Foliar application of plant-derived peptides decreases the severity of leaf rust (Puccinia triticina) infection in bread wheat (Triticum aestivum L.).

BACKGROUND: Screening and developing novel antifungal agents with minimal environmental impact are needed to maintain and increase crop production, which is constantly threatened by various pathogens. Small peptides with antimicrobial and antifungal activities have been known to play an important role in plant defense both at the pathogen level by suppressing its growth and proliferation as well as at the host level through activation or priming of the plant's immune system for a faster, more robust response against fungi. Rust fungi (Pucciniales) are plant pathogens that can infect key crops and overcome resistance genes introduced in elite wheat cultivars. RESULTS: We performed an in vitro screening of 18 peptides predominantly of plant origin with antifungal or antimicrobial activity for their ability to inhibit leaf rust (Puccinia triticina, CCDS-96-14-1 isolate) urediniospore germination. Nine peptides demonstrated significant fungicidal properties compared to the control. Foliar application of the top three candidates, ß-purothionin, Purothionin-α2 and Defensin-2, decreased the severity of leaf rust infection in wheat (Triticum aestivum L.) seedlings. Additionally, increased pathogen resistance was paralleled by elevated expression of defense-related genes. CONCLUSIONS: Identified antifungal peptides could potentially be engineered in the wheat genome to provide an alternative source of genetic resistance to leaf rust.

Identification of Sr67, a new gene for stem rust resistance in KU168-2 located close to the Sr13 locus in wheat.

KEY MESSAGE: Sr67 is a new stem rust resistance gene that represents a new resource for breeding stem rust resistant wheat cultivars Re-appearance of stem rust disease, caused by the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), in different parts of Europe emphasized the need to develop wheat varieties with effective resistance to local Pgt populations and exotic threats. A Kyoto University wheat (Triticum aestivum L.) accession KU168-2 was reported to carry good resistance to leaf and stem rust. To identify the genomic region associated with the KU168-2 stem rust resistance, a genetic study was conducted using a doubled haploid (DH) population from the cross RL6071 × KU168-2. The DH population was phenotyped with three Pgt races (TTKSK, TPMKC, and QTHSF) and genotyped using the Illumina 90 K wheat SNP array. Linkage mapping showed the resistance to all three Pgt races was conferred by a single stem rust resistance (Sr) gene on chromosome arm 6AL, associated with Sr13. Presently, four Sr13 resistance alleles have been reported. Sr13 allele-specific KASP and STARP markers, and sequencing markers all showed null alleles in KU168-2. KU168-2 showed a unique combination of seedling infection types for five Pgt races (TTKSK, QTHSF, RCRSF, TMRTF, and TPMKC) compared to Sr13 alleles. The phenotypic uniqueness of the stem rust resistance gene in KU168-2 and null alleles for Sr13 allele-specific markers showed the resistance was conferred by a new gene, designated Sr67. Since Sr13 is less effective in hexaploid background, Sr67 will be a good source of stem rust resistance in bread wheat breeding programs.

Uncovering the history of recombination and population structure in western Canadian stripe rust populations through mating type alleles.

BACKGROUND: The population structure of crop pathogens such as Puccinia striiformis f. sp. tritici (Pst), the cause of wheat stripe rust, is of interest to researchers looking to understand these pathogens on a molecular level as well as those with an applied focus such as disease epidemiology. Cereal rusts can reproduce sexually or asexually, and the emergence of novel lineages has the potential to cause serious epidemics such as the one caused by the 'Warrior' lineage in Europe. In a global context, Pst lineages in Canada were not well-characterized and the origin of foreign incursions was not known. Additionally, while some Pst mating type genes have been identified in published genomes, there has been no rigorous assessment of mating type diversity and distribution across the species. RESULTS: We used a whole-genome/transcriptome sequencing approach for the Canadian Pst population to identify lineages in their global context and evidence tracing foreign incursions. More importantly: for the first time ever, we identified nine alleles of the homeodomain mating type locus in the worldwide Pst population and show that previously identified lineages exhibit a single pair of these alleles. Consistently with the literature, we find only two pheromone receptor mating type alleles. We show that the recent population shift from the 'PstS1' lineage to the 'PstS1-related' lineage is also associated with the introduction of a novel mating type allele (Pst-b3-HD) to the Canadian population. We also show evidence for high levels of mating type diversity in samples associated with the Himalayan center of diversity for Pst, including a single Canadian race previously identified as 'PstPr' (probable recombinant) which we identify as a foreign incursion, most closely related to isolates sampled from China circa 2015. CONCLUSIONS: These data describe a recent shift in the population of Canadian Pst field isolates and characterize homeodomain-locus mating type alleles in the global Pst population which can now be utilized in testing several research questions and hypotheses around sexuality and hybridization in rust fungi.

