rust is a common disease of wheat, consistently reducing yields in many wheat-growing regions of the world. Although fungicides are commonly applied to wheat in the United States (US), genetic resistance can provide less expensive, yet effective control of the disease. Our objectives were to map leaf rust resistance genes in a large core collection of spring wheat accessions selected from the United States Department of Agriculture-Agricultural Research Service National Small Grains Collection (NSGC), determine whether previously characterized race-nonspecific resistance genes could be identified with our panel, and evaluate the use of targeted panels to identify seedling and adult plant resistance (APR) genes. Association mapping (AM) detected five potentially novel leaf rust resistance loci on chromosomes 2BL, 4AS, and 5DL at the seedling stage, and 2DL and 7AS that conditioned both seedling and adult plant resistance. In addition, ten potentially novel race-nonspecific resistance loci conditioned field resistance and lacked seedling resistance. Analyses of targeted subsets of the accessions identified additional loci not associated with resistance in the complete core panel. Using molecular markers, we also confirmed the presence and effectiveness of the race-nonspecific genes Lr34, Lr46, and Lr67 in our panel. Although most of the accessions in this study were susceptible to leaf rust in field and seedling tests, many resistance loci were identified with AM. Through the use of targeted subset panels, more loci were identified than in the larger core panels alone.

Genetic and Molecular Characterization of Leaf Rust Resistance in Two Durum Wheat Landraces.

Leaf rust, caused by Puccinia triticina, is a constraint to durum wheat (Triticum turgidum subsp. durum) production, and landraces are reported to be an important source of resistance. Two Portuguese landraces (Aus26582 and Aus26579) showed resistance against durum-specific P. triticina races and were crossed with a susceptible landrace (Bansi) to develop recombinant inbred line (RIL) populations. Monogenic segregation for leaf rust resistance was observed among both RIL populations. The underlying locus, temporarily named LrAW2, was mapped to the short arm of chromosome 6B in the Aus26582/Bansi population and five DArTseq markers cosegregated with LrAW2. Simple sequence repeat markers sun683 and sun684, developed from the chromosome survey sequence (CSS) contig 6BS_2963854, identified through BlastN search of cosegregating DArTseq markers in the International Wheat Genome Sequencing Consortium database, cosegregated with LrAW2. Comparison of the CSS contig 6BS_2963854-based sequences amplified from parental genotypes led to the development of marker sunKASP_60, which also showed close linkage with LrAW2. Markers sun684 and sunKASP_60 showed close association with LrAW2 in both RIL populations. The amplification of LrAW2-specific products by linked markers in Aus26582, Aus26579, and Guayacan (Lr61) indicated that LrAW2 may be Lr61. The alternate amplicon or haplotype produced with LrAW2-linked markers in Australian durum cultivars demonstrated their effectiveness in marker-assisted selection.

Inheritance and Bulked Segregant Analysis of Leaf Rust and Stem Rust Resistance in Durum Wheat Genotypes.

Leaf rust, caused by Puccinia triticina, and stem rust, caused by P. graminis f. sp. tritici, are important diseases of durum wheat. This study determined the inheritance and genomic locations of leaf rust resistance (Lr) genes to P. triticina race BBBQJ and stem rust resistance (Sr) genes to P. graminis f. sp. tritici race TTKSK in durum accessions. Eight leaf-rust-resistant genotypes were used to develop biparental populations. Accessions PI 192051 and PI 534304 were also resistant to P. graminis f. sp. tritici race TTKSK. The resulting progenies were phenotyped for leaf rust and stem rust response at seedling stage. The Lr and Sr genes were mapped in five populations using single-nucleotide polymorphisms and bulked segregant analysis. Five leaf-rust-resistant genotypes carried single dominant Lr genes whereas, in the remaining accessions, there was deviation from the expected segregation ratio of a single dominant Lr gene. Seven genotypes carried Lr genes different from those previously characterized in durum. The single dominant Lr genes in PI 209274, PI 244061, PI387263, and PI 313096 were mapped to chromosome arms 6BS, 2BS, 6BL, and 6BS, respectively. The Sr gene in PI 534304 mapped to 6AL and is most likely Sr13, while the Sr gene in PI 192051 could be uncharacterized in durum.

