Integration of genome-wide association study, linkage analysis, and population transcriptome analysis to reveal the TaFMO1-5B modulating seminal root growth in bread wheat.

Bread wheat, one of the keystone crops for global food security, is challenged by climate change and resource shortage. The root system plays a vital role in water and nutrient absorption, making it essential for meeting the growing global demand. Here, using an association-mapping population composed of 406 accessions, we identified QTrl.Rs-5B modulating seminal root development with a genome-wide association study and validated its genetic effects with two F5 segregation populations. Transcriptome-wide association study prioritized TaFMO1-5B, a gene encoding the flavin-containing monooxygenases, as the causal gene for QTrl.Rs-5B, whose expression levels correlate negatively with the phenotyping variations among our population. The lines silenced for TaFMO1-5B consistently showed significantly larger seminal roots in different genetic backgrounds. Additionally, the agriculture traits measured in multiple environments showed that QTrl.Rs-5B also affects yield component traits and plant architecture-related traits, and its favorable haplotype modulates these traits toward that of modern cultivars, suggesting the application potential of QTrl.Rs-5B for wheat breeding. Consistently, the frequency of the favorable haplotype of QTrl.Rs-5B increased with habitat expansion and breeding improvement of bread wheat. In conclusion, our findings identified and demonstrated the effects of QTrl.Rs-5B on seminal root development and illustrated that it is a valuable genetic locus for wheat root improvement.

Combined GWAS and eGWAS reveals the genetic basis underlying drought tolerance in emmer wheat (Triticum turgidum L.).

Drought is one of the major environmental constraints for wheat production world-wide. As the progenitor and genetic reservoir of common wheat, emmer wheat is considered as an invaluable gene pool for breeding drought-tolerant wheat. Combining GWAS and eGWAS analysis of 107 accessions, we identified 86 QTLs, 105 462 eQTLs as well as 68 eQTL hotspots associating with drought tolerance (DT) in emmer wheat. A complex regulatory network composed of 185 upstream regulator and 2432 downstream drought-responsive candidates was developed, of which TtOTS1 was found to play a negative effect in determining DT through affecting root development. This study sheds light on revealing the genetic basis underlying DT, which will provide the indispensable genes and germplasm resources for elite drought tolerance wheat improvement and breeding.

Complexity and regulation of age-dependent alternative splicing in Brachypodium distachyon.

Alternative splicing (AS) is a gene regulatory mechanism that generates multiple transcripts of the same gene precursor by the spliceosome complex, promoting messenger RNA complexity, and proteome diversity. Although AS is extensively studied in response to environmental stresses, whether it mediates age-dependent development and how it is adjusted by growth transitions are largely unknown. Here, we comprehensively explored the AS landscape at different developmental stages in the grass model plant Brachypodium (Brachypodium distachyon). We identified abundant coding genes and noncoding transcripts subject to dynamic AS regulation during juvenile, adult, and reproductive transitions. Moreover, we revealed that SC35-LIKE SPLICING FACTOR 33 (SCL33), a serine/arginine-rich splicing factor in spliceosomes, plays a redundant and antagonistic role with its putative paralog, SCL33L, in regulating intron assembly across distinct developmental stages. In addition, we determined global AS variations in microRNA156 (miR156)-overproducing plants, in which growth transitions are delayed, and found that SPLs were regulated by miR156 in intron retention alteration in addition to mRNA clearance and translation inhibition manners. Finally, we demonstrated a complex regulatory process of age-dependent AS events in B. distachyon that was coincidently or separately regulated by miR156 and SCL33/SCL33L. These results illustrate a substantial machinery of AS that mediates phase transitions in plants.

TaGSK3 regulates wheat development and stress adaptation through BR-dependent and BR-independent pathways.

