Lesion mimic mutants (LMMs) are ideal for dissecting plant immunity mechanisms. Here, we characterized lm10373, a stable LMM isolated from an EMS-induced library of wheat cultivar AK58. lm10373 developed light-dependent lesion spots on leaves from the late tillering stage, accompanied by programmed cell death (PCD) and reactive oxygen species (ROS) accumulation. Phenotypically, lm10373 showed reduced photosynthetic capacity and yield-related traits, but enhanced powdery mildew resistance at the heading stage. Genetic analysis revealed the lesion trait was controlled by a single semi-dominant nuclear gene, mapped to a 35 Mb interval on chromosome 3B via bulked segregant analysis coupled with exome capture sequencing (BSE-seq). Integrating exome sequencing and transcriptome data, we identified TaWSD1-3B (encoding an O-acyltransferase of the WSD1 family) as the causal gene. A G-to-A mutation (p.Ala79Thr) in its conserved acyltransferase domain introduced a new phosphorylation site, disrupting triacylglycerol biosynthesis. Two independent mutants (lm129, p.Arg207His; lm295, 3'UTR mutation) validated TaWSD1-3B function. Haplotype analysis of 183 wheat accessions identified three TaWSD1-3B haplotypes: Hap1 was associated with higher 1000-grain weight and lower leaf tip necrosis (LTN) severity, making it a favorable allele for breeding. This study characterized a wheat LMM mutant and candidate gene TaWSD1-3B, suggesting a lipid-ROS-PCD pathway regulating immunity, and provides valuable markers for stress-tolerant, high-yield wheat breeding.

Seed germination is a critical initial stage of the plant life cycle, regulated by signaling pathways such as phytohormones and reactive oxygen species (ROS). However, the low germination rate of immature grains is a key bottleneck limiting wheat speed breeding. This study used immature grains of the winter wheat cultivar Kenong 199 (KN199) collected 18 days post anthesis to establish an efficient germination protocol. By screening individual and combined treatments of hydrogen peroxide (H2O2, 1%), gibberellin (GA3, 20 μM), and varying concentrations of abscisic acid (ABA) synthesis inhibitor sodium tungstate (Na2WO4), alongside transcriptome analysis, we identified the optimal reagent combination and gained preliminary insight into its molecular basis. The triple reagent combination of 0.5 mM Na2WO4 + 20 μM GA3 + 1% H2O2 exhibited the highest germination rate of 80%, approximately sevenfold higher than single reagent treatments, with germination rate peaking after 4 days. Transcriptome profiling revealed that this combination modulated the expression of key genes related to dormancy release and germination, including upregulation of GA biosynthesis gene GA3ox2 and ABA catabolism gene TaCYP707A2, and downregulation of ABA biosynthesis and signaling genes (ABI5, TaNCED1, etc.). Additionally, genes associated with energy metabolism and transport pathways were enhanced. This optimized reagent combination significantly improves immature grain germination, shortens the breeding cycle, and provides a practical tool for achieving “five generations per year” speed breeding in winter wheat. Our findings contribute to seed biology by offering a chemical strategy to overcome dormancy in immature cereal grains.

Alternative splicing (AS) is a crucial post-transcriptional regulatory mechanism that enhances transcript and proteome diversity. However, AS in common wheat (Triticum aestivum) remains understudied due to the large and complex genome of this crop. Full-length transcriptome sequencing, which provides long, high-quality reads, offers a powerful tool for analyzing AS in wheat. In this study, we used the PacBio Sequel platform to sequence full-length transcripts from 5 wheat tissues (root, stem, leaf, spike, and grain) of the cultivar Aikang58 (AK58). We identified 560,631 isoforms from 86,073 genes, with 76.7% of genes producing multiple isoforms and 45.34% undergoing AS events (ASEs). Tissue-specific analysis revealed differences in the number and function of AS genes (ASGs), underscoring the potential role of AS in tissue differentiation. A comparison across the 3 wheat subgenomes showed similar numbers of ASGs and ASEs but distinct functional patterns, suggesting that AS is involved in subgenomic divergence. We also examined AS in genes linked to key agronomic traits, demonstrating association with trait regulation. These findings enhance our understanding of the adaptability and post-transcriptional gene regulation in wheat, offering insights for future research and breeding efforts.

