Characterization of Quantitative Trait Loci for Leaf Rust Resistance from CI 13227 in Three Winter Wheat Populations.

Leaf rust is a widespread foliar wheat disease causing substantial yield losses worldwide. Slow-rusting is "adult plant" resistance that significantly slows epidemic development and thereby reduces yield loss. Wheat accession CI 13227 was previously characterized as having slow-rusting resistance. To validate the quantitative trait loci (QTL) and develop diagnostic markers for slow rusting resistance in CI 13227, a new population of recombinant inbred lines (RILs) of CI 13227 × Everest was evaluated for latent period (LP), final severity (FS), area under disease progress curve (AUDPC), and infection type (IT) in greenhouses and genotyped using genotyping-by-sequencing (GBS). Four QTL were identified on chromosome arms 2BL, 2DS, 3BS, and 7BL, explaining 6.82 to 28.45% of the phenotypic variance for these traits. Seven kompetitive allele specific polymorphism (KASP) markers previously reported to be linked to the QTL in two other CI 13227 populations were validated. In addition, the previously reported QLr.hwwg-7AL was remapped to 2BL (renamed QLr.hwwg-2BL) after adding new markers in this study. Phenotypic data showed that the RILs harboring two or three of the QTL had a significantly longer LP. QLr.hwwg-2DS on 2DS showed a major effect on all rust resistance traits and was finely mapped to a 2.7 Mb interval by two newly developed flanking markers from exome capture. Three disease-resistance genes and two transporter genes were identified as the putative candidates for QLr.hwwg-2DS. The validated QTL can be used as slow rusting resistance resources and the markers developed in this study will be useful for marker-assisted selection.

Evaluating a worldwide wheat collection for resistance to Hessian fly biotype 'Great Plains'.

Hessian fly (HF), Mayetiola destructor, is a major insect pest that causes severe losses in grain yield and quality of wheat (Triticum aestivum). Growing resistant cultivars is the most cost-effective approach to minimize wheat yield losses caused by HF. In this study, 2,496 wheat accessions were screened for resistance to the HF biotype 'Great Plains' (GP) in the greenhouse experiments. To purify seeds from heterogeneous resistant accessions, we recovered single resistant plants from 331 accessions that had at least one resistant plant after HF infestation of a global collection of 1,595 accessions and confirmed 27 accessions with high resistance (HR), and 91 accessions with moderate resistance (MR) to the GP biotype using purified seeds. Screening of 203 U.S. winter wheat accessions in three experiments identified 63 HR and 28 MR accessions; and screening of three additional Asian panels identified 4 HR and 25 MR accessions. Together, this study identified 96 HR accessions and 144 MR accessions. Analysis of the geographic distribution of these HR and MR accessions revealed that these countries with HF as a major wheat pest usually showed higher frequencies of resistant accessions, with the highest frequency of HR (81.3%) and MR (30.6%) accessions identified from the U.S. In addition, phenotyping of 39 wheat accessions that carry known HF resistance genes showed that all the accessions except H1H2 remain effective against GP biotype. Some of these newly identified resistant accessions may contain new HF resistance genes and can be valuable sources for developing HF resistant wheat cultivars.

A glutathione S-transferase from Thinopyrum ponticum confers Fhb7 resistance to Fusarium head blight in wheat.

Fusarium head blight (FHB), mainly incited by Fusarium graminearum Schwabe, has caused great losses in grain yield and quality of wheat globally. Fhb7, a major gene from 7E chromosome of Thinopyrum ponticum, confers broad resistance to multiple Fusarium species in wheat, and has recently been cloned and identified as encoding a glutathione S-transferase (GST). However, some recent reports raised doubt about if GST is the causal gene of Fhb7. To resolve the discrepancy and validate the gene function of GST in wheat, we phenotyped Fhb7 near-isogenic lines (Jimai22-Fhb7 vs Jimai22) and GST over-expressed lines for FHB resistance. Jimai22-Fhb7 showed significantly higher FHB resistance with a lower percentage of symptomatic spikelets (PSS), Fusarium-damaged kernel (FDK) and Deoxynivalenol (DON) content than susceptible Jimai22 in three experiments. All the positive GST transgenic lines driven by either the maize ubiquitin promoter (MubiP) or its native promoter (NP) with high gene expression in the wheat cultivar 'Fielder' showed high FHB resistance. Only one MubiP-driven transgenic line showed low GST expression and similar susceptibility as Fielder, suggesting high GST expression confers Fhb7 resistance to FHB. Knockout of GST in Jimai22-Fhb7 line using CRISPR-Cas9-based gene-editing showed significantly higher FHB susceptibility compared with the non-edited control plants. Therefore, we confirmed GST as the causal gene of Fhb7 for FHB resistance. Considering its major effect on FHB resistance, pyramiding Fhb7 with other QTLs has a great potential to create highly FHB-resistant wheat cultivars.