The GSK3/SHAGGY-like kinase plays critical roles in plant development and response to stress, but its specific function remains largely unknown in wheat (Triticum aestivum L.). In this study, we investigated the function of TaGSK3, a GSK3/SHAGGY-like kinase, in wheat development and response to stress. Our findings demonstrated that TaGSK3 mutants had significant effects on wheat seedling development and brassinosteroid (BR) signalling. Quadruple and quintuple mutants showed amplified BR signalling, promoting seedling development, while a sextuple mutant displayed severe developmental defects but still responded to exogenous BR signals, indicating redundancy and non-BR-related functions of TaGSK3. A gain-of-function mutation in TaGSK3-3D disrupted BR signalling, resulting in compact and dwarf plant architecture. Notably, this mutation conferred significant drought and heat stress resistance of wheat, and enhanced heat tolerance independent of BR signalling, unlike knock-down mutants. Further research revealed that this mutation maintains a higher relative water content by regulating stomatal-mediated water loss and maintains a lower ROS level to reduces cell damage, enabling better growth under stress. Our study provides comprehensive insights into the role of TaGSK3 in wheat development, stress response, and BR signal transduction, offering potential for modifying TaGSK3 to improve agronomic traits and enhance stress resistance in wheat.

GenoBaits®WheatplusEE: a targeted capture sequencing panel for quick and accurate identification of wheat-Thinopyrum derivatives.

KEY MESSAGE: A total of 90,000 capture probes derived from wheat and Thinopyrum elongatum were integrated into one chip, which served as an economical genotype for explorating Thinopyrumspecies and their derivatives. Thinopyrum species play a crucial role as a source of new genetic variations for enhancing wheat traits, including resistance to both abiotic and biotic factors. Accurate identification of exogenous chromosome(s) or chromosome segments or genes is essential following the introduction of alien genetic material into wheat, but this task remains challenging. This study aimed to develop a high-resolution wheat-Thinopyrum elongatum array, named GenoBaits®WheatplusEE, to trace alien genetic information by genotyping using a target sequencing system. This GenoBaits®WheatplusEE array included 90,000 capture probes derived from two species and integrated into one chip, with 10,000 and 80,000 originating from wheat and Th. elongatum, respectively. The capture probes were strategically positioned in genes and evenly distributed across the genome, facilitating the development of a roadmap for identifying each alien gene. The array was applied to the high-throughput identification of the alien chromosomes or segments in Thinopyrum and distantly related species and their derivatives. Our results demonstrated that the GenoBaits®WheatplusEE array could be used for direct identification of the breakpoint of alien segments, determine copy number of alien chromosomes, and reveal variations in wheat chromosomes by a single round of target sequencing of the sample. Additionally, we could efficiently and cost-effectively genotype, supporting the exploration of subgenome composition, phylogenetic relationships, and polymorphisms in essential genes (e.g., Fhb7 gene) among Thinopyrum species and their derivatives. We hope that GenoBaits®WheatplusEE will become a widely adopted tool for exporting wild germplasm for wheat improvement in the future.

Genome-Wide Comparative Analysis of the Cytochrome P450 Monooxygenase Family in 19 Aphid Species and Their Expression Analysis in 4 Cereal Crop Aphids.

Cytochrome P450 monooxygenases (CYP450s) play a variety of physiological roles, including pesticide resistance, plant allelochemical detoxification, and hormone metabolism catalysis. However, limited information is available on the classification and expression profiles of the CYP450 gene family in aphid species. This is the first study to identify the cytochrome P450 gene family in 19 aphid species at the whole genome level. A total of 1100 CYP450 genes were identified in 19 aphid species. Three hundred CYP450 genes belonged to six cereal crop aphid species, which were further classified into four subfamilies according to the phylogenetic relationship. The conserved motifs, exon-intron structures, and genomic organization of the same subfamilies were similar. Predictions of subcellular localization revealed that the endoplasmic reticulum harbored the majority of CYP450 proteins. In Sitobion avenae and Rhopalosiphum maidis, the increase in the CYP450 gene was primarily caused by segmental duplication events. However, only tandem duplication occurred in the CYP450 gene family of Diuraphis noxia, Rhopalosiphum padi, Schizaphis graminum, and Sitobion miscanthi. Synteny analysis found three continuous colinear CYP450 gene pairs among six cereal crop aphid species. Furthermore, we obtained the expression profiles of four cereal crop aphids, including R. padi, D. noxia, S. graminum, and S. avenae. Differential expression analysis provided growth stage specificity genes, tissue specificity genes, organ specificity genes and some detoxification metabolic genes among these four cereal crop aphids. Meanwhile, their expression patterns were showed. The related functions and pathways of CYP450s were revealed by GO and KEGG enrichment analysis. Above all, we picked the differentially expressed CYP450 genes from all of the differentially expressed genes (DEGs). These differentially expressed CYP450 genes provided some new potential candidates for aphid control and management. This work establishes the foundation for further investigations into the regulatory functions of the CYP450 gene family in aphid species and beyond.

