A k-mer-based pangenome approach for cataloging seed-storage-protein genes in wheat to facilitate genotype-to-phenotype prediction and improvement of end-use quality.

Wheat is a staple food for more than 35% of the world's population, with wheat flour used to make hundreds of baked goods. Superior end-use quality is a major breeding target; however, improving it is especially time-consuming and expensive. Furthermore, genes encoding seed-storage proteins (SSPs) form multi-gene families and are repetitive, with gaps commonplace in several genome assemblies. To overcome these barriers and efficiently identify superior wheat SSP alleles, we developed "PanSK" (Pan-SSP k-mer) for genotype-to-phenotype prediction based on an SSP-based pangenome resource. PanSK uses 29-mer sequences that represent each SSP gene at the pangenomic level to reveal untapped diversity across landraces and modern cultivars. Genome-wide association studies with k-mers identified 23 SSP genes associated with end-use quality that represent novel targets for improvement. We evaluated the effect of rye secalin genes on end-use quality and found that removal of ω-secalins from 1BL/1RS wheat translocation lines is associated with enhanced end-use quality. Finally, using machine-learning-based prediction inspired by PanSK, we predicted the quality phenotypes with high accuracy from genotypes alone. This study provides an effective approach for genome design based on SSP genes, enabling the breeding of wheat varieties with superior processing capabilities and improved end-use quality.

Fine-mapping and validation of the major quantitative trait locus QFlANG-4B for flag leaf angle in wheat.

KEY MESSAGE: This study precisely mapped and validated a quantitative trait locus (QTL) located on chromosome 4B for flag leaf angle in wheat. Flag leaf angle (FLANG) is closely related to crop architecture and yield. We previously identified the quantitative trait locus (QTL) QFLANG-4B for FLANG on chromosome 4B, located within a 14-cM interval flanked by the markers Xbarc20 and Xzyh357, using a mapping population of recombinant inbred lines (RILs) derived from a cross between Nongda3331 (ND3331) and Zang1817. In this study, we fine-mapped QFLANG-4B and validated its associated genetic effect. We developed a BC3F3 population using ND3331 as the recurrent parent through marker-assisted selection, as well as near-isogenic lines (NILs) by selfing BC3F3 plants carrying different heterozygous segments for the QFLANG-4B region. We obtained eight recombinant types for QFLANG-4B, narrowing its location down to a 5.3-Mb region. This region contained 76 predicted genes, 7 of which we considered to be likely candidate genes for QFLANG-4B. Marker and phenotypic analyses of individual plants from the secondary mapping populations and their progeny revealed that the FLANG of the ND3331 allele is significantly higher than that of the Zang1817 allele in multiple environments. These results not only provide a basis for the map-based cloning of QFLANG-4B, but also indicate that QFLANG-4B has great potential for marker-assisted selection in wheat breeding programs designed to improve plant architecture and yield.

The potassium transporter TaNHX2 interacts with TaGAD1 to promote drought tolerance via modulating stomatal aperture in wheat.

Drought is a major global challenge in agriculture that decreases crop production. γ-Aminobutyric acid (GABA) interfaces with drought stress in plants; however, a mechanistic understanding of the interaction between GABA accumulation and drought response remains to be established. Here we showed the potassium/proton exchanger TaNHX2 functions as a positive regulator in drought resistance in wheat by mediating cross-talk between the stomatal aperture and GABA accumulation. TaNHX2 interacted with glutamate decarboxylase TaGAD1, a key enzyme that synthesizes GABA from glutamate. Furthermore, TaNHX2 targeted the C-terminal auto-inhibitory domain of TaGAD1, enhanced its activity, and promoted GABA accumulation under drought stress. Consistent with this, the tanhx2 and tagad1 mutants showed reduced drought tolerance, and transgenic wheat with enhanced TaNHX2 expression had a yield advantage under water deficit without growth penalty. These results shed light on the plant stomatal movement mechanism under drought stress and the TaNHX2-TaGAD1 module may be harnessed for amelioration of negative environmental effects in wheat as well as other crops.