Molecular mapping and improvement of leaf rust resistance in wheat breeding lines.

Leaf rust, caused by Puccinia triticina, is the most common and widespread disease of wheat (Triticum aestivum) worldwide. Deployment of host-plant resistance is one of the strategies to reduce losses due to leaf rust disease. The objective of this study was to map genes for adult-plant resistance to leaf rust in a recombinant inbred line (RIL) population originating from MN98550-5/MN99394-1. The mapping population of 139 RILs and five checks were evaluated in 2005, 2009, and 2010 in five environments. Natural infection occurred in the 2005 trials and trials in 2009 and 2010 were inoculated with leaf rust. Four quantitative trait loci (QTL) on chromosomes 2BS, 2DS, 7AL, and 7DS were detected. The QTL on 2BS explained up to 33.6% of the phenotypic variation in leaf rust response, whereas the QTL on 2DS, 7AL, and 7DS explained up to 15.7, 8.1, and 34.2%, respectively. Seedling infection type tests conducted with P. triticina races BBBD and SBDG confirmed that the QTL on 2BS and 2DS were Lr16 and Lr2a, respectively, and these genes were expressed in the seedling and field plot tests. The Lr2a gene mapped at the same location as Sr6. The QTL on 7DS was Lr34. The QTL on 7AL is a new QTL for leaf rust resistance. The joint effects of all four QTL explained 74% of the total phenotypic variation in leaf rust severity. Analysis of different combinations of QTL showed that the RILs containing all four or three of the QTL had the lowest average leaf rust severity in all five environments. Deployment of these QTL in combination or with other effective genes will lead to successful control of leaf rust.

Development of COS-SNP and HRM markers for high-throughput and reliable haplotype-based detection of Lr14a in durum wheat (Triticum durum Desf.).

Leaf rust (Puccinia triticina Eriks. & Henn.) is a major disease affecting durum wheat production. The Lr14a-resistant gene present in the durum wheat cv. Creso and its derivative cv. Colosseo is one of the best characterized leaf-rust resistance sources deployed in durum wheat breeding. Lr14a has been mapped close to the simple sequence repeat markers gwm146, gwm344 and wmc10 in the distal portion of the chromosome arm 7BL, a gene-dense region. The objectives of this study were: (1) to enrich the Lr14a region with single nucleotide polymorphisms (SNPs) and high-resolution melting (HRM)-based markers developed from conserved ortholog set (COS) genes and from sequenced Diversity Array Technology (DArT(®)) markers; (2) to further investigate the gene content and colinearity of this region with the Brachypodium and rice genomes. Ten new COS-SNP and five HRM markers were mapped within an 8.0 cM interval spanning Lr14a. Two HRM markers pinpointed the locus in an interval of <1.0 cM and eight COS-SNPs were mapped 2.1-4.1 cM distal to Lr14a. Each marker was tested for its capacity to predict the state of Lr14a alleles (in particular, Lr14-Creso associated to resistance) in a panel of durum wheat elite germplasm including 164 accessions. Two of the most informative markers were converted into KASPar(®) markers. Single assay markers ubw14 and wPt-4038-HRM designed for agarose gel electrophoresis/KASPar(®) assays and high-resolution melting analysis, respectively, as well as the double-marker combinations ubw14/ubw18, ubw14/ubw35 and wPt-4038-HRM-ubw35 will be useful for germplasm haplotyping and for molecular-assisted breeding.

Molecular Phylogenetic Relationships of the Brown Leaf Rust Fungi on Wheat, Rye, and Other Grasses.