Population-level transcriptomes reveal gene expression and splicing underlying cadmium accumulation in barley.

Genetic variation is an important determinant of gene transcription, which in turn contributes to functional and phenotypic diversity. Identification of the genetic variants controlling gene expression and alternative splicing in crops responding to cadmium (Cd), an important issue for food safety and human health, is of great value to improve our understanding of Cd accumulation-related genes. Here we report an in-depth survey of population-level transcriptome variation of barley (Hordeum vulgare) core accessions under Cd exposure. We reveal marked transcriptomic changes in response to Cd exposure, and these are largely independent of tissues. A genome-wide association study (GWAS) revealed 59 498 expression quantitative trait loci (eQTLs) and 23 854 splicing quantitative trait loci (sQTLs), leading to a complex network that covers 66.6% of the expressed genes, including 68 metal transporter genes. On average, 41.0% of sQTLs overlapped with eQTLs across different tissues, indicating that these two dimensions of transcript variation are largely independent. Moreover, we found that 34.5% of GWAS QTLs that underlie 10 Cd accumulation traits in barley are co-localized with eQTLs and sQTLs, which could imply a mechanistic role of different genetic variants affecting gene expression and alternative splicing in these traits. This study highlights the role of distal and proximal genetic effects on gene expression, splicing, and phenotypic plasticity. We anticipate that our results on the genetic control of expression and splicing underlying Cd accumulation provide a bridge to better understand genetic variation and phenotypic diversity to elucidate the mechanisms underlying Cd accumulation in plants.

Genome-wide identification and characterization of lipoxygenase genes related to the English grain aphid infestation response in wheat.

MAIN CONCLUSION: 44 wheat LOX genes were identified by silico genome-wide search method. TaLOX5, 7, 10, 24, 29, 33 were specifically expressed post aphid infestation, indicating their participation in wheat-aphid interaction. In plants, LOX genes play important roles in various biological progresses including seed germination, tuber development, plant vegetative growth and most crucially in plant signal transduction, stress response and plant defense against plant diseases and insects. Although LOX genes have been characterized in many species, the importance of the LOX family in wheat has still not been well understood, hampering further improvement of wheat under stress conditions. Here, we identified 44 LOX genes (TaLOXs) in the whole wheat genome and classified into three subfamilies (9-LOXs, Type I 13-LOXs and Type II 13-LOXs) according to phylogenetic relationships. The TaLOXs belonging to the same subgroup shared similar gene structures and motif organizations. Synteny analysis demonstrated that segmental duplication events mainly contributed to the expansion of the LOX gene family in wheat. The results of protein-protein interaction network (PPI) and miRNA-TaLOXs predictions revealed that three TaLOXs (TaLOX20, 22 and 37) interacted mostly with proteins related to methyl jasmonate (MeJA) signaling pathway. The expression patterns of TaLOXs in different tissues (root, stem, leaf, spike and grain) under diverse abiotic stresses (heat, cold, drought, drought and heat combined treatment, and salt) as well as under diverse biotic stresses (powdery mildew pathogen, Fusarium graminearum and stripe rust pathogen) were systematically analyzed using RNA-seq data. We obtained aphid-responsive candidate genes by RNA-seq data of wheat after the English grain aphid infestation. Aphid-responsive candidate genes, including TaLOX5, 7, 10, 24, 29 and 33, were up-regulated in the wheat aphid-resistant genotype (Lunxuan144), while they were little expressed in the susceptible genotype (Jimai22) during late response (48 h and 72 h) to the English grain aphid infestation. Meanwhile, qRT-PCR analysis was used to validate these aphid-responsive candidate genes. The genetic divergence and diversity of all the TaLOXs in bread wheat and its relative species were investigated by available resequencing data. Finally, the 3D structure of the TaLOX proteins was predicted based on the homology modeling method. This study not only systematically investigated the characteristics and evolutionary relationships of TaLOXs, but also provided potential candidate genes in response to the English grain aphid infestation and laid the foundation to further study the regulatory roles in the English grain aphid infestation of LOX family in wheat and beyond.