Positional cloning and characterization reveal the role of TaSRN-3D and TaBSR1 in the regulation of seminal root number in wheat.

Seminal roots play a critical role in water and nutrient absorption, particularly in the early developmental stages of wheat. However, the genes responsible for controlling SRN in wheat remain largely unknown. Genetic mapping and functional analyses identified a candidate gene (TraesCS3D01G137200, TaSRN-3D) encoding a Ser/Thr kinase glycogen synthase kinase 3 (STKc_GSK3) that regulated SRN in wheat. Additionally, experiments involving hormone treatment, nitrate absorption and protein interaction were conducted to explore the regulatory mechanism of TaSRN-3D. Results showed that the TaSRN-3D4332 allele inhibited seminal roots initiation and development, while loss-of-function mutants showed significantly higher seminal root number (SRN). Exogenous application of epi-brassinolide could increase the SRN in a HS2-allelic background. Furthermore, chlorate sensitivity and 15N uptake assays revealed that a higher number of seminal roots promoted nitrate accumulation. TaBSR1 (BIN2-related SRN Regulator 1, orthologous to OsGRF4/GL2 in rice) acts as an interactor of TaSRN-3D and promotes TaBSR1 degradation to reduce SRN. This study provides valuable insights into understanding the genetic basis and regulatory network of SRN in wheat, highlighting their roles as potential targets for root-based improvement in wheat breeding.

Innovative computational tools provide new insights into the polyploid wheat genome.

Bread wheat (Triticum aestivum) is an important crop and serves as a significant source of protein and calories for humans, worldwide. Nevertheless, its large and allopolyploid genome poses constraints on genetic improvement. The complex reticulate evolutionary history and the intricacy of genomic resources make the deciphering of the functional genome considerably more challenging. Recently, we have developed a comprehensive list of versatile computational tools with the integration of statistical models for dissecting the polyploid wheat genome. Here, we summarize the methodological innovations and applications of these tools and databases. A series of step-by-step examples illustrates how these tools can be utilized for dissecting wheat germplasm resources and unveiling functional genes associated with important agronomic traits. Furthermore, we outline future perspectives on new advanced tools and databases, taking into consideration the unique features of bread wheat, to accelerate genomic-assisted wheat breeding.

Genomic insights into the origin and evolution of spelt (Triticum spelta L.) as a valuable gene pool for modern wheat breeding.

Spelt (Triticum aestivum ssp. spelta) is an important wheat subspecies mainly cultivated in Europe before the 20th century that has contributed to modern wheat breeding as a valuable genetic resource. However, relatively little is known about the origins and maintenance of spelt populations. Here, using resequencing data from 416 worldwide wheat accessions, including representative spelt wheat, we demonstrate that European spelt emerged when primitive hexaploid wheat spread to the west and hybridized with pre-settled domesticated emmer, the putative maternal donor. Genomic introgression regions from domesticated emmer confer spelt's primitive morphological characters used for species taxonomy, such as tenacious glumes and later flowering. We propose a haplotype-based "spelt index" to identify spelt-type wheat varieties and to quantify utilization of the spelt gene pool in modern wheat cultivars. This study reveals the genetic basis for the establishment of the spelt wheat subspecies in a specific ecological niche and the vital role of the spelt gene pool as a unique germplasm resource in modern wheat breeding.

Natural variation of STKc_GSK3 kinase TaSG-D1 contributes to heat stress tolerance in Indian dwarf wheat.