The classification of brown leaf rust fungi (Puccinia recondita complex and allied species) on wheat (Triticum aestivum), rye (Secale cereale), and other grasses in the family Poaceae has experienced a long history of controversy and uncertainty due to the reduced morphological characteristics available for taxonomy and difficulty of conducting interfertility experiments. However, because these are pathogens on important crops, it is important to clarify the species delimitations reflecting the natural lineages. In this study, phylogenetic analyses were conducted with DNA sequence data from the ribosomal DNA internal transcribed spacer region and elongation factor 1-α to elucidate this species complex. Three phylogenetic lineages were recovered within the complex of rye leaf rust fungi, P. recondita sensu stricto, which is congruent with existing classifications based on DNA content, sexual compatibility, and morphological studies. The brown leaf rust fungus on wheat (P. triticina) grouped with the related species P. persistens on Elymus repens and E. intermedia as a strongly supported clade. Collections on other Elymus spp. were separated into six clades. Based on the phylogenetic affinities of nine type specimens and aecial host associations, potential taxonomic names were evaluated for selected lineages.

Identification of leaf rust resistance loci in a geographically diverse panel of wheat using genome-wide association analysis.

Leaf rust, caused by Puccinia triticina (Pt) is among the most devastating diseases posing a significant threat to global wheat production. The continuously evolving virulent Pt races in North America calls for exploring new sources of leaf rust resistance. A diversity panel of 365 bread wheat accessions selected from a worldwide population of landraces and cultivars was evaluated at the seedling stage against four Pt races (TDBJQ, TBBGS, MNPSD and, TNBJS). A wide distribution of seedling responses against the four Pt races was observed. Majority of the genotypes displayed a susceptible response with only 28 (9.8%), 59 (13.5%), 45 (12.5%), and 29 (8.1%) wheat accessions exhibiting a highly resistant response to TDBJQ, TBBGS, MNPSD and, TNBJS, respectively. Further, we conducted a high-resolution multi-locus genome-wide association study (GWAS) using a set of 302,524 high-quality single nucleotide polymorphisms (SNPs). The GWAS analysis identified 27 marker-trait associations (MTAs) for leaf rust resistance on different wheat chromosomes of which 20 MTAs were found in the vicinity of known Lr genes, MTAs, or quantitative traits loci (QTLs) identified in previous studies. The remaining seven significant MTAs identified represent genomic regions that harbor potentially novel genes for leaf rust resistance. Furthermore, the candidate gene analysis for the significant MTAs identified various genes of interest that may be involved in disease resistance. The identified resistant lines and SNPs linked to the QTLs in this study will serve as valuable resources in wheat rust resistance breeding programs.

Adult plant leaf rust resistance QTL derived from wheat line CI13227 maps to chromosomes 2AL, 4BS, and 7AL.

The winter wheat (Triticum aestivum L.) line CI13227 has been characterized as having adult plant resistance to leaf rust caused by Puccinia triticina Eriks (Pt). Line CI13227 was crossed with the susceptible spring wheat 'Thatcher' (Tc) and a Tc*2/CI13227 F6 line with adult plant leaf rust resistance designated as 411A was derived. Line 411A was crossed with Tc to develop an F6 recombinant inbred line (RIL) population. The parents and 120 F6 lines were assessed for leaf rust severity at the flag leaf stage in five field plot tests from 2011 through 2015 and were genotyped for single-nucleotide polymorphism (SNP) markers with the Illumina iSelect 90K wheat bead array. A total of 2,384 SNP markers segregated among the RILs. Completely linked SNPs were removed, and 474 markers that covered 2,605 centimorgans (cM) were used for linkage map construction. Quantitative trait loci (QTL) on chromosome 2AL with logarithm of odds (LOD) values 2.34-7.88, on chromosome 4BS with LOD values 1.35- 4.66, and on chromosome 7AL with LOD values 2.92-7.81 were associated with significant reduction in leaf rust severity in the field plot tests. Recombinant inbred lines that had combinations of two or three of the QTL had significantly lower leaf rust severity than RILs that lacked any resistance QTL. Kompetitive allele specific polymerase chain reaction (KASP) markers were developed for the SNPs that were closely linked with the three QTL to facilitate marker-based selection of the leaf rust resistance in breeding programs.

Genome-Wide Association Studies Reveal All-Stage Rust Resistance Loci in Elite Durum Wheat Genotypes.