The selection and application of peduncle length QTL QPL_6D.1 in modern wheat (Triticum aestivum L.) breeding.

KEY MESSAGE: QPL_6D.1b displayed an additive effect with Rht-B1b and Rht-D1b in reducing wheat plant height and peduncle length, which confers shorter peduncle length and more kernels per spike, and had been broadly selected by Chinese modern wheat cultivars. Peduncle length (PL), as the key component of wheat plant height (PH), plays critical role in determining wheat lodging resistance and wheat pathogen resistance; then, its breeding selection and genetic basis remain largely unclear. Here the PH and PL were investigated in 406 wheat accessions in eight environments. In this study, a PL preferentially QTL QPL_6D.1 was identified in six environments by GWAS, which explained 13.6-24.2% of wheat PL variations in natural population. The allele QPL_6D.1b displayed a significantly additive effect with Rht-B1b and Rht-D1b in controlling PH and PL and could freely combined with Rht-B1b and Rht-D1b in current wheat cultivars. Haplotypic analysis demonstrates the QPL_6D.1b has been selected by Chinese modern wheat cultivar and confers shorter PL and more kernels per spike, highlighting its potentials in wheat breeding.

An intron-located single nucleotide variation of TaGS5-3D is related to wheat grain size through accumulating intron retention transcripts.

KEY MESSAGE: Thirty-three stable QTL for 13 yield-related traits across ten environments were identified in the PD34/MY47 RIL population, and a candidate gene TaGS5-3D in Qmt.nwafu.3D was preliminarily identified to affect grain-related traits through accumulation of specific transcripts. Dissecting the genetic basis of yield-related traits is pivotal for improvement of wheat yield potential. In this study, a recombinant inbred line (RIL) population genotyped by SNP markers was used to detect quantitative trait loci (QTL) related to yield-related traits in ten environments. A total of 102 QTL were detected, including 33 environmentally stable QTL and 69 putative QTL. Among them, Qmt.nwafu.3D was identified as a pleiotropic QTL in the physical interval of 149.77-154.11 Mb containing a potential candidate gene TaGS5-3D. An SNP (T > C) was detected in its ninth intron, and TaGS5-3D-C was validated as a superior allele associated with larger grains using a CAPS marker. Interestingly, we found that TaGS5-3D-C was closely related to significantly up-regulated expression of intron-retained transcript (TaGS5-3D-PD34.1), while TaGS5-3D-T was related to dominant expression of normal splicing transcript (TaGS5-3D-MY47.1). Our results indicated that alternative splicing associated with the SNP T/C could be involved in the regulation of grain-related traits, laying a foundation for the functional analysis of TaGS5-3D and its greater potential application in high-yield wheat breeding.

Host Specificity of Soilborne Pathogens in Hordeum Species and Their Relatives.