Heat stress threatens global wheat (Triticum aestivum) production, causing dramatic yield losses worldwide. Identifying heat tolerance genes and comprehending molecular mechanisms are essential. Here, we identify a heat tolerance gene, TaSG-D1E286K, in Indian dwarf wheat (Triticum sphaerococcum), which encodes an STKc_GSK3 kinase. TaSG-D1E286K improves heat tolerance compared to TaSG-D1 by enhancing phosphorylation and stability of downstream target TaPIF4 under heat stress condition. Additionally, we reveal evolutionary footprints of TaPIF4 during wheat selective breeding in China, that is, InDels predominantly occur in the TaPIF4 promoter of Chinese modern wheat cultivars and result in decreased expression level of TaPIF4 in response to heat stress. These sequence variations with negative effect on heat tolerance are mainly introduced from European germplasm. Our study provides insight into heat stress response mechanisms and proposes a potential strategy to improve wheat heat tolerance in future.

Methyltransferase TaSAMT1 mediates wheat freezing tolerance by integrating brassinosteroid and salicylic acid signaling.

Cold injury is a major environmental stress affecting the growth and yield of crops. Brassinosteroids (BRs) and salicylic acid (SA) play important roles in plant cold tolerance. However, whether or how BR signaling interacts with the SA signaling pathway in response to cold stress is still unknown. Here, we identified an SA methyltransferase, TaSAMT1, that converts SA to methyl SA (MeSA) and confers freezing tolerance in wheat (Triticum aestivum). TaSAMT1 overexpression greatly enhanced wheat freezing tolerance, with plants accumulating more MeSA and less SA, whereas Tasamt1 knockout lines were sensitive to freezing stress and accumulated less MeSA and more SA. Spraying plants with MeSA conferred freezing tolerance to Tasamt1 mutants, but SA did not. We revealed that BRASSINAZOLE-RESISTANT 1 (TaBZR1) directly binds to the TaSAMT1 promoter and induces its transcription. Moreover, TaBZR1 interacts with the histone acetyltransferase TaHAG1, which potentiates TaSAMT1 expression via increased histone acetylation and modulates the SA pathway during freezing stress. Additionally, overexpression of TaBZR1 or TaHAG1 altered TaSAMT1 expression and improved freezing tolerance. Our results demonstrate a key regulatory node that connects the BR and SA pathways in the plant cold stress response. The regulatory factors or genes identified could be effective targets for the genetic improvement of freezing tolerance in crops.

Reshaped DNA methylation cooperating with homoeolog-divergent expression promotes improved root traits in synthesized tetraploid wheat.

Polyploidization is a major event driving plant evolution and domestication. However, how reshaped epigenetic modifications coordinate gene transcription to generate phenotypic variations during wheat polyploidization is currently elusive. Here, we profiled transcriptomes and DNA methylomes of two diploid wheat accessions (SlSl and AA) and their synthetic allotetraploid wheat line (SlSlAA), which displayed elongated root hair and improved root capability for nitrate uptake and assimilation after tetraploidization. Globally decreased DNA methylation levels with a reduced difference between subgenomes were observed in the roots of SlSlAA. DNA methylation changes in first exon showed strong connections with altered transcription during tetraploidization. Homoeolog-specific transcription was associated with biased DNA methylation as shaped by homoeologous sequence variation. The hypomethylated promoters showed significantly enriched binding sites for MYB, which may affect gene transcription in response to root hair growth. Two master regulators in root hair elongation pathway, AlCPC and TuRSL4, exhibited upregulated transcription levels accompanied by hypomethylation in promoter, which may contribute to the elongated root hair. The upregulated nitrate transporter genes, including NPFs and NRTs, also are significantly associated with hypomethylation, indicating an epigenetic-incorporated regulation manner in improving nitrogen use efficiency. Collectively, these results provided new insights into epigenetic changes in response to crop polyploidization and underscored the importance of epigenetic regulation in improving crop traits.

Type I MADS-box transcription factor TaMADS-GS regulates grain size by stabilizing cytokinin signalling during endosperm cellularization in wheat.