Genetic mapping of the wheat leaf rust resistance gene Lr2a and its importance in Canadian wheat cultivars.

KEY MESSAGE: Leaf rust resistance gene Lr2a was located to chromosome arm 2DS in three mapping populations, which will facilitate map-based cloning and marker-assisted selection of Lr2a in wheat breeding programs. Incorporating effective leaf rust resistance (Lr) genes into high-yielding wheat cultivars has been an efficient method of disease control. One of the most widely used genes in Canada is the multi-allelic resistance gene Lr2, with alleles Lr2a, Lr2b, Lr2c, and Lr2d. The Lr2a allele confers complete resistance to a large portion of the Puccinia triticina (Pt) population in Canada. In this study, Lr2a was genetically mapped in two doubled haploid populations developed from the crosses Superb/BW278 and Superb/86ISMN 2137, and an F2 population developed from the cross Chinese Spring/RL6016. Seedlings were tested with the Lr2a avirulent Pt races 74-2 MGBJ (Superb/BW278) and 12-3 MBDS (Superb/86ISMN 2137 and Chinese Spring/RL6016) in greenhouse assays and were genotyped with 90K wheat Infinium SNP and kompetitive allele-specific PCR (KASP) markers. Lr2a was mapped to a collinear position on chromosome arm 2DS in all three populations, within a 1.00 cM genetic interval between KASP markers kwm1620 and kwm1623. This corresponded to a 305 kb genomic region of chromosome 2D in Chinese Spring RefSeq v2.1. The KASP marker kwh740 was predictive of Lr2a in all mapping populations. A panel of 260 wheats were tested with three Pt isolates, which revealed that Lr2a is common in Canadian wheats. The KASP markers kwh740 and kwm1584 were highly associated with resistance at the Lr2 locus, while kwm1622 was slightly less correlated. Genetic mapping of the leaf rust resistance gene Lr2a and DNA markers developed here will facilitate its use in wheat breeding programs.

Characterization of Brazilian spring wheat germplasm and its potential for increasing wheat genetic diversity in Canada.

In the present era of climate instability, Canadian wheat production has been frequently affected by abiotic stresses and by dynamic populations of pathogens and pests that are more virulent and aggressive over time. Genetic diversity is fundamental to guarantee sustainable and improved wheat production. In the past, the genetics of Brazilian cultivars, such as Frontana, have been studied by Canadian researchers and consequently, Brazilian germplasm has been used to breed Canadian wheat cultivars. The objective of this study was to characterize a collection of Brazilian germplasm under Canadian growing conditions, including the reaction of the Brazilian germplasm to Canadian isolates/pathogens and to predict the presence of certain genes in an effort to increase genetic diversity, improve genetic gain and resilience of Canadian wheat. Over 100 Brazilian hard red spring wheat cultivars released from 1986 to 2016 were evaluated for their agronomic performance in eastern Canada. Some cultivars showed good adaptability, with several cultivars being superior or statistically equal to the highest yielding Canadian checks. Several Brazilian cultivars had excellent resistance to leaf rust, even though only a few of these tested positive for the presence of either Lr34 or Lr16, two of the most common resistance genes in Canadian wheat. Resistance for stem rust, stripe rust and powdery mildew was variable among the Brazilian cultivars. However, many Brazilian cultivars had high levels of resistance to Canadian and African - Ug99 strains of stem rust. Many Brazilian cultivars had good Fusarium head blight (FHB) resistance, which appears to be derived from Frontana. In contrast FHB resistance in Canadian wheat is largely based on the Chinese variety, Sumai-3. The Brazilian germplasm is a valuable source of semi-dwarf (Rht) genes, and 75% of the Brazilian collection possessed Rht-B1b. Many cultivars in the Brazilian collection were found to be genetically distinct from Canadian wheat, making them a valuable resource to increase the disease resistance and genetic variability in Canada and elsewhere.