Leaf width is an important component of plant architecture that strongly affects light capture during photosynthesis and thus grain yield, particularly under dense planting conditions. However, the genetic and molecular mechanisms regulating leaf width in wheat (Triticum aestivum L.) remain unclear. Here, we identified the narrow-leaf mutant nl1 with fewer small veins than the wild-type and isolated the narrow-leaf gene Narrow Leaf 1 (NL1) through a combination of map-based cloning and bulked segregant exome capture sequencing (BSE-seq). NL1 encodes CELL DIVISION CYCLE 48-like (CDC48-like). A single Ser-to-Phe amino acid substitution in this protein led to a narrow-leaf phenotype. Transcriptomic analysis and measurement of endogenous phytohormone levels in nl1 vs. the wild-type suggested that NL1 might regulate cell division and the cytokinin pathway to control leaf width. Haplotype analysis showed that Hap2 of NL1 has been selected during wheat breeding. These findings provide insights into the genetic and molecular mechanisms underlying the role of NL1 in regulating leaf width and point to the potential of Hap2 for improving wheat plant architecture.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), threatens global wheat production. Breeding resistant varieties is a key to disease control. In this study, 198 modern wheat varieties were phenotyped with the prevalent Pst races CYR33 and CYR34 at the seedling stage and with mixed Pst races at the adult-plant stage. Seven stable resistance varieties with infection type (IT) ≤ 2 and disease severity (DS) ≤ 20% were found, including five Chinese accessions (Zhengpinmai8, Zhengmai1860, Zhoumai36, Lantian36, and Chuanmai32), one USA accession (GA081628-13E16), and one Pakistani accession (Pa12). The genotyping applied a 55K wheat single-nucleotide polymorphism (SNP) array. A genome-wide association study (GWAS) identified 14 QTL using a significance threshold of p ≤ 0.001, which distributed on chromosomes 1B (4), 1D (2), 2B (4), 6B, 6D, 7B, and 7D (4 for CYR33, 7 for CYR34, 3 for mixed Pst races), explaining 6.04% to 18.32% of the phenotypic variance. Nine of these QTL were potentially novel, as they did not overlap with the previously reported Yr or QTL loci within a ±5.0 Mb interval (consistent with genome-wide LD decay). The haplotypes and resistance effects were evaluated to identify the favorable haplotype for each QTL. Candidate genes within the QTL regions were inferred based on their transcription levels following the stripe rust inoculation. These resistant varieties, QTL haplotypes, and favorable alleles will aid in wheat breeding for stripe rust resistance.

Polyploidization is a fundamental evolutionary process in plants, including bread wheat. In the present study, we performed a comprehensive genome-wide analysis of dynamic homoeologous gene divergence in Aikang58 (AK58), a modern elite polyploid wheat cultivar with a recently released reference genome, and in other wheat genomes, including landraces, synthetic wheat, and several breeding lines. Over 40% of transposable element (TE) families exhibit biased distribution across the three wheat subgenomes. Approximately 95.0% (113 421) of genes are co-located with TEs, and these variable TEs significantly contribute to homoeologous divergence. We found that about 80% of triad homoeologs are divergent due to differences in expression or sub-functionalization. In addition, subgenome divergence potentially promote polyploid wheat domestication and improvement by increasing favorable homoeoallele combinations. Our findings suggest that homoeolog divergence contributes to the adaptation, domestication, and improvement of hexaploid wheat. The contribution of subgenomic divergence to polyploid heterosis is also discussed. This study provides a valuable resource for the investigation of how TEs drive homoeologous divergence during wheat domestication and improvement.

The Q gene is a key domestication gene in wheat (Triticum aestivum) that regulates free-threshing habit, spike morphology, height, and other critical agronomic traits. However, the precise molecular mechanisms underlying its function remain unclear. In this study, we identified a Q allele with a missense mutation (G to A) in the fifth exon of the Q gene, resulting in reduced plant height and spike length. Further investigation revealed that this mutation causes a Gly-229-Ser amino acid substitution, which enhances Q protein stability. Furthermore, we discovered that Q directly binds to the promoter region of Gibberellin 3-oxidase 2 gene (TaGA3ox2) and represses its expression. Moreover, Q interacts with both REDUCED HEIGHT1 (RHT1) and GIBBERELLIN INSENSITIVE 2 (TaGID2), which may disrupt GID2-triggered RHT1 degradation. Collectively, these findings reveal the dual roles of Q in regulating both GA biosynthesis and signaling, providing insights into the molecular mechanisms through which Q modulates plant height and spike length in wheat.