Soilborne pathogens destabilize the yields of Triticeae crops, including barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). Although genetic resistance derived from relatives of these species has been utilized to prevent rust diseases (i.e., in the wheat-rye 1BL-1RS translocation line), research on resistance against soilborne pathogens remains limited. Here, we performed field trials using 76 genotypes representing 28 Hordeum, six Triticum, and two Aegilops species to examine resistance against three soilborne bymoviruses: barley yellow mosaic virus (BaYMV), barley mild mosaic virus (BaMMV), and wheat yellow mosaic virus (WYMV). We also performed greenhouse tests using the soilborne fungal pathogen Fusarium pseudograminearum, which causes Fusarium crown rot (FCR). Using RT-PCR, we detected BaMMV and BaYMV in several Hordeum species, whereas WYMV induced systemic infection in the Triticum and Aegilops species. The identification of FCR susceptibility in all species examined suggests that F. pseudograminearum is a facultative fungal pathogen in Triticeae. Intraspecies variation in FCR disease severity was observed for several species, pointing to the possibility of exploring host resistance mechanisms. Therefore, by unlocking the host specificity of four soilborne pathogens in Hordeum species and their relatives, we obtained insights for the further exploration of wild sources of soilborne pathogen resistance for future wheat and barley improvement programs.

TaSYP137 and TaVAMP723, the SNAREs Proteins from Wheat, Reduce Resistance to Blumeria graminis f. sp. tritici.

SNARE protein is an essential factor driving vesicle fusion in eukaryotes. Several SNAREs have been shown to play a crucial role in protecting against powdery mildew and other pathogens. In our previous study, we identified SNARE family members and analyzed their expression pattern in response to powdery mildew infection. Based on quantitative expression and RNA-seq results, we focused on TaSYP137/TaVAMP723 and hypothesized that they play an important role in the interaction between wheat and Blumeria graminis f. sp. Tritici (Bgt). In this study, we measured the expression patterns of TaSYP132/TaVAMP723 genes in wheat post-infection with Bgt and found that the expression pattern of TaSYP137/TaVAMP723 was opposite in resistant and susceptible wheat samples infected by Bgt. The overexpression of TaSYP137/TaVAMP723 disrupted wheat's defense against Bgt infection, while silencing these genes enhanced its resistance to Bgt. Subcellular localization studies revealed that TaSYP137/TaVAMP723 are present in both the plasma membrane and nucleus. The interaction between TaSYP137 and TaVAMP723 was confirmed using the yeast two-hybrid (Y2H) system. This study offers novel insights into the involvement of SNARE proteins in the resistance of wheat against Bgt, thereby enhancing our comprehension of the role of the SNARE family in the pathways related to plant disease resistance.

Molecular Cytological Analysis and Specific Marker Development in Wheat-Psathyrostachys huashanica Keng 3Ns Additional Line with Elongated Glume.

Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) is an excellent gene resource for wheat breeding, which is characterized by early maturity, low plant height, and disease resistance. The wheat-P. huashanica derivatives were created by the elite genes of P. huashanica and permeate into common wheat through hybridization. Among them, a long-glume material 20JH1155 was identified, with larger grains and longer spike than its parents. In the present study, the methods of cytological observation, GISH, and sequential FISH analysis showed that 20JH1155 contained 21 pairs of wheat chromosomes and a pair of P. huashanica. There were some differences in 5A and 7B chromosomes between 20JH1155 and parental wheat 7182. Molecular marker, FISH, and sequence cloning indicated 20JH1155 alien chromosomes were 3Ns of P. huashanica. In addition, differentially expressed genes during immature spikelet development of 20JH1155 and 7182 and predicted transcription factors were obtained by transcriptome sequencing. Moreover, a total of 7 makers derived from Ph#3Ns were developed from transcriptome data. Taken together, the wheat-P. huashanica derived line 20JH1155 provides a new horizon on distant hybridization of wheat and accelerates the utilization of genes of P. huashanica.

Humic acid chelated selenium is suitable for wheat biofortification.