Genetic mapping of leaf rust (Puccinia triticina Eriks) resistance genes in six Canadian spring wheat cultivars.

The Canada Western Red Spring wheat (Triticum aestivum L.) cultivars AAC Concord, AAC Prevail, CDC Hughes, Lillian, Glenlea, and elite line BW961 express a spectrum of resistance to leaf rust caused by Puccinia triticina Eriks. This study aimed to identify and map the leaf rust resistance of the cultivars using three doubled haploid populations, AAC Prevail/BW961 (PB), CDC Hughes/AAC Concord (HC), and Lillian/Glenlea (LG). The populations were evaluated for seedling resistance in the greenhouse and adult plant disease response in the field at Morden, MB for 3 years and genotyped with the 90K wheat Infinium iSelect SNP array. Genetic maps were constructed to perform QTL analysis on the seedling and field leaf rust data. A total of three field leaf rust resistance QTL segregated in the PB population, five in the HC, and six in the LG population. In the PB population, BW961 contributed two QTL on chromosomes 2DS and 7DS, and AAC Prevail contributed a QTL on 4AL consistent across trials. Of the five QTL in HC, AAC Concord contributed two QTL on 4AL and 7AL consistent across trials and a QTL on 3DL.1 that provided seedling resistance only. CDC Hughes contributed two QTL on 1DS and 3DL.2. Lillian contributed four QTL significant in at least two of the three trials on 2BS, 4AL, 5AL, and 7AL, and Glenlea two QTL on 4BL and 7BL. The 1DS QTL from CDC Hughes, the 2DS from BW961, the 4AL from the AAC Prevail, AAC Concord, and Lillian, and the 7AL from AAC Concord and Lillian conferred seedling leaf rust resistance. The QTL on 4AL corresponded with Lr30 and was the same across cultivars AAC Prevail, AAC Concord, and Lillian, whereas the 7AL corresponding with LrCen was coincident between AAC Concord and Lillian. The 7DS and 2DS QTL in BW961 corresponded with Lr34 and Lr2a, respectively, and the 1DS QTL in CDC Hughes with Lr21. The QTL identified on 5AL could represent a novel gene. The results of this study will widen our knowledge of leaf rust resistance genes in Canadian wheat and their utilization in resistance breeding.

Systematic Characterization of Multi-Rust Resistance Genes from a 'Parula × Thatcher' Population with a High-Density Genetic Map.

Pyramiding multiple resistant genes has been proposed as the most effective way to control wheat rust diseases globally. Identifying the most effective pyramids is challenged by the large pool of rust resistance genes and limited information about their mechanisms of resistance and interactions. Here, using a high-density genetic map, a double haploid population, and multi-rust field testing, we aimed to systematically characterize the most effective gene pyramids for rust resistance from the durable multi-rust resistant CIMMYT cultivar Parula. We revealed that the Parula resistance gene pyramid contains Lr34/Yr18/Sr57 (Lr34), Lr46/Yr29/Sr58 (Lr46), Lr27/Yr30/Sr2 (Sr2), and Lr68. The efficacy, magnitude of effect, and interactions varied for the three rust diseases. A subpopulation mapping approach was applied to characterize the complex interactions of the resistance genes by controlling for the effect of Lr34. Using this approach, we found that Lr34 and Lr68 have a strong additive effect for leaf rust, whereas no additive effects were observed for any rusts between Lr34 and Lr46. Lr34 combined synergistically with Sr12 from Thatcher for stem rust, whereas the additive effect of Lr34 and Sr2 was dependent on the type of rust and environment. Two novel leaf rust quantitative trait loci (QTLs) from Parula were identified in this study, a stable QTL QLr-7BS and QLr-5AS, which showed Lr34 dependent expression. With these findings, we propose combining two to three high-value genes from Canadian wheat (e.g., Sr12 from Thatcher) with a foundational multi-adult plant resistance cassette for desirable and durable resistance to all three rusts in Canadian wheat.

