The ERECTA family of receptor-like kinases (RLKs) plays a crucial regulatory role in plant stomatal development and responses to abiotic stresses. This study therefore focused on investigating the barley HvERECTA gene, aiming to clarify its regulatory roles in these biological processes and provide potential targets for barley drought resistance breeding. We first cloned the HvERECTA gene from barley and conducted comprehensive bioinformatics analyses to characterize its sequence and protein properties. Subsequently, we constructed a heterologous overexpression vector carrying the HvERECTA gene and transformed it into Arabidopsis thaliana. For the transgenic Arabidopsis lines, we systematically determined stomatal phenotypic traits, expression levels of the key related genes, and physiological indicators under drought stress conditions to evaluate the gene function. The bioinformatics analysis results showed that the full-length coding sequence (CDS) of HvERECTA is 2 934 bp, encoding a protein of 977 amino acids. The HvERECTA protein is localized to the plasma membrane and contains a typical serine/threonine protein kinase domain, which is a hallmark of the receptor-like kinase family. Sequence alignment and phylogenetic analysis revealed that HvERECTA is highly conserved among gramineous plants, with the highest homology to those from wheat (Triticum aestivum) and ryegrass (Lolium perenne). Additionally, analysis of cis-acting elements in the HvERECTA promoter region indicated that it is rich in regulatory elements associated with abiotic stress responses and plant hormone signaling, such as abscisic acid (ABA)-responsive elements (ABRE) and drought-responsive elements (MBS). Phenotypic observations on the transgenic Arabidopsis lines showed that overexpression of HvERECTA significantly reduced stomatal density compared with the wild-type control. Molecular analysis demonstrated that the expression levels of genes encoding key stomatal development transcription factors, namely SPCH (SPEECHLESS), MUTE, and FAMA, were all downregulated by more than 50% in the transgenic lines. In contrast, the expression level of YODA, a core gene in the mitogen-activated protein kinase (MAPK) cascade pathway that is involved in stomatal development regulation, was upregulated by 50%-80% in the transgenic plants. Under drought stress, the transgenic Arabidopsis lines showed enhanced drought resistance-related physiological performance. Specifically, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the transgenic lines were increased by 25%-40% compared with the control. Meanwhile, the content of malondialdehyde (MDA), which is an indicator of lipid peroxidation and cellular damage under stress, was decreased by approximately 30% in the transgenic lines. Additionally, the water loss rate of the transgenic lines was reduced by 20%, and their relative water content was higher than that of the control. Furthermore, the expression levels of stress-responsive genes, including AtKIN2, AtABF3, and AtRD22, showed no abnormal changes under normal growth conditions but were significantly upregulated under drought stress in the transgenic lines. In conclusion, the barley HvERECTA gene enhances plant drought resistance by regulating the MAPK signaling pathway to reduce stomatal density and by improving the activity of the plant antioxidant defense system. These two regulatory pathways collectively enhance the water retention capacity of plants under drought stress, thereby improving their stress tolerance. This study clarifies the important regulatory role of HvERECTA in stomatal development and abiotic stress response, and provides an important functional gene resource for drought-resistant molecular breeding of barley.

Abstract:Agrobacterium-mediated transformation is an important approach for genetic modification of wheat, but it still suffers from several unresolved critical technical bottlenecks, including low transformation efficiency, strong genotype dependence, severe browning of calli, and low regeneration efficiency. In this study, immature embryos of the model cultivar Fielder were used as explants to systematically investigate the effects of embryo age, acetosyringone (AS) concentration and Agrobacterium infection duration, phosphinothricin (PPT) selection pressure, and exogenous hormone (abscisic acid, ABA; kinetin, KT) ratios on callus induction, regeneration and transformation efficiency. Using the optimized protocol, applicability was evaluated in the cultivars Yangmai 20, Zhengmai 7698, and Kenong 199. The results showed that embryos harvested 12 days after anthesis exhibited the lowest browning rate (4.40%) and were most suitable as recipients for transformation. When the Agrobacterium suspension was adjusted to OD₆₀₀=0.5, supplementation with 200 μmol·L^-1 AS and a 10 min infection produced the highest induction rate of resistant green callus spots (34.29%). Addition of 0.5 mg·L^-1 ABA to the induction medium and 1.5 mg·L^-1 KT to the regeneration medium significantly enhanced embryogenic callus formation and plant regeneration. The optimal PPT selection concentration was determined to be 10 mg·L^-1. Under the optimized conditions, transformation efficiency of Fielder increased significantly from 8.16% before optimization to 14.83%; transformation efficiencies of Yangmai 20, Zhengmai 7698, and Kenong 199 reached 9.03%, 7.32% and 8.70%, respectively, approaching or exceeding the pre-optimization level of Fielder. In summary, the optimized system established in this study improves wheat genetic transformation efficiency and is broadly applicable across different genetic backgrounds, providing a technical platform for gene functional analysis and molecular breeding in wheat.