Grain size is one of the important traits in wheat breeding programs aimed at improving yield, and cytokinins, mainly involved in cell division, have a positive impact on grain size. Here, we identified a novel wheat gene TaMADS-GS encoding type I MADS-box transcription factor, which regulates the cytokinins signalling pathway during early stages of grain development to modulate grain size and weight in wheat. TaMADS-GS is exclusively expressed in grains at early stage of seed development and its knockout leads to delayed endosperm cellularization, smaller grain size and lower grain weight. TaMADS-GS protein interacts with the Polycomb Repressive Complex 2 (PRC2) and leads to repression of genes encoding cytokinin oxidase/dehydrogenases (CKXs) stimulating cytokinins inactivation by mediating accumulation of the histone H3 trimethylation at lysine 27 (H3K27me3). Through the screening of a large wheat germplasm collection, an elite allele of the TaMADS-GS exhibits higher ability to repress expression of genes inactivating cytokinins and a positive correlation with grain size and weight, thus representing a novel marker for breeding programs in wheat. Overall, these findings support the relevance of TaMADS-GS as a key regulator of wheat grain size and weight.

Mutation of a highly conserved amino acid in RPM1 causes leaf yellowing and premature senescence in wheat.

KEY MESSAGE: A point mutation of RPM1 triggers persistent immune response that induces leaf premature senescence in wheat, providing novel information of immune responses and leaf senescence. Leaf premature senescence in wheat (Triticum aestivum L.) is one of the most common factors affecting the plant's development and yield. In this study, we identified a novel wheat mutant, yellow leaf and premature senescence (ylp), which exhibits yellow leaves and premature senescence at the heading and flowering stages. Consistent with the yellow leaves phenotype, ylp had damaged and collapsed chloroplasts. Map-based cloning revealed that the phenotype of ylp was caused by a point mutation from Arg to His at amino acid 790 in a plasma membrane-localized protein resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). The point mutation triggered excessive immune responses and the upregulation of senescence- and autophagy-associated genes. This work provided the information for understanding the molecular regulatory mechanism of leaf senescence, and the results would be important to analyze which mutations of RPM1 could enable plants to obtain immune activation without negative effects on plant growth.

Paternally imprinted LATE-FLOWERING2 transcription factor contributes to paternal-excess interploidy hybridization barriers in wheat.

Interploidy hybridization between hexaploid and tetraploid genotypes occurred repeatedly during genomic introgression events throughout wheat evolution, and is commonly employed in wheat breeding programs. Hexaploid wheat usually serves as maternal parent because the reciprocal cross generates progeny with severe defects and poor seed germination, but the underlying mechanism is poorly understood. Here, we performed detailed analysis of phenotypic variation in endosperm between two interploidy reciprocal crosses arising from tetraploid (Triticum durum, AABB) and hexaploid wheat (Triticum aestivum, AABBDD). In the paternal- versus the maternal-excess cross, the timing of endosperm cellularization was delayed and starch granule accumulation in the endosperm was repressed, causing reduced germination percentage. The expression profiles of genes involved in nutrient metabolism differed strongly between these endosperm types. Furthermore, expression patterns of parental alleles were dramatically disturbed in interploidy versus intraploidy crosses, leading to increased number of imprinted genes. The endosperm-specific TaLFL2 showed a paternally imprinted expression pattern in interploidy crosses partially due to allele-specific DNA methylation. Paternal TaLFL2 binds to and represses a nutrient accumulation regulator TaNAC019, leading to reduced storage protein and starch accumulation during endosperm development in paternal-excess cross, as confirmed by interploidy crosses between tetraploid wild-type and clustered regularly interspaced palindromic repeats (CRISPR) - CRISPR-associated protein 9 generated hexaploid mutants. These findings reveal a contribution of genomic imprinting to paternal-excess interploidy hybridization barriers during wheat evolution history and explains why experienced breeders preferentially exploit maternal-excess interploidy crosses in wheat breeding programs.

Deciphering the evolution and complexity of wheat germplasm from a genomic perspective.