A complete reference genome assembly is crucial for biological research and genetic improvement. Owing to its large size and highly repetitive nature, there are numerous gaps in the globally used wheat Chinese Spring (CS) genome assembly. In this study, we generated a 14.46 Gb near-complete assembly of the CS genome, with a contig N50 of over 266 Mb and an overall base accuracy of 99.9963%. Among the 290 gaps that remained (26, 257, and 7 gaps from the A, B, and D subgenomes, respectively), 278 were extremely high-copy tandem repeats, whereas the remaining 12 were transposable-element-associated gaps. Four chromosome assemblies were completely gap-free, including chr1D, chr3D, chr4D, and chr5D. Extensive annotation of the near-complete genome revealed 151 405 high-confidence genes, of which 59 180 were newly annotated, including 7602 newly assembled genes. Except for the centromere of chr1B, which has a gap associated with superlong GAA repeat arrays, the centromeric sequences of all of the remaining 20 chromosomes were completely assembled. Our near-complete assembly revealed that the extent of tandem repeats, such as simple-sequence repeats, was highly uneven among different subgenomes. Similarly, the repeat compositions of the centromeres also varied among the three subgenomes. With the genome sequences of all six types of seed storage proteins (SSPs) fully assembled, the expression of ω-gliadin was found to be contributed entirely by the B subgenome, whereas the expression of the other five types of SSPs was most abundant from the D subgenome. The near-complete CS genome will serve as a valuable resource for genomic and functional genomic research and breeding of wheat as well as its related species.

INTRODUCTION:High-density Wheat 660K and 90K SNP arrays are powerful tools for understanding the genetic basis of wheat traits. However, their inconsistantly physical positions that were caused by different versions of Chinese Spring genome during developing arrays are confused and inconvenient for further application. OBJECTIVE:With the repid development of wheat geonome sequencing, we aim to reconciliate Wheat 660K and 90K SNP arrays in modern cultivar and reveal the genetic basis of dough rheological properties in bread wheat. METHODS:We refined physical positions of Wheat 660K and 90K SNP arrays in the currently popular wheat cultivar AK58 genome that was released more recently. We next performed genome-wide association studies (GWAS) and linkage analysis to identify important genetic loci related to quality traits using updated and un-updated arrays, respectively. RESULTS:Refining results showed that 92.3% and 83% of SNPs in the Wheat 660K and 90K SNP arrays were precisely mapped to the AK58 genome, respective. GWAS results by the updated 660K and 90K arrays indicated that 26 intervals composed of 1032 significant SNPs were associated with 9 quality traits in multiple environments. The significant interval for stability time on 1D was narrowed into an 8.4-Mb region using the updated arrays, whereas the interval is 405 Mb using the un-updated arrays. Linkage analysis revealed an important QTL QST.henau-1D.2 for stability time with 1.64 Mb. Integration of GWAS and QTL results narrowed the significant interval into 6.46 Mb containing 35 annotation genes by collinearity analysis. After T-test, gene expression analysis, seven of them are potential candidate genes and thus favorable haplotypes are identified to benefit marker-assisted selection. CONCLUSION:A reconciliation of Wheat 660K and 90K arrays promote their efficient applications. Important genetic loci and favorable haplotypes identified in this study provided valuable information for wheat quality breeding.

Although numerous studies have focused on phytohormones in specific organs or tissues at different development stages or under various abiotic and biotic stress conditions, our understanding of the distribution and relative abundance of phytohormones throughout the entire life cycle of plants remains insufficient. Here, we present a phytohormone atlas resource obtained from the quantitative analysis of eight major classes of phytohormones, comprising a total of 40 hormone-related compounds, throughout the complete life cycle of wheat. In combination with transcriptome analysis, we established a wheat phytohormone metabolic regulatory network (WPMRN). Using the WPMRN dataset and Gene Ontology enrichment analysis, we swiftly characterized the function of TaLOG5-B1 in cytokinin biosynthesis. Furthermore, a detailed investigation of the WPMRN dataset uncovered transcription factor-mediated co-regulatory mechanisms among different classes of phytohormones. We focused specifically on the metabolic regulation of cytokinin and jasmonic acid, and functionally characterized the genes TaLOG3-D1 and TaAOS-D1 that are involved in the biosynthesis of these phytohormones, respectively, along with their regulatory transcription factor genes TaDOF3A and TaDOF5.6B. The functions of these genes were validated in transgenic plants, revealing their ability to co-regulate radicle length. These findings serve as a case study that highlights the utility of this resource for studying phytohormone metabolic regulatory networks in cereal crops and for gaining insights into the roles of phytohormones in enhancing agronomic traits.