Fine mapping and marker development for the wheat leaf rust resistance gene Lr32.

Wheat leaf rust is caused by the fungal pathogen Puccinia triticina and is one of the wheat diseases of concern globally. Among the known leaf rust resistance genes (Lr) genes, Lr32 is a broadly effective gene derived from the diploid species Aegilops tauschii coss. accession RL5497-1 and has been genetically mapped to chromosome arm 3DS. However, Lr32 resistance has not been utilized in current cultivars in part due to the lack of modern, predictive DNA markers. The goals of this study were to fine map the Lr32 region and develop SNP-based kompetitive allele-specific polymerase chain reaction markers. The genomic analysis was conducted by using doubled haploid and F2-derived mapping populations. For marker development, a 90K wheat chip array, 35K and 820K Axiom R SNPs, A. tauschii pseudomolecules v4.0 and International Wheat Genome Sequencing Consortium ReqSeq v2.1 reference genomes were used. Total 28 kompetitive allele-specific polymerase chain reaction and 2 simple sequence repeat markers were developed. The Lr32 region was fine mapped between kompetitive allele-specific polymerase chain reaction markers Kwh142 and Kwh355 that flanked 34-35 Mb of the diploid and hexaploid reference genomes. Leaf rust resistance mapped as a Mendelian trait that cosegregated with 20 markers, recombination restriction limited the further resolution of the Lr32 region. A total of 10-11 candidate genes associated with disease resistance were identified between the flanking regions on both reference genomes, with the majority belonging to the nucleotide-binding domain and leucine-rich repeat gene family. The validation analysis selected 2 kompetitive allele-specific polymerase chain reaction markers, Kwh147 and Kwh722, for marker-assisted selection. The presence of Lr32 along with other Lr genes such as Lr67 and Lr34 would increase the resistance in future wheat breeding lines and have a high impact on controlling wheat leaf rust.

Dual RNA-sequencing of Fusarium head blight resistance in winter wheat.

Fusarium head blight (FHB) is a devastating fungal disease responsible for significant yield losses in wheat and other cereal crops across the globe. FHB infection of wheat spikes results in grain contamination with mycotoxins, reducing both grain quality and yield. Breeding strategies have resulted in the production of FHB-resistant cultivars, however, the underlying molecular mechanisms of resistance in the majority of these cultivars are still poorly understood. To improve our understanding of FHB-resistance, we performed a transcriptomic analysis of FHB-resistant AC Emerson, FHB-moderately resistant AC Morley, and FHB-susceptible CDC Falcon in response to Fusarium graminearum. Wheat spikelets located directly below the point of inoculation were collected at 7-days post inoculation (dpi), where dual RNA-sequencing was performed to explore differential expression patterns between wheat cultivars in addition to the challenging pathogen. Differential expression analysis revealed distinct defense responses within FHB-resistant cultivars including the enrichment of physical defense through the lignin biosynthesis pathway, and DON detoxification through the activity of UDP-glycosyltransferases. Nucleotide sequence variants were also identified broadly between these cultivars with several variants being identified within differentially expressed putative defense genes. Further, F. graminearum demonstrated differential expression of mycotoxin biosynthesis pathways during infection, leading to the identification of putative pathogenicity factors.

Identification of Disease Resistance Parents and Genome-Wide Association Mapping of Resistance in Spring Wheat.