To elucidate the molecular mechanism of miRNA in wheat's response to dry-hot wind stress, a sensitive germplasm Xinchun 14 and a tolerant variety Xinchun 46 were selected as materials. During the grain-filling stage, artificial simulation of dry-hot wind stress (daytime temperature 35 ℃/nighttime temperature 25 ℃, relative humidity 20%, wind speed 5 m·s^-1, continuous treatment for 5 days) was applied. High-throughput sequencing technology was used to screen differentially expressed miRNAs, followed by validation via qRT-PCR. The results showed that 203 miRNAs were involved in the expression under dry-hot wind stress, with 136 co-expressed miRNAs. After differential screening, six core differentially expressed miRNAs were ultimately identified, namely, novel_miR_537, novel_miR_443, novel_miR_469, novel_miR_393, novel_miR_390, and novel_miR_339. These miRNAs were significantly enriched in four pathways: the biosynthesis of mannose O-glycans, the degradation of polysaccharides, peroxisomes, and the metabolism of selenium compounds. Target gene predictions revealed that these miRNAs targeted and regulated key membrane protein-encoding genes, including β-1, 2-xyltransferase, α-1, 3-arabinosyltransferase, GDSL esterase/lipase, α-L-fucosidase, and peroxidase, and are involved in the maintenance of cell wall stability, reactive oxygen species clearance, and osmotic regulation processes. This indicates there is a miRNA regulatory network in the response of dry-hot wind in wheat, and it jointly participates in the resistance process of wheat to dry-hot wind stress in the form of a 'miRNA-targeted gene' molecular module.

Wheat lines carrying the 1B/1R translocation have been widely utilized in breeding due to their enhanced disease resistance and yield stability. However, its impact on the three-dimensional (3D) chromatin architecture and transcriptional regulation of non-translocated subgenomes remains largely unexplored. Here, we used a 1B/1R translocation line, Wanmai 270, and its corresponding near-isogenic line lacking the 1B/1R segment to investigate chromatin spatial remodeling in the wheat A subgenome and its relationship with gene expression. By integrating Hi-C, RNA-seq, and qRT-PCR, we systematically characterized the effects of the 1B/1R translocation on 3D genome organization. A substantial number of A/B compartment switching regions were identified in the A subgenome, with a predominance of B-to-A transitions. Notably, compartment reorganization exhibited pronounced heterogeneity across chromosomes. To assess the association between compartment switching and transcriptional changes, 20 differentially expressed genes located within translocation regions were selected for qRT-PCR. Genes in A-to-B regions showed reduced expression in Wanmai 270 relative to those in the near-isogenic line, whereas genes in B-to-A regions exhibited increased expression, consistent with the direction of compartment transitions. Subcompartment analysis revealed a genome-wide decrease in A1/A2 proportions and an increase in B1/B2 proportions in the A subgenome of Wanmai 270, with the most prominent reorganization observed on chromosomes 1A and 3A. Topologically associating domains (TAD) analysis showed that the overall TAD framework was largely conserved between the two lines, although local TAD merging and boundary reorganization occurred in specific regions of Wanmai 270. Integration with transcriptome data further indicated that compartment-translocation regions were enriched for differentially expressed genes involved in chromatin organization, DNA replication, carbohydrate metabolism, and amino acid metabolism. GO, KEGG, and GSEA analyses revealed that transcriptional differences in Wanmai 270 were not characterized by global activation of basal metabolic pathways, but rather by fine-tuned regulation within specific pathways. In summary, the 1B/1R translocation preserves the overall 3D chromatin framework of the A subgenome while inducing multiscale spatial remodeling at the levels of compartments, subcompartments, and local TADs, thereby modulating transcriptional responses through changes in the chromatin regulatory landscape.