Aegilops speltoides, the closest ancestor of the wheat B subgenome, has been well studied genomically. However, the epigenetic landscape of Ae. speltoides and the effects of epigenetics on its growth and development remain poorly understood. Here, we present a comprehensive multi-omics atlas of leaves and roots in Ae. speltoides, encompassing transcriptome, DNA methylation, histone modifications, and small RNA profiling. Divergent DNA methylation levels were detected between leaves and roots, and were associated with differences in accumulated 24-nt siRNAs. DNA methylation changes in promoters and gene bodies showed strong connections with altered expression between leaves and roots. Transcriptional regulatory networks (TRN) reconstructed between leaves and roots were driven by tissue-specific TF families. DNA methylation and histone modification act together as switches that shape root and leaf morphogenesis by modulating the binding of tissue-specific TFs to their target genes. The TRNs in leaves and roots reshaped during wheat polyploidization were associated with alterations in epigenetic modifications. Collectively, these results not only shed light on the critical contribution of epigenetic regulation in the morphogenesis of leaves and roots in Ae. speltoides but also provide new insights for future investigations into the complex interplay of genetic and epigenetic factors in the developmental biology of common wheat.

BACKGROUND:Winter wheat undergoes vernalization, a process activated by prolonged exposure to low temperatures. During this phase, flowering signals are generated and transported to the apical meristems, stimulating the transition to the inflorescence meristem while inhibiting tiller bud elongation. Although some vernalization genes have been identified, the key cis-regulatory elements and precise mechanisms governing this process in wheat remain largely unknown. RESULTS:In this study, we construct extensive epigenomic and transcriptomic profiling across multiple tissues-leaf, axillary bud, and shoot apex-during the vernalization of winter wheat. Epigenetic modifications play a crucial role in eliciting tissue-specific responses and sub-genome-divergent expressions during vernalization. Notably, we observe that H3K27me3 primarily regulates vernalization-induced genes and has limited influence on vernalization-repressed genes. The integration of these datasets enables the identification of 10,600 putative vernalization-related regulatory elements including distal accessible chromatin regions (ACRs) situated 30Kb upstream of VRN3, contributing to the construction of a comprehensive regulatory network. Furthermore, we discover that TaSPL7/15, integral components of the aging-related flowering pathway, interact with the VRN1 promoter and VRN3 distal regulatory elements. These interactions finely regulate their expressions, consequently impacting the vernalization process and flowering. CONCLUSIONS:Our study offers critical insights into wheat vernalization's epigenomic dynamics and identifies the putative regulatory elements crucial for developing wheat germplasm with varied vernalization characteristics. It also establishes a vernalization-related transcriptional network, and uncovers that TaSPL7/15 from the aging pathway participates in vernalization by directly binding to the VRN1 promoter and VRN3 distal regulatory elements.

QKl/Tgw/Gns.yaas-2D associates with KL, TGW, and GNS, and QKl/Tgw.yaas-5A associates with KL and TGW. Significantly pleiotropic and additive effects of these two QTL were validated. The YM5 allele both at QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A was proved to be the best allelic combination for improving yield potential. Kernel length (KL), kernel width (KW), thousand grain weight (TGW), and grain number per spike (GNS) play important roles in the yield improvement of wheat. In this study, one recombinant inbred line (RIL) derived from a cross between Yangmai 5 (YM5) and Yanzhan 1 (YZ1) was used to identify quantitative trait loci (QTL) associated with KL, KW, TGW, and GNS across three years. Two pleiotropic QTL namely QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A were located in two genomic regions on chromosomes 2D and 5A, respectively. Breeder-friendly Kompetitive Allele-Specific PCR (KASP) markers for QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A were developed and validated in a set of 246 wheat cultivars/lines. Analysis of allelic combinations indicated that the YM5 allele both at QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A is probably the best one to promote TGW, GNS, and grain weight per spike. Based on the analysis of gene annotation, sequence variations, expression patterns, and GO enrichment, twenty-five and twenty-four candidate genes of QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A, respectively, were identified. These results provide the basis of fine-mapping the target QTL and marker-assisted selection in wheat yield-breeding programs.