Fhb7-GST catalyzed glutathionylation effectively detoxifies the trichothecene family.

Trichothecene (TCN) contamination in food and feed is a serious challenge due to the negative health and economic impacts. Here, we confirmed that the glutathione S-transferase (GST) Fhb7-GST could broadly catalyze type A, type B and type D TCNs into glutathione epoxide adducts (TCN-13-GSHs). To evaluate the toxicity of TCN-13-GSH adducts, we performed cell proliferation assays in vitro, which demonstrated decreased cytotoxicity of the adducts. Moreover, in vivo assays (repeated-dose treatment in mice) confirmed that TCN-13-GSH adducts were dramatically less toxic than the corresponding TCNs. To establish whether TCN-13-GSH was metabolized back to free toxin during digestion, single-dose metabolic tests were performed in rats; DON-13-GSH was not hydrolyzed in vivo, but rather was quickly metabolized to another low-toxicity compound, DON-13-N-acetylcysteine. These results demonstrate the promise of Fhb7-GST as a candidate of detoxification enzyme potentially applied in TCN-contaminated agricultural samples, minimizing the detrimental effects of the mycotoxin.

The Black Necrotic Lesion Enhanced Fusarium graminearum Resistance in Wheat.

Fusarium head blight, mainly incited by Fusarium graminearum, is a devastating wheat disease worldwide. Diverse Fusarium head blight (FHB) resistant sources have been reported, but the resistance mechanisms of these sources remain to be investigated. FHB-resistant wheat germplasm often shows black necrotic lesions (BNLs) around the infection sites. To determine the relationship between BNL and FHB resistance, leaf tissue of a resistant wheat cultivar Sumai 3 was inoculated with four different F. graminearum isolates. Integrated metabolomic and transcriptomic analyses of the inoculated samples suggested that the phytohormone signaling, phenolamine, and flavonoid metabolic pathways played important roles in BNL formation that restricted F. graminearum extension. Exogenous application of flavonoid metabolites on wheat detached leaves revealed the possible contribution of flavonoids to BNL formation. Exogenous treatment of either salicylic acid (SA) or methyl jasmonate (MeJA) on wheat spikes significantly reduced the FHB severity. However, exogenous MeJA treatment prevented the BNL formation on the detached leaves of FHB-resistant wheat Sumai 3. SA signaling pathway influenced reactive oxygen species (ROS) burst to enhance BNL formation to reduce FHB severity. Three key genes in SA biosynthesis and signal transduction pathway, TaICS1, TaNPR1, and TaNPR3, positively regulated FHB resistance in wheat. A complex temporal interaction that contributed to wheat FHB resistance was detected between the SA and JA signaling pathways. Knowledge of BNLs extends our understanding of the molecular mechanisms of FHB resistance in wheat and will benefit the genetic improvement of wheat FHB resistance.

Precise mapping of QTL for Hessian fly resistance in the hard winter wheat cultivar 'Overland'.

KEY MESSAGE: A major QTL for Hessian fly resistance was precisely mapped to a 2.32 Mb region on chromosome 3B of the US hard winter wheat cultivar 'Overland'. The Hessian fly (HF, Mayetiola destructor) is a destructive insect pest of wheat in the USA and worldwide. Deploying HF-resistant cultivars is the most effective and economical approach to control this insect pest. A population of 186 recombinant inbred lines (RILs) was developed from 'Overland' × 'Overley' and phenotyped for responses to HF attack using the HF biotype 'Great Plains'. A high-density genetic linkage map was constructed using 1,576 single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS). Two quantitative trait loci (QTLs) with a significant epistatic effect on HF resistance were mapped to chromosomes 3B (QHf.hwwg-3B) and 7A (QHf.hwwg-7A) in Overland, which are located in similar chromosome regions as found for H35 and H36 in the cultivar 'SD06165', respectively. QHf.hwwg-3B showed a much larger effect on HF resistance than QHf.hwwg-7A. Five and four GBS-SNPs, respectively, in the QHf.hwwg-3B and QHf.hwwg-7A QTL intervals were converted into Kompetitive allele specific polymerase chain reaction (KASP) markers. QHf.hwwg-3B was precisely mapped to a 2.32 Mb interval (2,479,314-4,799,538 bp) using near-isogenic lines (NILs) and RILs that have recombination within the QTL interval. The US winter wheat accessions carrying contrasting alleles at KASP markers KASP-3B4525164, KASP-7A47772047 and KASP-7A65090410 showed significant difference in HF resistance. The combination of the two KASP markers KASP-3B3797431 and KASP-3B4525164 is near-diagnostic for the detection of QHf.hwwg-3B in a US winter wheat panel and can be potentially used for screening the QTL in breeding programs.