The red coleoptile in wheat confers antioxidant, photoprotective, and seedling stress-tolerance enhancing physiological functions. To provide key support for clarifying the molecular regulatory mechanism of the red coleoptile in wheat and improving the theory and technical system of morphological marker-assisted breeding, this study used a population of 127 F₁₂ recombinant inbred lines (RILs) derived from the cross between a common wheat Ning 7840 (white coleoptile, female parent) and Clark (red coleoptile, male parent) as materials. Quantitative trait loci (QTLs) controlling the red coleoptile were mapped using the inclusive composite interval mapping-additive effect (ICIM-ADD) method combined with a high-density genetic map containing 593 SNP and 402 SSR markers. Comparative genomics was used to align the collinear chromosomal regions between wheat and Brachypodium distachyon, sorghum, and rice to screen candidate genes related to anthocyanin regulation. Candidate genes were cloned, and sequence differences between red and white coleoptile lines were analyzed. CAPS markers were developed to verify gene-phenotype associations. The gene characteristics and functions were examined through subcellular localization and transient expression in wheat calli. The results showed that the ratio of red to white coleoptile lines in the RIL population conformed to 1∶1. A major QTL (qRc-7A) was detected on chromosome 7A, located between markers Xsnp7205 and Xsnp5258 (1.69 cM), explaining 66.4% of the phenotypic variation. Collinearity analysis revealed a collinear relationship between this mapped interval and the corresponding regions on chromosome 1 of Brachypodium distachyon, chromosome 10 of Sorghum bicolor, and chromosome 6 of Oryza sativa. Within the collinear regions of Sorghum bicolor and Oryza sativa, anthocyanin regulatory R2R3-MYB transcription factor-encoding genes, namely Sb10g06800 and Os06g10350 (OsC1), were identified, which shared high sequence similarity with the TraesCS7A02G165700 gene on wheat chromosome 7A. It was thus preliminarily inferred that TraesCS7A02G165700 is a key candidate gene regulating anthocyanin biosynthesis in the red coleoptile of wheat, and this gene was designated as TaC1. TaC1a in red coleoptile lines (cDNA length 774 bp, encoding 258 amino acids) contains a complete R2R3 domain and motif 5 domain, while TaC1b in white coleoptile lines has a frameshift mutation due to a 714 bp deletion, resulting in the loss of motif 5. TaC1a is localized in the nucleus and can induce anthocyanin accumulation in transient expression experiments in wheat calli, whereas TaC1b lacks this function. CAPS marker detection showed that TaC1a was significantly associated with the red trait, but 4 red coleoptile materials carried TaC1b. In conclusion, TaC1 is the core gene regulating anthocyanin synthesis in wheat red coleoptiles, and TaC1b loses its function due to base deletion. The CAPS marker based on this variation can be used for molecular identification of red coleoptite, but other regulatory genes may be involved in some red materials, requiring further analysis of multi-gene interaction mechanisms.

To investigate the occurrence of autumn wheat stripe rust and the transmission pathways of pathogen sources in the over-summering areas of Puccinia striiformis f. sp. tritici in Qinghai, systematic field disease surveys were conducted on the main over-summering host wheat in eastern Qinghai from August to November in 2023 and 2024. The results showed that overlapping host growth stages occurred in the eastern wheat-growing regions of Qinghai from August to October, allowing the pathogen to complete infection across different hosts. Puccinia striiformis f. sp. tritici in Qinghai is unaffected by summer high temperatures and can successfully survive over summer in nearly all wheat-growing areas, with pathogen reproduction lasting until December. From August to October, abundant precipitation in eastern Qinghai led to severe disease occurrence in autumn-sown wheat seedlings. The average diseased leaf rate showed a linear positive correlation with precipitation, and a significant positive correlation was observed with precipitation in September of 2023 (P≤0.05). Based on airflow trajectory simulations, it is inferred that during the occurrence of stripe rust on autumn-sown wheat seedlings in the eastern wheat-growing areas of Qinghai, the disease can directly impact autumn seedling infections in Gansu and Ningxia. Moreover, through a single long-distance dispersal event, the pathogen can reach the Guanzhong Plain and even the wheat-growing regions of southern Henan, with the potential to threaten northwestern Hubei.