Grain hardness (GH) plays an important role in wheat quality evaluation. Identification of new genes or quantitative trait loci (QTL) for GH is an effective strategy for wheat quality breeding. Here, we used a recombinant inbred line (RIL) population derived from a cross between two hard wheat Yangmai 4 (YM4) and Yanzhan 1 (YZ1) to identify QTL for GH. No QTL was detected on 5D chromosome, as parents YM4 and YZ1 possessed the two hard alleles Pinb-D1b and Pinb-D1p at the Hardness-5D (Ha-5D) locus, respectively. A total of three GH QTL were identified, among which QGh.yaas-4B and QGh.yaas-7D could be detected in all experiments and for mean value, explaining 8.69%-15.07% of the phenotypic variances. QGh.yaas-4D, co-located with Rht-D1, was detected in one experiment and for mean value, explaining 9.94%-11.39% of the phenotypic variances. We were not able to precisely validate QGh.yaas-4B due to its large mapping interval. Kompetitive allele-specific PCR (KASP) markers for QGh.yaas-7D were successfully developed, and then QGh.yaas-4D and QGh.yaas-7D were validated in a panel of 101 wheat cultivars/lines (all carrying Pina-D1a and Pinb-D1a alleles). Cultivars/lines harbouring the positive alleles of QGh.yaas-4D and QGh.yaas-7D increased GH by 85.16% relative to the ones without any positive allele. These results provide new loci and resources in molecular breeding for wheat hardness.

Wheat is the second largest food crop with a very good breeding system and pedigree record in China. Investigating the genomic footprints of wheat cultivars will unveil potential avenues for future breeding efforts1,2. Here we report chromosome-level genome assemblies of 17 wheat cultivars that chronicle the breeding history of China. Comparative genomic analysis uncovered a wealth of structural rearrangements, identifying 249,976 structural variations with 49.03% (122,567) longer than 5 kb. Cultivars developed in 1980s displayed significant accumulations of structural variations, a pattern linked to the extensive incorporation of European and American varieties into breeding programmes of that era. We further proved that structural variations in the centromere-proximal regions are associated with a reduction of crossover events. We showed that common wheat evolved from spring to winter types via mutations and duplications of the VRN-A1 gene as an adaptation strategy to a changing environment. We confirmed shifts in wheat cultivars linked to dietary preferences, migration and cultural integration in Northwest China. We identified large presence or absence variations of pSc200 tandem repeats on the 1RS terminal, suggesting its own rapid evolution in the wheat genome. The high-quality genome assemblies of 17 representatives developed and their good complementarity to the 10+ pan-genomes offer a robust platform for future genomics-assisted breeding in wheat. The pan-genome of 17 wheat cultivars grown in China is explored, providing insights into the breeding history of wheat in East Asia.

Five QTL for wheat grain protein content were identified, and the effects of two dwarfing genes Rht-B1b and Rht-D1b on grain protein content were validated in multiple populations. Grain protein content (GPC) plays an important role in wheat quality. Here, a recombinant inbred line (RIL) population derived from a cross between Yangmai 12 (YM12) and Yanzhan 1 (YZ1) was used to identify quantitative trait loci (QTL) for GPC. Two hundred and five RILs and their parents were grown in three years in randomized complete blocks each with two replications, and genotyped using the wheat 55 K SNP array. Five QTL were identified for GPC on chromosomes 1A, 1B, 2D, 4B, and 4D. Notably, QGpc.yaas-4B (co-located with Rht-B1) and QGpc.yaas-4D (co-located with Rht-D1) were consistently detected across all experiments and best linear unbiased estimating, accounting for 6.61–8.39

Fusarium head blight (FHB) is a major concern for wheat production and food safety, globally. Resistance to infection (type I resistance) is important for breeding varieties with good FHB resistance. To better understand the genetic architecture underlying type I FHB resistance, quantitative trait loci (QTL) mapping was conducted using two recombinant inbred line populations derived from the crosses Yangmai 4 (YM4)/Yanzhan 1 (YZ1) and Yangmai 5 (YM5)/YZ1. A total of five resistance QTL, including QFhb.yaas-2D, QFhb.yaas-4D, QFhb.yaas-5B, QFhb.yaas-6B, and QFhb.yaas-3A, were detected from these two populations. Except for QFhb.yaas-2D and QFhb.yaas-5B, the resistance effects of all the other QTLs are derived from YM4 or YM5. QFhb.yaas-4D is a common QTL detected both in YM4/YZ1 and YM5/YZ1 populations. QFhb.yaas-2D, QFhb.yaas-4D, and QFhb.yaas-5B were colocalized with known plant height (PH) genes Rht8, Rht-D1, and flowering date (FD) gene Vrn-B1. However, QFhb.yaas-6B and QFhb.yaas-3A were not associated with PH and FD. Kompetitive Allele-Specific PCR markers for QFhb.yaas-6B and QFhb.yaas-3A had been developed and validated in an additional panel of 211 wheat cultivars/lines. The result showed that these two resistance alleles decreased 46.65% of the percentage of diseased spikelets. These results provide valuable information for the fine mapping of QFhb.yaas-6B and QFhb.yaas-3A in the future.