Bread wheat provides an essential fraction of the daily calorific intake for humanity. Due to its huge and complex genome, progress in studying on the wheat genome is substantially trailed behind those of the other two major crops, rice and maize, for at least a decade. With rapid advances in genome assembling and reduced cost of high-throughput sequencing, emerging de novo genome assemblies of wheat and whole-genome sequencing data are leading to a paradigm shift in wheat research. Here, we review recent progress in dissecting the complex genome and germplasm evolution of wheat since the release of the first high-quality wheat genome. New insights have been gained in the evolution of wheat germplasm during domestication and modern breeding progress, genomic variations at multiple scales contributing to the diversity of wheat germplasm, and complex transcriptional and epigenetic regulations of functional genes in polyploid wheat. Genomics databases and bioinformatics tools meeting the urgent needs of wheat genomics research are also summarized. The ever-increasing omics data, along with advanced tools and well-structured databases, are expected to accelerate deciphering the germplasm and gene resources in wheat for future breeding advances.

Thermosensitive SUMOylation of TaHsfA1 defines a dynamic ON/OFF molecular switch for the heat stress response in wheat.

Dissecting genetic components in crop plants associated with heat stress (HS) sensing and adaptation will facilitate the design of modern crop varieties with improved thermotolerance. However, the molecular mechanisms underlying the ON/OFF switch controlling HS responses (HSRs) in wheat (Triticum aestivum) remain largely unknown. In this study, we focused on the molecular action of TaHsfA1, a class A heat shock transcription factor, in sensing dynamically changing HS signals and regulating HSRs. We show that the TaHsfA1 protein is modified by small ubiquitin-related modifier (SUMO) and that this modification is essential for the full transcriptional activation activity of TaHsfA1 in triggering downstream gene expression. During sustained heat exposure, the SUMOylation of TaHsfA1 is suppressed, which partially reduces TaHsfA1 protein activity, thereby reducing the intensity of downstream HSRs. In addition, we demonstrate that TaHsfA1 interacts with the histone acetyltransferase TaHAG1 in a thermosensitive manner. Together, our findings emphasize the importance of TaHsfA1 in thermotolerance in wheat. In addition, they define a highly dynamic SUMOylation-dependent "ON/OFF" molecular switch that senses temperature signals and contributes to thermotolerance in crops.

Alternative splicing of TaHSFA6e modulates heat shock protein-mediated translational regulation in response to heat stress in wheat.

Heat stress greatly threatens crop production. Plants have evolved multiple adaptive mechanisms, including alternative splicing, that allow them to withstand this stress. However, how alternative splicing contributes to heat stress responses in wheat (Triticum aestivum) is unclear. We reveal that the heat shock transcription factor gene TaHSFA6e is alternatively spliced in response to heat stress. TaHSFA6e generates two major functional transcripts: TaHSFA6e-II and TaHSFA6e-III. TaHSFA6e-III enhances the transcriptional activity of three downstream heat shock protein 70 (TaHSP70) genes to a greater extent than does TaHSFA6e-II. Further investigation reveals that the enhanced transcriptional activity of TaHSFA6e-III is due to a 14-amino acid peptide at its C-terminus, which arises from alternative splicing and is predicted to form an amphipathic helix. Results show that knockout of TaHSFA6e or TaHSP70s increases heat sensitivity in wheat. Moreover, TaHSP70s are localized in stress granule following exposure to heat stress and are involved in regulating stress granule disassembly and translation re-initiation upon stress relief. Polysome profiling analysis confirms that the translational efficiency of stress granule stored mRNAs is lower at the recovery stage in Tahsp70s mutants than in the wild types. Our finding provides insight into the molecular mechanisms by which alternative splicing improves the thermotolerance in wheat.

Pangenome-based trajectories of intracellular gene transfers in Poaceae unveil high cumulation in Triticeae.