The Analysis of Trajectory Control of Non-holonomic Mobile Robots Based on Internet of Things Target Image Enhancement Technology and Backpropagation Neural Network.

The trajectory tracking and control of incomplete mobile robots are explored to improve the accuracy of the trajectory tracking of the robot controller. First, the mathematical kinematics model of the non-holonomic mobile robot is studied. Then, the improved Backpropagation Neural Network (BPNN) is applied to the robot controller. On this basis, a mobile robot trajectory tracking controller combining the fuzzy algorithm and the neural network is designed to control the linear velocity and angular velocity of the mobile robot. Finally, the robot target image can be analyzed effectively based on the Internet of Things (IoT) image enhancement technology. In the MATLAB environment, the performances of traditional BPNN and improved BPNN in mobile robots' trajectory tracking are compared. The tracking accuracy before and after the improvement shows no apparent differences; however, the training speed of improved BPNN is significantly accelerated. The fuzzy-BPNN controller presents significant improvements in tracking speed and tracking accuracy compared with the improved BPNN. The trajectory tracking controller of the mobile robot is designed and improved based on the fuzzy BPNN. The designed controller combining the fuzzy algorithm and the improved BPNN can provide higher accuracy and tracking efficiency for the trajectory tracking and control of the non-holonomic mobile robots.

Characterization of wheat curl mite resistance gene Cmc4 in OK05312.

KEY MESSAGE: Cmc4, a wheat curl mite resistance gene, was delimited to a 523 kb region and a diagnostic marker haplotype was identified for selecting Cmc4 in breeding programs. Wheat curl mite (WCM, Aceria tosichella Keifer) is a disastrous wheat pest in many wheat-growing regions worldwide. WCM not only directly affects wheat yield, but also transmits wheat streak mosaic virus. Growing WCM resistant cultivars is the most economical and sustainable method to reduce its damage. A hard winter wheat breeding line OK05312 (PI 670019) carries Cmc4 gene resistance to A. tosichella and has many desirable agronomic traits. To finely map Cmc4 in OK05312, two recombinant inbred line populations were developed from crosses between OK05312 and two susceptible cultivars, SD06165 and Jerry, genotyped using single nucleotide polymorphism (SNP) markers generated from genotyping-by-sequencing (GBS), and phenotyped for WCM resistance. Gene mapping using the two SNP maps confirmed Cmc4 in OK05312 that explained up to 68% of the phenotypic variation. Further analysis delimited Cmc4 to a ~ 523 kb region between SNPs SDOKSNP6314 and SDOKSNP2805 based on the Ae. tauschii reference genome. We developed 18 polymorphic Kompetitive Allele Specific PCR (KASP) markers using the sequences of GBS-SNPs in this region and 23 additional KASP markers based on the SNPs between the parents derived from 90K SNP chips. The KASP markers SDOKSNP6314 and SDOKSNP9699 are closest to Cmc4 and can be used to diagnose the presence of Cmc4 in wheat breeding programs. Haplotype analysis suggested that CmcTAM112 in TAM112 might be the same gene as Cmc4.

Integrated metabolo-transcriptomics and functional characterization reveals that the wheat auxin receptor TIR1 negatively regulates defense against Fusarium graminearum.

Fusarium head blight (FHB) caused by Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schw.) Perch) results in large yield losses in annual global wheat production. Although studies have identified a number of wheat FHB resistance genes, a deeper understanding of the mechanisms underlying host plant resistance to F. graminearum is required for the control of FHB. Here, an integrated metabolomics and transcriptomics analysis of infected wheat plants (Triticum aestivum L.) enabled identification of 789 differentially accumulated metabolites, including flavonoids, phenolamides, tryptamine derivatives, and phytohormones, and revealed altered expression of more than 100 genes that function in the biosynthesis or regulation of these pathways. Our data regarding the effects of F. graminearum infection on flavonoids and auxin signaling led to follow-up experiments that showed that exogenous kaempferide and apigenin application on spikes increased wheat resistance to FHB, while exogenous auxin treatment increased FHB susceptibility. RNAi-mediated knockdown of the gene encoding the auxin receptor, TaTIR1, increased FHB resistance. Our data supported the use of TaTIR1 knockdown in controlling FHB. Our study provides insights on the wheat response to F. graminearum infection and its FHB resistance mechanisms while illustrating the potential of TaTIR1 knockdown in increasing FHB resistance during crop improvement programs.