Leaf rust, caused by Puccinia triticina (Pt), stripe rust caused by Puccinia striiformis f. sp. tritici (Pst), and stem rust caused by Puccinia graminis f. sp. tritici (Pgt) are major diseases to wheat production globally. Host resistance is the most suitable approach to manage these fungal pathogens. We investigated the phenotypic and genotypic structure of resistance to leaf rust, stem rust, and stripe rust pathogen races at the seedling stage in a collection of advanced durum wheat breeding lines and cultivars adapted to Upper Mid-West region of the United States. Phenotypic evaluation showed that the majority of the durum wheat genotypes were susceptible to Pt isolates adapted to durum wheat, whereas all the genotypes were resistant to common wheat type-Pt isolate. The majority of genotypes were resistant to stripe rust and stem rust pathogen races. The durum panel genotyped using Illumina iSelect 90 K wheat SNP assay was used for genome-wide association mapping (GWAS). The GWAS revealed 64 marker-trait associations (MTAs) representing six leaf rust resistance loci located on chromosome arms 2AS, 2AL, 5BS, 6AL, and 6BL. Two of these loci were identified at the positions of Lr52 and Lr64 genes, whereas the remaining loci are most likely novel. A total of 46 MTAs corresponding to four loci located on chromosome arms 1BS, 5BL, and 7BL were associated with stripe rust response. None of these loci correspond to designated stripe rust resistance genes. For stem rust, a total of 260 MTAs, representing 22 loci were identified on chromosome arms 1BL, 2BL, 3AL, 3BL, 4AL, 5AL, 5BL, 6AS, 6AL, 6BL, and 7BL. Four of these loci were located at the positions of known genes/alleles (Sr7b, Sr8155B1, Sr13a, and Sr13b). The discovery of known and novel rust resistance genes and their linked SNPs will help diversify rust resistance in durum wheat.

Whole-genome sequencing of multiple isolates of Puccinia triticina reveals asexual lineages evolving by recurrent mutations.

The wheat leaf rust fungus, Puccinia triticina Erikss., is a worldwide pathogen of tetraploid durum and hexaploid wheat. Many races of P. triticina differ for virulence to specific leaf rust resistance genes and are found in most wheat-growing regions of the world. Wheat cultivars with effective leaf rust resistance exert selection pressure on P. triticina populations for virulent race types. The objectives of this study were to examine whole-genome sequence data of 121 P. triticina isolates and to gain insight into race evolution. The collection included isolates comprising of many different race phenotypes collected worldwide from common and durum wheat. One isolate from wild wheat relative Aegilops speltoides and two from Ae. cylindrica were also included for comparison. Based on 121,907 informative variants identified relative to the reference Race 1-1 genome, isolates were clustered into 11 major lineages with 100% bootstrap support. The isolates were also grouped based on variation in 1311 predicted secreted protein genes. In gene-coding regions, all groups had high ratios of nonsynonymous to synonymous mutations and nonsense to readthrough mutations. Grouping of isolates based on two main variation principle components for either genome-wide variation or variation just within the secreted protein genes, indicated similar groupings. Variants were distributed across the entire genome, not just within the secreted protein genes. Our results suggest that recurrent mutation and selection play a major role in differentiation within the clonal lineages.

A five-transgene cassette confers broad-spectrum resistance to a fungal rust pathogen in wheat.

Breeding wheat with durable resistance to the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), a major threat to cereal production, is challenging due to the rapid evolution of pathogen virulence. Increased durability and broad-spectrum resistance can be achieved by introducing more than one resistance gene, but combining numerous unlinked genes by breeding is laborious. Here we generate polygenic Pgt resistance by introducing a transgene cassette of five resistance genes into bread wheat as a single locus and show that at least four of the five genes are functional. These wheat lines are resistant to aggressive and highly virulent Pgt isolates from around the world and show very high levels of resistance in the field. The simple monogenic inheritance of this multigene locus greatly simplifies its use in breeding. However, a new Pgt isolate with virulence to several genes at this locus suggests gene stacks will need strategic deployment to maintain their effectiveness.

Virulence Phenotypes and Molecular Genotypes in Collections of Puccinia triticina from Italy.