Intracellular gene transfers (IGTs) between the nucleus and organelles, including plastids and mitochondria, constantly reshape the nuclear genome during evolution. Despite the substantial contribution of IGTs to genome variation, the dynamic trajectories of IGTs at the pangenomic level remain elusive. Here, we developed an approach, IGTminer, that maps the evolutionary trajectories of IGTs using collinearity and gene reannotation across multiple genome assemblies. We applied IGTminer to create a nuclear organellar gene (NOG) map across 67 genomes covering 15 Poaceae species, including important crops. The resulting NOGs were verified by experiments and sequencing data sets. Our analysis revealed that most NOGs were recently transferred and lineage specific and that Triticeae species tended to have more NOGs than other Poaceae species. Wheat (Triticum aestivum) had a higher retention rate of NOGs than maize (Zea mays) and rice (Oryza sativa), and the retained NOGs were likely involved in photosynthesis and translation pathways. Large numbers of NOG clusters were aggregated in hexaploid wheat during 2 rounds of polyploidization, contributing to the genetic diversity among modern wheat accessions. We implemented an interactive web server to facilitate the exploration of NOGs in Poaceae. In summary, this study provides resources and insights into the roles of IGTs in shaping interspecies and intraspecies genome variation and driving plant genome evolution.

Reducing brassinosteroid signalling enhances grain yield in semi-dwarf wheat.

Modern green revolution varieties of wheat (Triticum aestivum L.) confer semi-dwarf and lodging-resistant plant architecture owing to the Reduced height-B1b (Rht-B1b) and Rht-D1b alleles1. However, both Rht-B1b and Rht-D1b are gain-of-function mutant alleles encoding gibberellin signalling repressors that stably repress plant growth and negatively affect nitrogen-use efficiency and grain filling2-5. Therefore, the green revolution varieties of wheat harbouring Rht-B1b or Rht-D1b usually produce smaller grain and require higher nitrogen fertilizer inputs to maintain their grain yields. Here we describe a strategy to design semi-dwarf wheat varieties without the need for Rht-B1b or Rht-D1b alleles. We discovered that absence of Rht-B1 and ZnF-B (encoding a RING-type E3 ligase) through a natural deletion of a haploblock of about 500 kilobases shaped semi-dwarf plants with more compact plant architecture and substantially improved grain yield (up to 15.2%) in field trials. Further genetic analysis confirmed that the deletion of ZnF-B induced the semi-dwarf trait in the absence of the Rht-B1b and Rht-D1b alleles through attenuating brassinosteroid (BR) perception. ZnF acts as a BR signalling activator to facilitate proteasomal destruction of the BR signalling repressor BRI1 kinase inhibitor 1 (TaBKI1), and loss of ZnF stabilizes TaBKI1 to block BR signalling transduction. Our findings not only identified a pivotal BR signalling modulator but also provided a creative strategy to design high-yield semi-dwarf wheat varieties by manipulating the BR signal pathway to sustain wheat production.

The translational landscape of bread wheat during grain development.

The dynamics of gene expression in crop grains has typically been investigated at the transcriptional level. However, this approach neglects translational regulation, a widespread mechanism that rapidly modulates gene expression to increase the plasticity of organisms. Here, we performed ribosome profiling and polysome profiling to obtain a comprehensive translatome data set of developing bread wheat (Triticum aestivum) grains. We further investigated the genome-wide translational dynamics during grain development, revealing that the translation of many functional genes is modulated in a stage-specific manner. The unbalanced translation between subgenomes is pervasive, which increases the expression flexibility of allohexaploid wheat. In addition, we uncovered widespread previously unannotated translation events, including upstream open reading frames (uORFs), downstream open reading frames (dORFs), and open reading frames (ORFs) in long noncoding RNAs, and characterized the temporal expression dynamics of small ORFs. We demonstrated that uORFs act as cis-regulatory elements that can repress or even enhance the translation of mRNAs. Gene translation may be combinatorially modulated by uORFs, dORFs, and microRNAs. In summary, our study presents a translatomic resource that provides a comprehensive and detailed overview of the translational regulation in developing bread wheat grains. This resource will facilitate future crop improvements for optimal yield and quality.