The likelihood of success in developing modern cultivars depend on multiple factors, including the identification of suitable parents to initiate new crosses, and characterizations of genomic regions associated with target traits. The objectives of the present study were to (a) determine the best economic weights of four major wheat diseases (leaf spot, common bunt, leaf rust, and stripe rust) and grain yield for multi-trait restrictive linear phenotypic selection index (RLPSI), (b) select the top 10% cultivars and lines (hereafter referred as genotypes) with better resistance to combinations of the four diseases and acceptable grain yield as potential parents, and (c) map genomic regions associated with resistance to each disease using genome-wide association study (GWAS). A diversity panel of 196 spring wheat genotypes was evaluated for their reaction to stripe rust at eight environments, leaf rust at four environments, leaf spot at three environments, common bunt at two environments, and grain yield at five environments. The panel was genotyped with the Wheat 90K SNP array and a few KASP SNPs of which we used 23,342 markers for statistical analyses. The RLPSI analysis performed by restricting the expected genetic gain for yield displayed significant (p < 0.05) differences among the 3125 economic weights. Using the best four economic weights, a subset of 22 of the 196 genotypes were selected as potential parents with resistance to the four diseases and acceptable grain yield. GWAS identified 37 genomic regions, which included 12 for common bunt, 13 for leaf rust, 5 for stripe rust, and 7 for leaf spot. Each genomic region explained from 6.6 to 16.9% and together accounted for 39.4% of the stripe rust, 49.1% of the leaf spot, 94.0% of the leaf rust, and 97.9% of the common bunt phenotypic variance combined across all environments. Results from this study provide valuable information for wheat breeders selecting parental combinations for new crosses to develop improved germplasm with enhanced resistance to the four diseases as well as the physical positions of genomic regions that confer resistance, which facilitates direct comparisons for independent mapping studies in the future.

Interactions Between Lr67 or Lr34 and Other Leaf Rust Resistance Genes in Wheat (Triticum aestivum).

The wheat multi-pest resistance genes Lr67 and Lr34 are similar in that they both condition resistance to many diseases, in a non-race-specific manner, and code for cellular transporters. Lr34 plays a critical role in breeding wheat for disease resistance in large part because it interacts with other resistance genes to result in effective and durable resistance. To determine if Lr67 interacts with other resistance genes in a similar manner as Lr34 six different doubled haploid populations were developed which segregated for either Lr67 or Lr34 along with a second resistance gene, either Lr13, Lr16, or Lr32. The presence or absence of each of these genes in the progeny lines was determined by molecular marker analysis. These six populations were tested for leaf rust field resistance in the same environments to compare the effects of Lr34 and Lr67 alone, and in combination with Lr13, Lr16 or Lr32. Lr67 and Lr34 significantly reduced the levels of rust severity, Lr34 showed a significant interaction with Lr13 but Lr67 did not. Both genes interacted with Lr16, and Lr67 had a significant interaction with Lr32. This analysis demonstrates the similar effect of Lr67, as seen with Lr34, on the interaction with other resistance genes to give a better level of resistance than with single resistance genes. While Lr67 is not widely deployed in agriculture, it could play an important role in disease resistance in future wheat cultivars.

Comparison of single-trait and multi-trait genomic predictions on agronomic and disease resistance traits in spring wheat.

KEY MESSAGE: This study performed comprehensive analyses on the predictive abilities of single-trait and two multi-trait models in three populations. Our results demonstrated the superiority of multi-traits over single-trait models across seven agronomic and four to seven disease resistance traits of different genetic architecture. The predictive ability of multi-trait and single-trait prediction models has not been investigated on diverse traits evaluated under organic and conventional management systems. Here, we compared the predictive abilities of 25% of a testing set that has not been evaluated for a single trait (ST), not evaluated for multi-traits (MT1), and evaluated for some traits but not others (MT2) in three spring wheat populations genotyped either with the wheat 90K single nucleotide polymorphisms array or DArTseq. Analyses were performed on seven agronomic traits evaluated under conventional and organic management systems, four to seven disease resistance traits, and all agronomic and disease resistance traits simultaneously. The average prediction accuracies of the ST, MT1, and MT2 models varied from 0.03 to 0.78 (mean 0.41), from 0.05 to 0.82 (mean 0.47), and from 0.05 to 0.92 (mean 0.67), respectively. The predictive ability of the MT2 model was significantly greater than the ST model in all traits and populations except common bunt with the MT1 model being intermediate between them. The MT2 model increased prediction accuracies over the ST and MT1 models in all traits by 9.0-82.4% (mean 37.3%) and 2.9-82.5% (mean 25.7%), respectively, except common bunt that showed up to 7.7% smaller accuracies in two populations. A joint analysis of all agronomic and disease resistance traits further improved accuracies within the MT1 and MT2 models on average by 21.4% and 17.4%, respectively, as compared to either the agronomic or disease resistance traits, demonstrating the high potential of the multi-traits models in improving prediction accuracies.