The Hessian fly recessive resistance gene h4 mapped to chromosome 1A of the wheat cultivar 'Java' using genotyping-by-sequencing.

KEY MESSAGE: The recessive Hessian fly resistance gene h4 and flanking SNP markers were located to a 642 kb region in chromosome 1A of the wheat cultivar 'Java.' Hessian fly (HF), Mayetiola destructor, is one of the most destructive insect pests in wheat worldwide. The wheat cultivar 'Java' was reported to carry a recessive gene (h4) for HF resistance; however, its chromosome location has not been determined. To map the HF resistance gene in Java, two populations of recombinant inbred lines (RILs) were developed from 'Bobwhite' × Java and 'Overley' × Java, respectively, and were phenotyped for responses to infestation of HF Great Plains biotype. Analysis of phenotypic data from the F1 and the RIL populations confirmed that one recessive gene conditioned HF resistance in Java. Two linkage maps were constructed using single-nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS). The h4 gene was mapped to the distal end of the short arm of chromosome 1A, which explained 60.4 to 70.5% of the phenotypic variation for HF resistance in the two populations. The GBS-SNPs in the h4 candidate interval were converted into Kompetitive Allele-Specific Polymerase Chain Reaction (KASP) markers to eliminate the missing data points in GBS-SNPs. Using the revised maps with KASP markers, h4 was further located to a 642 kb interval (6,635,984-7,277,935 bp). The two flanking KASP markers, KASP3299 and KASP1871, as well as four other closely linked KASP markers, may be useful for pyramiding h4 with other HF resistance genes in breeding.

Identification of two novel Hessian fly resistance genes H35 and H36 in a hard winter wheat line SD06165.

KEY MESSAGE: Two new Hessian fly resistance QTLs (H35 and H36) and tightly linked SNP markers were identified in a US hard winter wheat SD06165. Hessian fly (HF), Mayetiola destructor (Say), is one of the most destructive pests in wheat (Triticum aestivum L.) worldwide. Growing resistant cultivars is the most effective approach to minimize Hessian fly damage. To identify new quantitative trait loci (QTLs) for HF resistance, a recombinant inbred line population was developed by crossing HF resistant wheat line SD06165 to a susceptible line OK05312. The population was genotyped with 1709 single-nucleotide polymorphisms (SNPs) generated from genotyping-by-sequencing and phenotyped for HF resistance in greenhouses. Two novel QTLs for HF resistance were identified from SD06165. The major QTL, designated as H35, was closely linked to SNP marker SDOKSNP7679 on chromosome 3BS that explained 23.8% and 36.0% of the phenotypic variations; the minor QTL, designated as H36, was flanked by SNP markers SDOKSNP1618 and SDOKSNP8089 on chromosome 7AS and explained 8.5% and 13.1% of the phenotypic variation in the two experiments. Significant interaction was detected between the two QTLs. Seventeen SNPs that tightly link to H35 and eight SNPs that tightly link to H36 were converted to kompetitive allele specific polymerase chain reaction markers for selecting these QTLs in breeding programs.

Horizontal gene transfer of Fhb7 from fungus underlies Fusarium head blight resistance in wheat.

Fusarium head blight (FHB), a fungal disease caused by Fusarium species that produce food toxins, currently devastates wheat production worldwide, yet few resistance resources have been discovered in wheat germplasm. Here, we cloned the FHB resistance gene Fhb7 by assembling the genome of Thinopyrum elongatum, a species used in wheat distant hybridization breeding. Fhb7 encodes a glutathione S-transferase (GST) and confers broad resistance to Fusarium species by detoxifying trichothecenes through de-epoxidation. Fhb7 GST homologs are absent in plants, and our evidence supports that Th. elongatum has gained Fhb7 through horizontal gene transfer (HGT) from an endophytic Epichloë species. Fhb7 introgressions in wheat confers resistance to both FHB and crown rot in diverse wheat backgrounds without yield penalty, providing a solution for Fusarium resistance breeding.

Application of Brachypodium genotypes to the analysis of type II resistance to Fusarium head blight (FHB).