BACKGROUND: Selenium rich bread is a good carrier of selenium, but the inorganic selenium used in the actual production process is toxic. It is necessary to develop a new green bread production technology. The extraction and utilization of humic acid chelated selenium from selenium-rich soil is beneficial for reducing resource waste and pollution without destroying the soil ecosystem in selenium-deficient areas. Sodium selenite and nanoselenium were selected as controls because they are commonly used as selenium agronomic enhancers in production. RESULTS: Humic acid chelated selenium can be absorbed and accumulated by wheat leaves, and humic acid chelated selenium had no significant effect on wheat yield, which was also shown in the treatments with nanoselenium and sodium selenite. Excessive accumulation of selenium in wheat grains can lead to a deterioration of processing quality. Among them, the use of excessive nanoselenium at the filling stage inhibited the accumulation of wheat grain protein, whereas humic acid chelated selenium is beneficial to grain protein accumulation and has the least negative effect on the processing quality. The accumulation of excessive selenium in wheat seeds had a negative effect on seed germination and growth; specifically, the seed vigor of wheat treated with humic acid chelated selenium was higher than that of untreated wheat. CONCLUSION: Humic acid chelated selenium is particularly suitable for the whole process of Se-enriched bread wheat production. The seed vigour of wheat treated with humic acid chelated selenium, which supplied a moderate amount of selenium, was higher than that of untreated wheat. Conversely, the accumulation of excessive selenium in wheat seeds reduced germination and seedling growth. © 2023 Society of Chemical Industry.

Genetic analysis of resistance to powdery mildew on 7Mg chromosome of wheat-Aegilops geniculata, development and utilization of specific molecular markers.

BACKGROUND: Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is prevalent in the main wheat-producing regions of China, resulting in severe yield losses in recent years. Mining and utilization of resistant genes from wild relatives of wheat is the most environmentally sound measure to control disease. Aegilops geniculata Roth (2n = 2x = 28, UgUgMgMg) is an essential and valuable disease-resistance gene donor for wheat improvement as a close relative species. RESULTS: In this study, to validate powdery mildew resistance locus on chromosome 7Mg, two genetic populations were constructed and through crossing wheat - Ae. geniculata 7Mg disomic addition line NA0973-5-4-1-2-9-1 and 7Mg (7 A) alien disomic substitution line W16998 with susceptible Yuanfeng175 (YF175, authorized varieties from Shaanxi province in 2005), respectively. Cytological examination, in situ hybridization (ISH), and functional molecular markers analysis revealed that the plants carrying chromosome 7Mg showed high resistance to powdery mildew in both F1 and F2 generation at the seedling stage. Besides, 84 specific markers were developed to identify the plants carrying chromosome 7Mg resistance based on the specific-locus amplified fragment sequencing (SLAF-seq) technique. Among them, four markers were selected randomly to check the reliability in F2 segregating populations derived from YF175/NA0973-5-4-1-2-9-1 and YF175/W16998. In summary, the above analysis confirmed that a dominant high powdery mildew resistance gene was located on chromosome 7Mg of Ae. geniculata. CONCLUSION: The results provide a basis for mapping the powdery mildew resistance gene mapping on chromosome 7Mg and specific markers for their utilization in the future.

Molecular cytogenetics and development of St-chromosome-specific molecular markers of novel stripe rust resistant wheat-Thinopyrum intermedium and wheat-Thinopyrum ponticum substitution lines.

BACKGROUND: Owing to their excellent resistance to abiotic and biotic stress, Thinopyrum intermedium (2n = 6x = 42, JJJsJsStSt) and Th. ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs. Disomic substitution lines (DSLs) carrying one pair of alien chromosomes are valuable bridge materials for transmission of novel genes, fluorescence in situ hybridization (FISH) karyotype construction and specific molecular marker development. RESULTS: Six wheat-Thinopyrum DSLs derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), were characterized by sequential FISH-genome in situ hybridization (GISH), multicolor GISH (mc-GISH), and an analysis of the wheat 15 K SNP array combined with molecular marker selection. ES-9 (DS2St (2A)) and ES-10 (DS3St (3D)) are wheat-Th. ponticum DSLs, while ES-23 (DS2St (2A)), ES-24 (DS3St (3D)), ES-25(DS2St (2B)), and ES-26 (DS2St (2D)) are wheat-Th. intermedium DSLs. ES-9, ES-23, ES-25 and ES-26 conferred high thousand-kernel weight and stripe rust resistance at adult stages, while ES-10 and ES-24 were highly resistant to stripe rust at all stages. Furthermore, cytological analysis showed that the alien chromosomes belonging to the same homoeologous group (2 or 3) derived from different donors carried the same FISH karyotype and could form a bivalent. Based on specific-locus amplified fragment sequencing (SLAF-seq), two 2St-chromosome-specific markers (PTH-005 and PTH-013) and two 3St-chromosome-specific markers (PTH-113 and PTH-135) were developed. CONCLUSIONS: The six wheat-Thinopyrum DSLs conferring stripe rust resistance can be used as bridging parents for transmission of valuable resistance genes. The utility of PTH-113 and PTH-135 in a BC1F2 population showed that the newly developed markers could be useful tools for efficient identification of St chromosomes in a common wheat background.