Genomic Predictions for Common Bunt, FHB, Stripe Rust, Leaf Rust, and Leaf Spotting Resistance in Spring Wheat.

Some studies have investigated the potential of genomic selection (GS) on stripe rust, leaf rust, Fusarium head blight (FHB), and leaf spot in wheat, but none of them have assessed the effect of the reaction norm model that incorporated GE interactions. In addition, the prediction accuracy on common bunt has not previously been studied. Here, we investigated within-population prediction accuracies using the baseline M1 model and two reaction norm models (M2 and M3) with three random cross-validation (CV1, CV2, and CV0) schemes. Three Canadian spring wheat populations were evaluated in up to eight field environments and genotyped with 3158, 5732, and 23,795 polymorphic markers. The M3 model that incorporated GE interactions reduced residual variance by an average of 10.2% as compared with the main effect M2 model and increased prediction accuracies on average by 2-6%. In some traits, the M3 model increased prediction accuracies up to 54% as compared with the M2 model. The average prediction accuracies of the M3 model with CV1, CV2, and CV0 schemes varied from 0.02 to 0.48, from 0.25 to 0.84, and from 0.14 to 0.87, respectively. In both CV2 and CV0 schemes, stripe rust in all three populations, common bunt and leaf rust in two populations, as well as FHB severity, FHB index, and leaf spot in one population had high to very high (0.54-0.87) prediction accuracies. This is the first comprehensive genomic selection study on five major diseases in spring wheat.

Variations in exons 11 and 12 of the multi-pest resistance wheat gene Lr34 are independently additive for leaf rust resistance.

Introduction: Characterization of germplasm collections for the wheat leaf rust gene Lr34 previously defined five haplotypes in spring wheat. All resistant lines had a 3-bp TTC deletion (null) in exon 11, resulting in the absence of a phenylalanine residue in the ABC transporter, as well as a single nucleotide C (Tyrosine in Lr34+) to T (Histidine in Lr34-) transition in exon 12. A rare haplotype present in Odesskaja 13 and Koktunkulskaja 332, both of intermediate rust resistance, had the 3-bp deletion typical of Lr34+ in exon 11 but the T nucleotide of Lr34- in exon 12. Methods: To quantify the role of each mutation in leaf rust resistance, Odesskaja 13 and Koktunkulskaja 332 were crossed to Thatcher and its near-isogenic line Thatcher-Lr34 (RL6058). Single seed descent populations were generated and evaluated for rust resistance in six different rust nurseries. Results: The Odesskaja 13 progeny with the TTC/T haplotype were susceptible with an average severity rating of 62.3%, the null/T haplotype progeny averaged 39.7% and the null/C haplotype was highly resistant, averaging 13.3% severity. The numbers for the Koktunkulskaja 332 crosses were similar with 63.5%, 43.5% and 23.7% severity ratings, respectively. Differences between all classes in all crosses were statistically significant, indicating that both mutations are independently additive for leaf rust resistance. The three-dimensional structural models of LR34 were used to analyze the locations and putative interference of both amino acids with the transport channel. Koktunkulskaja 332 also segregated for marker csLV46 which is linked to Lr46. Rust severity in lines with Lr34+ and csLV46+ had significantly lower rust severity ratings than those without, indicating the additivity of the two loci. Discussion: This has implications for the deployment of Lr34 in wheat cultivars and for the basic understanding of this important wheat multi-pest durable resistance gene.

Capturing Multiple Disease Resistance in Wheat through Intergeneric Hybridization.

Derivatives from 4 species from the secondary gene pool of wheat-1 diploid (T. monococcum), 2 tetraploid (T. carthlicum; T. timopheevi), and 1 hexaploid (T. miguschovae)-were screened for resistance to Fusarium head blight, leaf rust, stem rust, and stripe rust. Where screening, genetic studies, and mapping were completed it was shown that all species carried resistance to multiple plant diseases. Some derived lines carried resistance to up to four different diseases. Where mapping was completed, it was shown that different diseases mapped to different chromosomes within any one accession.