The resistance to Fusarium head blight (FHB) in wheat is mainly via the restrain of fungal expansion through spike rachis (type II resistance). In order to unravel the resistance mechanisms, Brachypodium distachyon 21 (Bd21), a monocotyledonous model plant, was previously proved to interact with F. graminearum, while the disease development in spike still needs to be explored in detail. Herein, it is found that the fungal spores mainly germinate on pistil of Bd21, then the hyphae rapidly extend to the bottom of floret and enter spike rachis, similar with the infection progress in wheat. However, structural difference of spike rachis was found between Brachypodium and wheat. It was found that the spread of the fungus through the rachis node of inoculated spikelets is an important index for the evaluation of type II FHB resistance in Brachypodium under optimal conditions at 28 °C and 50%-70% humidity. To verify the feasibility of this strategy, the transcription factor TaTGA2 was overexpressed in Bd21, and transgenic plants were found to show improved resistance to F. graminearum in both spikes and detached leaves, which was further supported by the increased disease severity when silencing TaTGA2 in the wheat cultivar "Sumai 3" or in tilling "Kronos" mutants. Except for Bd21, another 49 Brachypodium germplasms were further screened for FHB resistance, and three moderately susceptible germplasms, namely, PI 317418, W6-39284, and PI 254868, feasible for transformation, were determined to be better hosts than Bd21 when evaluating heterologous genes that positively regulate FHB resistance. The present study also observed variations in the levels of FHB resistance between coleoptiles and spikes or transgenic plants and natural germplasms.

Cloning and characterization of a specific UDP-glycosyltransferase gene induced by DON and Fusarium graminearum.

KEY MESSAGE: TaUGT5: can reduce the proliferation and destruction of F. graminearum and enhance the ability of FHB resistance in wheat. Deoxynivalenol (DON) is one of the most important toxins produced by Fusarium species that enhances the spread of the pathogen in the host. As a defense, the UDP-glycosyltransferase (UGT) family has been deduced to transform DON into the less toxic form DON-3-O-glucoside (D3G), but the specific gene member in wheat that is responsible for Fusarium head blight (FHB) resistance has been little investigated and proved. In this study, a DON and Fusarium graminearum responsive gene TaUGT5, which is specific for resistant cultivars, was cloned with a 1431 bp open reading frame (ORF) encoding 476 amino acids in Sumai3. TaUGT5 is located on chromosome 2B, which has been confirmed in nulli-tetrasomic lines of Chinese Spring (CS) and is solely expressed among three homologs on the A, B and D genomes. Over-expression of this gene in Arabidopsis conferred enhanced tolerance when grown on agar plates that contain DON. Similarly, the coleoptiles of wheat over-expressing TaUGT5 showed more resistance to F. graminearum, evidencing reduced proliferation and destruction of plant tissue by the pathogen. However, the disease resistance in spikes was not as significant as that on coleoptile compared with wild-type plants. A subcellular localization analysis revealed that TaUGT5 was localized on the plasma membrane of tobacco leaf epidermal cells. It is possible that TaUGT5 could enhance tolerance to DON, protect the plant cell from the pathogen infection and result in better maintenance of the cell structure, which slows down pathogen proliferation in plant tissue.

Identification of wheat non-specific lipid transfer proteins involved in chilling tolerance.

KEY MESSAGE: Three TaLTPs were found to enhance chilling tolerance of transgenic Arabidopsis, which were characterized by analyzes of promoter-GUS activity, subcellular localization, chromosomal location and transcriptional profile. Non-specific lipid transfer proteins (nsLTP) are abundantly expressed in plants, however, their functions are still unclear. In this study, we primarily characterized the functions of 3 type I TaLTP genes that were localized on chromosomes 3A, 3B, and 5D, respectively. The transcripts of TaLTPIb.1 and TaLTPIb.5 were induced under chilling, wound, and drought conditions, while TaLTPId.1 was only up-regulated by dark treatment. All the 3 TaLTP genes could be stimulated by the in vitro treatment of salicylic acid, while TaLTPId.1 was also positively regulated by methyljasmonic acid. Furthermore, the promoter-reporter assay of TaLTPIb.1 in the transgenic brachypodium showed a typical epidermis-specific expression pattern of this gene cluster. When fused with EGFP, all the 3 proteins were shown to localize on the plasma membrane in transgenic tobacco, although a signal in chloroplasts was also observed for TaLTPId.1. Heterogeneous overexpression of each of the TaLTP genes in Arabidopsis resulted in longer root length compared with wild type plants under chilling condition. These results suggest that type I TaLTPs may have a conserved functionality in chilling tolerance by lipid permeation in the plasma membrane of epidermal cells. On the other hand, the type I TaLTPs may exert functional divergence mainly through regulatory subfunctionalization.