A truncated CC-NB-ARC gene TaRPP13L1-3D positively regulates powdery mildew resistance in wheat via the RanGAP-WPP complex-mediated nucleocytoplasmic shuttle.

MAIN CONCLUSION: A wheat RPP13-like isoform interacting with WPP1 contributes to quantitative and/or basal resistance to powdery mildew (Blumeria graminis f. sp. tritici) by restricting the development of Bgt conidia. Plant disease resistance (R) genes confer an ability to resist infection by pathogens expressing specific avirulence genes. Recognition of Peronospora parasitica 13-like (RPP13-like) genes belong to the nucleotide-binding site and leucine-rich repeat (NBS-LRR) superfamily and play important roles in resistance to various plant diseases. Previously, we detected a TaRPP13-like gene located on chromosome 3D (TaRPP13L1-3D) in the TaSpl1 resided region, which is strongly induced by the cell death phenotype (Zhang et al. 2021). Here, we investigated the expression and functional role of TaRPP13L1-3D in wheat responding to fungal stress. TaRPP13L1-3D encoded a typical NB-ARC structure characterized by Rx-N and P-loop NTPase domains. TaRPP13L1-3D transcripts were strongly upregulated in wheat by powdery mildew (Blumeria graminis f. sp. tritici; Bgt) and stripe rust (Puccinia striiformis f. sp. tritici; Pst) infection although opposing expression patterns were observed in response to wheat-Bgt in incompatible and compatible backgrounds. Overexpression of TaRPP13L1-3D enhanced disease resistance to Bgt, accompanied by upregulation of the defense-related marker genes encoding phytoalexin-deficient4 (PAD4), thaumatin-like protein (TLP) and chitinase 8-like protein (Chi8L), while silencing of TaRPP13L1-3D disrupted the resistance to Bgt infection. Subcellular localization studies showed that TaRPP13L1-3D is located in both the plasma membrane and nucleus, while yeast-two-hybrid (Y2H) assays indicated that TaRPP13L1-3D interacts with WPP domain-containing protein 1 (TaWPP1). This indicates that TaRPP13L1-3D shuttles between the nucleus and cytoplasm membrane via a mechanism that is mediated by the RanGAP-WPP complex in nuclear pores. This insight into TaRPP13L1-3D will be useful in dissecting the mechanism of fungal resistance in wheat, and understanding the interaction between R gene expression and pathogen defense.

An alternative splicing isoform of wheat TaYRG1 resistance protein activates immunity by interacting with dynamin-related proteins.

Wheat (Triticum aestivum) is a commercially important crop and its production is seriously threatened by the fungal pathogen Puccinia striiformis f. sp. tritici West (Pst). Resistance (R) genes are critical factors that facilitate plant immune responses. Here, we report a wheat R gene NB-ARC-LRR ortholog, TaYRG1, that is associated with distinct alternative splicing events in wheat infected by Pst. The native splice variant, TaYRG1.6, encodes internal-motif-deleted polypeptides with the same N- and C-termini as TaYRG1.1, resulting in gain of function. Transient expression of protein variants in Nicotiana benthamiana showed that the NB and ARC domains, and TaYRG1.6 (half LRR domain), stimulate robust elicitor-independent cell death based on a signal peptide, although the activity was negatively modulated by the CC and complete LRR domains. Furthermore, molecular genetic analyses indicated that TaYRG1.6 enhanced resistance to Pst in wheat. Moreover, we provide multiple lines of evidence that TaYRG1.6 interacts with a dynamin-related protein, TaDrp1. Proteome profiling suggested that the TaYRG1.6-TaDrp1-DNM complex in the membrane trafficking systems may trigger cell death by mobilizing lipid and kinase signaling in the endocytosis pathway. Our findings reveal a unique mechanism by which TaYRG1 activates cell death and enhances disease resistance by reconfiguring protein structure through alternative splicing.