A Combination of Leaf Rust Resistance Genes, Including Lr34 and Lr46, Is the Key to the Durable Resistance of the Canadian Wheat Cultivar, Carberry.

The hexaploid spring wheat cultivar, Carberry, was registered in Canada in 2009, and has since been grown over an extensive area on the Canadian Prairies. Carberry has maintained a very high level of leaf rust (Puccinia triticina Eriks.) resistance since its release. To understand the genetic basis of Carberry's leaf rust resistance, Carberry was crossed with the susceptible cultivar, Thatcher, and a doubled haploid (DH) population of 297 lines was generated. The DH population was evaluated for leaf rust in seven field environments at the adult plant stage. Seedling and adult plant resistance (APR) to multiple virulence phenotypes of P. triticina was evaluated on the parents and the progeny population in controlled greenhouse studies. The population was genotyped with the wheat 90 K iSelect single nucleotide polymorphism (SNP) array, and quantitative trait loci (QTL) analysis was performed. The analysis using field leaf rust response indicated that Carberry contributed nine QTL located on chromosomes 1B, 2B (2 loci), 2D, 4A, 4B, 5A, 5B, and 7D. The QTL located on 1B, 2B, 5B, and 7D chromosomes were observed in two or more environments, whereas the remainder were detected in single environments. The resistance on 1B, detected in five environments, was attributed to Lr46 and on 7D, detected in seven environments to Lr34. The first 2B QTL corresponded with the adult plant gene, Lr13, while the second QTL corresponded with Lr16. The seedling analysis showed that Carberry carries Lr2a, Lr16, and Lr23. Five epistatic effects were identified in the population, with synergistic interactions being observed for Lr34 with Lr46, Lr16, and Lr2a. The durable rust resistance of Carberry is attributed to Lr34 and Lr46 in combination with these other resistance genes, because the resistance has remained effective even though the P. triticina population has evolved virulent to Lr2a, Lr13, Lr16, and Lr23.

Identification of New Leaf Rust Resistance Loci in Wheat and Wild Relatives by Array-Based SNP Genotyping and Association Genetics.

Leaf rust caused by Puccinia triticina is the most widespread rust disease of wheat. As pathogen populations are constantly evolving, identification of novel sources of resistance is necessary to maintain disease resistance and stay ahead of this plant-pathogen evolutionary arms race. The wild genepool of wheat is a rich source of genetic diversity, accounting for 44% of the Lr genes identified. Here we performed a genome-wide association study (GWAS) on a diverse germplasm of 385 accessions, including 27 different Triticum and Aegilops species. Genetic characterization using the wheat 90 K array and subsequent filtering identified a set of 20,501 single nucleotide polymorphic (SNP) markers. Of those, 9,570 were validated using exome capture and mapped onto the Chinese Spring reference sequence v1.0. Phylogenetic analyses illustrated four major clades, clearly separating the wild species from the T. aestivum and T. turgidum species. GWAS was conducted using eight statistical models for infection types against six leaf rust isolates and leaf rust severity rated in field trials for 3-4 years at 2-3 locations in Canada. Functional annotation of genes containing significant quantitative trait nucleotides (QTNs) identified 96 disease-related loci associated with leaf rust resistance. A total of 21 QTNs were in haplotype blocks or within flanking markers of at least 16 known Lr genes. The remaining significant QTNs were considered loci that putatively harbor new Lr resistance genes. Isolation of these candidate genes will contribute to the elucidation of their role in leaf rust resistance and promote their usefulness in marker-assisted selection and introgression.

A phenylpropanoid diglyceride associates with the leaf rust resistance Lr34res gene in wheat.