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), severely influences wheat yield and grain quality. Mining novel powdery mildew resistance (Pm) genes and developing functional markers is of great significance for molecular breeding of disease-resistant wheat. In this study, an F₂ segregating population was first constructed using Hongyoumai as the resistant parent and Ningmaizi 119 as the susceptible parent. By integrating phenotypic data from resistance identification, diagnostic molecular marker development, and recombinant analysis, the wheat powdery mildew resistance gene PmHYM was finely mapped. Subsequently, sequence characteristics of the candidate genes were analyzed through sequence alignment, flow sorting and chromosome sequencing, expression pattern analysis, and copy number variation detection. Finally, KASP markers were developed based on functional SNP, and the breeding potential of the gene was evaluated via backcross breeding. The results showed that PmHYM was fine-mapped to a 0.5 cM genetic interval between markers Xmp1410 and Xmp1396 on chromosome 7BL, corresponding to a 310 kb physical interval in the Chinese Spring reference genome. Two coiled-coil nucleotide-binding leucine-rich repeat (CNL)-type proteins were predicted within this region. The sequences of these two genes in Hongyoumai were identical to those in Fuzhuang 30. The difference in the resistance spectrum to Bgt isolate B16, between Hongyoumai and Fuzhuang 30 was independent of gene sequence variations or copy number variations. A high-throughput diagnostic KASP marker Xmp1394 for PmHYM was successfully developed, enabling rapid tracking of PmHYM. Backcross-derived lines carrying PmHYM exhibited no significant differences in agronomic traits compared with the recurrent parent, suggesting as high potential for breeding application.

To gain an deep understanding of the genetic characteristics of domestic and foreign wheat germplasm and to screen superior resources for rational parental combinations, 490 wheat germplasm resources were used as experimental materials. Genetic diversity analysis, correlation analysis, clustering analysis, principal component analysis, subordinate function analysis and comprehensive evaluation were conducted on 22 major agronomic traits (including flag leaf length, plant height, number of fertile spikelets, thousand-kernel weight, kernels per spike, yield and et al.) and 7 quality traits (including grain crude protein content, wet gluten content, sedimentation value, volume weight and et al.). The results showed that the 490 wheat germplasm resources had abundant phenotypic diversity. The coefficient of variation (CV) for major agronomic traits ranged from 1.65% to 16.75%, being lowest for heading period and highest for kernels per spike. The genetic diversity index ranged from 1.74 to 2.07, with an average of 1.97, being highest for the number of fertile spikelets and lowest for growth period. The CV for grain quality traits ranged from 1.45% to 15.93%, being highest for sedimentation value and lowest for bulk weight. The genetic diversity index of grain quality traits ranged from 2.01 to 2.07, with an average of 2.04. Correlation analysis results indicated that: flag leaf length, plant height, number of fertile spikelets, kernels per spike, and thousand-kernel weight showed highly significant correlations with yield. Grain crude protein content, wet gluten content, and sedimentation value showed highly significantly positive pairwise correlations with extensibility. Principal component analysis (PCA) extracted three principal components, with a cumulative contribution rate of 64.64%. Among these: the first and second principal components mainly reflected information related to yield-related traits, and the first and third principal components mainly reflected information related to quality traits. Through cluster analysis, the 490 wheat germplasm resources were divided into five groups. Group Ⅰ had the highest average yield; Group Ⅱ had the highest average kernels per spike; Group Ⅳ had the shortest average growth period, and Group V exhibited significantly higher crude protein content, wet gluten content, sedimentation value, and extensibility compared to the other groups. Overall, Groups Ⅰ and Ⅳ demonstrated superior yield-related traits, while Group V showed outstanding quality-related traits. Comprehensive evaluation based on subordinative function analysis indicated that 22 wheat germplasms, including xf19200, Xikemai 3, Shannong 71, and Flanders exhibited better comprehensive traits. These can be selectively utilized based on the characteristics of their respective clusters in conjunction with the cluster analysis results.