Twenty-four isolates of Puccinia triticina from Italy were characterized for virulence to seedlings of 22 common wheat Thatcher isolines, each with a different leaf rust resistance gene, and for molecular genotypes at 15 simple sequence repeat (SSR) loci. The isolates were compared to a set of 13 previously characterized P. triticina isolates from either durum or common wheat. Clustering based on virulence phenotypes and SSR genotypes grouped the Italian P. triticina isolates into three groups. In the first group, the isolates had virulence phenotypes and SSR genotypes that were similar to the isolates collected from durum wheat. Isolates in the second group were unique because they had virulence similar to the isolates from common wheat but were distinct for SSR genotypes compared to the isolates from durum wheat and from common wheat. Isolates in the third group had virulence phenotypes and SSR genotypes closely related to the isolates from common wheat. The isolates were grouped based on the known or assumed host of origin, virulence phenotype, and SSR genotypes. Measures of FST and RST for SSR genotypes, and ΦST for virulence phenotype were significant, which indicated differentiation among the three groups of isolates. Virulence phenotypes and molecular genotypes were highly correlated with r = 0.74.

Mapping of Novel Leaf Rust and Stem Rust Resistance Genes in the Portuguese Durum Wheat Landrace PI 192051.

Leaf rust caused by Puccinia triticina Erikss. (Pt) and stem rust caused by Puccinia graminis f. sp. tritici Erikss. & E. Henn (Pgt) are serious constraints to production of durum wheat (Triticum turgidum L). The objective of this study was to identify leaf rust resistance (Lr) and stem rust resistance (Sr) genes/QTL in Portuguese durum landrace PI 192051. Four Pt-isolates, representing three virulence phenotypes (BBBQJ, BBBSJ & EEEEE) and six Pgt-races TTKSK, JRCQC, TKTTF, QFCFC, TPMKC and TMLKC were used to evaluate 180 recombinant inbred lines (RILs) derived from the cross Rusty (rust susceptible) × PI 192051-1 (rust resistant) at the seedling stage. The RILs were also phenotyped at the adult-plant stage in a stem rust nursery in Ethiopia in 2017. The RILs were genotyped using the Illumina iSelect 9K wheat SNP array. PI 192051-1 carries a previously unidentified major Sr gene designated as QSr.ace-7A on chromosome arm 7AS and Lr gene Lr.ace-4A in the pericentromeric region of chromosome 4A. In addition, three minor Sr QTL QSr.ace-1A, QSr.ace-2B and QSr.ace-4A were mapped in PI 192051-1 on chromosomes 1AL, 2BL, and 4A, respectively Lr.ace-4A could be co-located or tightly linked to QSr.ace-4A Markers linked to the identified QTL/genes can be used for marker assisted selection. These findings enrich the genetic basis of rust resistance in both durum and common wheat.

Lr34-mediated leaf rust resistance in wheat: transcript profiling reveals a high energetic demand supported by transient recruitment of multiple metabolic pathways.

The wheat gene Lr34 confers partial resistance to all races of Puccinia triticina, the causal agent of wheat leaf rust. However, the biological basis for the exceptional durability of Lr34 is unclear. We used the Affymetrix GeneChip Wheat Genome Array to compare transcriptional changes of near-isogenic lines of Thatcher wheat in a compatible interaction, an incompatible interaction conferred by the resistance gene Lr1, and the race-nonspecific response conditioned by Lr34 3 and 7 days postinoculation (dpi) with P. triticina. No differentially expressed genes were detected in Lr1 plants at either timepoint whereas, in the compatible Thatcher interaction, differentially expressed genes were detected only at 7 dpi. In contrast, differentially expressed genes were identified at both timepoints in P. triticina-inoculated Lr34 plants. At 3 dpi, upregulated genes associated with Lr34-mediated resistance encoded various defense and stress-related proteins, secondary metabolism enzymes, and transcriptional regulation and cellular-signaling proteins. Further, coordinated upregulation of key genes in several metabolic pathways that can contribute to increased carbon flux through the tricarboxylic cycle was detected. This indicates that Lr34-mediated resistance imposes a high energetic demand that leads to the induction of multiple metabolic responses to support cellular energy requirements. These metabolic responses were not sustained through 7 dpi, and may explain why Lr34 fails to inhibit the pathogen fully but does increase the latent period.