The gene Lr34res is one of the most long-lasting sources of quantitative fungal resistance in wheat. It is shown to be effective against leaf, stem, and stripe rusts, as well as powdery mildew and spot blotch. Recent biochemical characterizations of the encoded ABC transporter have outlined a number of allocrites, including phospholipids and abscisic acid, consistent with the established general promiscuity of ABC transporters, but ultimately leaving its mechanism of rust resistance unclear. Working with flag leaves of Triticum aestivum L. variety 'Thatcher' (Tc) and a near-isogenic line of 'Thatcher' into which the Lr34res allele was introgressed (Tc+Lr34res; RL6058), a comparative semi-targeted metabolomics analysis of flavonoid-rich extracts revealed virtually identical profiles with the exception of one metabolite accumulating in Tc+Lr34res, which was not present at comparable levels in Tc. Structural characterization of the purified metabolite revealed a phenylpropanoid diglyceride structure, 1-O-p-coumaroyl-3-O-feruloylglycerol (CFG). Additional profiling of CFG across a collection of near-isogenic lines and representative Lr34 haplotypes highlighted a broad association between the presence of Lr34res and elevated accumulations of CFG. Depletion of CFG upon infection, juxtaposed to its relatively lower anti-fungal activity, suggests CFG may serve as a storage form of the more potent anti-microbial hydroxycinnamic acids that are accessed during defense responses. Altogether these findings suggest a role for the encoded LR34res ABC transporter in modifying the accumulation of CFG, leading to increased accumulation of anti-fungal metabolites, essentially priming the wheat plant for defense.

Genetic analyses of native Fusarium head blight resistance in two spring wheat populations identifies QTL near the B1, Ppd-D1, Rht-1, Vrn-1, Fhb1, Fhb2, and Fhb5 loci.

KEY MESSAGE: QTL analyses of two bi-parental mapping populations with AC Barrie as a parent revealed numerous FHB-resistance QTL unique to each population and uncovered novel variation near Fhb1. Fusarium head blight (FHB) is a destructive disease of wheat worldwide, leading to severe yield and quality losses. The genetic basis of native FHB resistance was examined in two populations: a recombinant inbred line population from the cross Cutler/AC Barrie and a doubled haploid (DH) population from the cross AC Barrie/Reeder. Numerous QTL were detected among the two mapping populations with many being cross-specific. Photoperiod insensitivity at Ppd-D1 and dwarfing at Rht-B1 and Rht-D1 was associated with increased FHB susceptibility. Anthesis date QTL at or near the Vrn-A1 and Vrn-B1 loci co-located with major FHB-resistance QTL in the AC Barrie/Reeder population. The loci were epistatic for both traits, such that DH lines with both late alleles were considerably later to anthesis and had reduced FHB symptoms (i.e., responsible for the epistatic interaction). Interestingly, AC Barrie contributed FHB resistance near the Fhb1 locus in the Cutler population and susceptibility in the Reeder population. Analyses of the Fhb1 candidate genes PFT and TaHRC confirmed that AC Barrie, Cutler, and Reeder do not carry the Sumai-3 Fhb1 gene. Resistance QTL were also detected at the expected locations of Fhb2 and Fhb5. The native FHB-resistance QTL detected near Fhb1, Fhb2, and Fhb5 do not appear to be as effective as Fhb1, Fhb2, and Fhb5 from Sumai-3. The presence of awns segregated at the B1 awn inhibitor locus in both populations, but was only associated with FHB resistance in the Cutler/AC Barrie population suggesting linkage caused the association rather than pleiotropy.

The role of reactive oxygen species in the virulence of wheat leaf rust fungus Puccinia triticina.

Reactive oxygen species (ROS) play an important role during host-pathogen interactions and are often an indication of induced host defence responses. In this study, we demonstrate for the first time that Puccinia triticina (Pt) generates ROS, including superoxide, H2 O2 and hydroxyl radicals, during wheat infection. Through pharmacological inhibition, we found that ROS are critical for both Pt urediniospore germination and pathogenic development on wheat. A comparative RNA-Seq analysis of different stages of Pt infection process revealed 291 putative Pt genes associated with the oxidation-reduction process. Thirty-seven of these genes encode known proteins. The expressions of five Pt genes, including PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod, were subsequently verified using RT-qPCR analysis. The results show that the expressions of PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod are up-regulated during urediniospore germination. In comparison, the expressions of PtNoxA, PtNoxB, PtNoxR and PtCat are down-regulated during wheat infection from 12 to 120 h after inoculation (HAI), whereas the expression of PtSod is up-regulated with a peak of expression at 120 HAI. We conclude that ROS are critical for the full virulence of Pt and a coordinate down-regulation of PtNox genes may be important for successful infection in wheat.