Selenium-enriched wheat is a viable strategy to address dietary selenium deficiencies in selenium-deficient regions. To explore the selenium uptake, translocation, and distribution patterns in various wheat tissues, a wheat line Chuanwen 2401 was used as the material. Four foliar spraying treatments were set up: three nano-selenium application rates of 7.5 g·hm^-2 (C₁), 15.0 g·hm^-2 (C₂) and 30.0 g·hm^-2 (C₃), with clean water as the control (CK), to analyze the effects on selenium absorption and translocation, selenium speciation in grains forms, nutrient element contents, ecological risks, dietary selenium intake, yield and yield components. The results showed that, foliar nano-selenium spraying significantly increased selenium content in various wheat tissues, and selenium content in wheat grain significantly increased by 25.5-126.8 fold. Compared with CK, selenium translocation coefficients showed significant changes, for example, TF of flag leaf and the 1st stem node, the 2nd leaf and 2nd stem node, the 3rd leaf and 3rd stem node, and the 4th leaf and 4th stem node were significantly reduced by 85.5%-91.3%, 64.7%-82.4%, 75.0%-75.0%, and 50.0%-83.3%, respectively. TF of rachis and glume, rachis and grain were significantly increased by 209.4%-718.8% and 111.4%-640.0%, respectively. Only selenocysteine (SeCys) and selenomethionine (SeMet) were detected in CK grains, with SeCys accounting for the higher proportion. An additional selenium speciation, methylselenocysteine (MeSeCys), was identified in grains under C₁, C₂, and C₃ treatments, with SeMet being the dominant selenium form. Compared with CK, contents of Mn, Zn, Cu, and grain protein significantly decreased by 12.7%-15.6%, 4.1%-18.2%, 6.8%-16.1%, and 6.9%-22.7%, respectively. Fe content significantly increased by 46.4%-111.1%. The ecological risk index (Er) of the C₁ treatment was 51.6, which was closest to the safety threshold (40.0), and the estimation of daily selenium intake (EDI) was 297.2 μg, falling within the recommended range (60-400 μg). The Er value of CK was within the safety threshold, but its EDI was far below the lower limit of the recommended range. Both Er and EDI values of C₂ and C₃ treatments exceeded the safety/recommended ranges. Compared with CK, yield thous and graiin weight of C₁, C₂, and C₃ treatments significantly increased by 3.5%-8.5% and 2.8%-4.4%, respectively. Effective spikes and grains per spike showed no significant changes. In conclusion, foliar nano-selenium spraying significantly increased the selenium content and yield of wheat grains, and improved the wheat grains quality by regulating the selenium speciation, proportion, and nutrient element content. Among them, C₁ treatment (7.5 g·hm^-2) was the optimal foliar nano-selenium spraying concentration.

To investigate the differences in grain yield and nitrogen use efficiency among different wheat varieties and their physiological mechanisms, this study was conducted at the experimental station of Henan Agricultural University in Yuanyang County, Xinxiang City, Henan Province, during the 2023—2024 wheat growing season. Forty approved wheat varieties were used as experimental materials. Principal component analysis and comprehensive scoring were performed on four key nitrogen efficiency indices: nitrogen recovery efficiency, partial factor productivity of nitrogen, physiological nitrogen use efficiency, and agronomic nitrogen use efficiency. Combined with grain yield, wheat varieties were screened for yield and nitrogen efficiency levels, and physiological indices of different yield-nitrogen efficiency types were further compared to reveal the physiological basis underlying the differences in yield and nitrogen use efficiency. The results showed that grain yield and the four nitrogen efficiency indices varied significantly among varieties, with coefficients of variation ranging from 11.61% to 46.73%. Based on principal component analysis, comprehensive scores of nitrogen use efficiency indices, and yield levels, the 40 wheat varieties were classified into four types: high-yield and high-efficiency (11 varieties), high-yield and low-efficiency (5 varieties), low-yield and high-efficiency (10 varieties), and low-yield and low-efficiency (14 varieties). Analysis of dry matter accumulation and translocation revealed that high-yield and high-efficiency varieties had significantly higher aboveground dry matter and nitrogen accumulation at anthesis and maturity than other types. Compared with high-yield and low-efficiency varieties, they also exhibited greater preanthesis nitrogen accumulation and preanthesis nitrogen translocation. In addition, the leaf area index (LAI) attenuation rate from anthesis to grain filling was lower in high-yield and high-efficiency varieties (18.77%) than in high-yield and low-efficiency varieties (22.67%). Meanwhile, high-yield types maintained significantly higher SPAD values during grain filling. Under the experimental cultivation conditions, high-yield and high-efficiency varieties maintained high dry matter and nitrogen accumulation at anthesis. By optimizing leaf spatial distribution and prolonging functional leaf longevity, they sustained dry matter production during grain filling, thereby achieving higher grain yield and nitrogen use efficiency.