Fine mapping and distribution analysis of hybrid necrosis genes Ne1 and Ne2 in wheat in China.

KEY MESSAGE: Flanking markers useful for identifying hybrid necrosis alleles were identified by fine mapping Ne1 and Ne2 and the distribution of the two necrosis genes was investigated in Chinese elite wheat varieties. Hybrid necrosis of wheat is caused by the interaction of two dominant complementary genes Ne1 and Ne2 present separately in normal parents and is regarded as a barrier to gene transfer in wheat breeding. However, the necrosis alleles still occur at a high frequency in modern wheat varieties. In this study, we constructed two high-density genetic maps of Ne1 and Ne2 in winter wheat. In these cultivars, Ne1 was found to be located in a span interval of 0.50 centimorgan (cM) on chromosome 5BL delimited by markers Nwu_5B_4137 and Nwu_5B_5114, while Ne2 co-segregated with markers Lseq102 and TC67744 on 2BS. Statistical analysis confirmed that the dosage effect of Ne1 and Ne2 also existed in moderate and severe hybrid necrosis systems, and the symptoms of necrosis can also be affected by the genetic background. Furthermore, we clarified the discrete distribution and proportion of the Ne1 and Ne2 in the 10 China's agro-ecological production zones. We concluded that 26.2% and 33.2% of the 1364 cultivars (lines) were genotyped with Ne1Ne1ne2ne2 and ne1ne1Ne2Ne2, respectively and introduced modern cultivars should directly affect the frequencies of necrosis genes in modern Chinese cultivars (lines), especially that of Ne2. Taking investigations in spring wheat together, we proposed that hybrid necrosis alleles could positively affect breeding owing to their linked excellent genes such as Lr13. Additionally, based on the pedigrees and hybridization tests, we speculated that the Ne1 and Ne2 in winter wheat may directly originate from wild emmer and introduced cultivars or hexaploid triticale, respectively.

Development and Molecular Cytogenetic Identification of Two Wheat-Aegilops geniculata Roth 7Mg Chromosome Substitution Lines with Resistance to Fusarium Head Blight, Powdery Mildew and Stripe Rust.

Fusarium head blight (Fhb), powdery mildew, and stripe rust are major wheat diseases globally. Aegilops geniculata Roth (UgUgMgMg, 2n = 4x = 28), a wild relative of common wheat, is valuable germplasm of disease resistance for wheat improvement and breeding. Here, we report the development and characterization of two substitution accessions with high resistance to powdery mildew, stripe rust and Fhb (W623 and W637) derived from hybrid progenies between Ae. geniculata and hexaploid wheat Chinese Spring (CS). Fluorescence in situ hybridization (FISH), Genomic in situ hybridizations (GISH), and sequential FISH-GISH studies indicated that the two substitution lines possess 40 wheat chromosomes and 2 Ae. geniculata chromosomes. Furthermore, compared that the wheat addition line parent W166, the 2 alien chromosomes from W623 and W637 belong to the 7Mg chromosomes of Ae. geniculata via sequential FISH-GISH and molecular marker analysis. Nullisomic-tetrasomic analysis for homoeologous group-7 of wheat and FISH revealed that the common wheat chromosomes 7A and 7B were replaced in W623 and W637, respectively. Consequently, lines W623, in which wheat chromosomes 7A were replaced by a pair of Ae. geniculata 7Mg chromosomes, and W637, which chromosomes 7B were substituted by chromosomes 7Mg, with resistance to Fhb, powdery mildew, and stripe rust. This study has determined that the chromosome 7Mg from Ae. geniculata exists genes resistant to Fhb and powdery mildew.