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.

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.

The roles of DNA methylation on pH dependent i-motif (iM) formation in rice.

I-motifs (iMs) are four-stranded non-B DNA structures containing C-rich DNA sequences. The formation of iMs is sensitive to pH conditions and DNA methylation, although the extent of which is still unknown in both humans and plants. To investigate this, we here conducted iMab antibody-based immunoprecipitation and sequencing (iM-IP-seq) along with bisulfite sequencing using CK (original genomic DNA without methylation-related treatments) and hypermethylated or demethylated DNA at both pH 5.5 and 7.0 in rice, establishing a link between pH, DNA methylation and iM formation on a genome-wide scale. We found that iMs folded at pH 7.0 displayed higher methylation levels than those formed at pH 5.5. DNA demethylation and hypermethylation differently influenced iM formation at pH 7.0 and 5.5. Importantly, CG hypo-DMRs (differentially methylated regions) and CHH (H = A, C and T) hyper-DMRs alone or coordinated with CG/CHG hyper-DMRs may play determinant roles in the regulation of pH dependent iM formation. Thus, our study shows that the nature of DNA sequences alone or combined with their methylation status plays critical roles in determining pH-dependent formation of iMs. It therefore deepens the understanding of the pH and methylation dependent modulation of iM formation, which has important biological implications and practical applications.

R-loops act as regulatory switches modulating transcription of COLD-responsive genes in rice.

COLD is a major naturally occurring stress that usually causes complex symptoms and severe yield loss in crops. R-loops function in various cellular processes, including development and stress responses, in plants. However, how R-loops function in COLD responses is largely unknown in COLD susceptible crops like rice (Oryza sativa L.). We conducted DRIP-Seq along with other omics data (RNA-Seq, DNase-Seq and ChIP-Seq) in rice with or without COLD treatment. COLD treatment caused R-loop reprogramming across the genome. COLD-biased R-loops had higher GC content and novel motifs for the binding of distinct transcription factors (TFs). Moreover, R-loops can directly/indirectly modulate the transcription of a subset of COLD-responsive genes, which can be mediated by R-loop overlapping TF-centered or cis-regulatory element-related regulatory networks and lncRNAs, accounting for c. 60% of COLD-induced expression of differential genes in rice, which is different from the findings in Arabidopsis. We validated two R-loop loci with contrasting (negative/positive) roles in the regulation of two individual COLD-responsive gene expression, as potential targets for enhanced COLD resistance. Our study provides detailed evidence showing functions of R-loop reprogramming during COLD responses and provides some potential R-loop loci for genetic and epigenetic manipulation toward breeding of rice varieties with enhanced COLD tolerance.

Adulthood status and its associations with stress and well-being among Chinese emerging adults: A person-centred approach.

With the implementation of China's one-child policy and the increasing living costs, a large number of young people are expected to postpone reaching adulthood. However, little research has explored the patterns of Chinese young people's transition to adulthood. Using a person-centred approach, the current study examined the commonly accepted markers of adulthood status endorsed and attained by 1203 Chinese emerging adults aged 18-29 years. We further compared the distinct adulthood statuses with regard to perceptions of stress and well-being. Latent profile analysis identified four adulthood status profiles: diffused (16.0%), moratorium (45.3%), transitional (23.3%) and achieved (15.4%). Females, those who had no job or came from low family socioeconomics, were more likely to be in the moratorium status. Compared to the other two profiles, participants in diffused and moratorium profiles reported higher levels of perceived stress and lower well-being. These results suggest that there are distinct profiles of Chinese emerging adults on the transition path to adulthood, with a large proportion of them not yet reaching adulthood, and that different statuses of adulthood may be associated with different psychological outcomes.

TaACTIN7-D regulates plant height and grain shape in bread wheat.

Exploitation of new gene resources and genetic networks contributing to the control of crop yield-related traits, such as plant height, grain size, and shape, may enable us to breed modern high-yielding wheat varieties through molecular methods. In this study, via ethylmethanesulfonate mutagenesis, we identify a wheat mutant plant, mu-597, that shows semi-dwarf plant architecture and round grain shape. Through bulked segregant RNA-seq and map-based cloning, the causal gene for the semi-dwarf phenotype of mu-597 is located. We find that a single-base mutation in the coding region of TaACTIN7-D (TaACT7-D), leading to a Gly-to-Ser (G65S) amino acid mutation at the 65th residue of the deduced TaACT7-D protein, can explain the semi-dwarfism and round grain shape of mu-597. Further evidence shows that the G65S mutation in TaACT7-D hinders the polymerization of actin from monomeric (G-actin) to filamentous (F-actin) status while attenuates wheat responses to multiple phytohormones, including brassinosteroids, auxin, and gibberellin. Together, these findings not only define a new semi-dwarfing gene resource that can be potentially used to design plant height and grain shape of bread wheat but also establish a direct link between actin structure modulation and phytohormone signal transduction.

Epigenomic Features and Potential Functions of K+ and Na+ Favorable DNA G-Quadruplexes in Rice.

DNA G-quadruplexes (G4s) are non-canonical four-stranded DNA structures involved in various biological processes in eukaryotes. Molecularly crowded solutions and monovalent cations have been reported to stabilize in vitro and in vivo G4 formation. However, how K+ and Na+ affect G4 formation genome-wide is still unclear in plants. Here, we conducted BG4-DNA-IP-seq, DNA immunoprecipitation with anti-BG4 antibody coupled with sequencing, under K+ and Na+ + PEG conditions in vitro. We found that K+-specific IP-G4s had a longer peak size, more GC and PQS content, and distinct AT and GC skews compared to Na+-specific IP-G4s. Moreover, K+- and Na+-specific IP-G4s exhibited differential subgenomic enrichment and distinct putative functional motifs for the binding of certain trans-factors. More importantly, we found that K+-specific IP-G4s were more associated with active marks, such as active histone marks, and low DNA methylation levels, as compared to Na+-specific IP-G4s; thus, K+-specific IP-G4s in combination with active chromatin features facilitate the expression of overlapping genes. In addition, K+- and Na+-specific IP-G4 overlapping genes exhibited differential GO (gene ontology) terms, suggesting they may have distinct biological relevance in rice. Thus, our study, for the first time, explores the effects of K+ and Na+ on global G4 formation in vitro, thereby providing valuable resources for functional G4 studies in rice. It will provide certain G4 loci for the biotechnological engineering of rice in the future.

Transcriptome-based gene regulatory network analyses of differential cold tolerance of two tobacco cultivars.

BACKGROUND: Cold is one of the main abiotic stresses that severely affect plant growth and development, and crop productivity as well. Transcriptional changes during cold stress have already been intensively studied in various plant species. However, the gene networks involved in the regulation of differential cold tolerance between tobacco varieties with contrasting cold resistance are quite limited. RESULTS: Here, we conducted multiple time-point transcriptomic analyses using Tai tobacco (TT, cold susceptibility) and Yan tobacco (YT, cold resistance) with contrasting cold responses. We identified similar DEGs in both cultivars after comparing with the corresponding control (without cold treatment), which were mainly involved in response to abiotic stimuli, metabolic processes, kinase activities. Through comparison of the two cultivars at each time point, in contrast to TT, YT had higher expression levels of the genes responsible for environmental stresses. By applying Weighted Gene Co-Expression Network Analysis (WGCNA), we identified two main modules: the pink module was similar while the brown module was distinct between the two cultivars. Moreover, we obtained 100 hub genes, including 11 important transcription factors (TFs) potentially involved in cold stress, 3 key TFs in the brown module and 8 key TFs in the pink module. More importantly, according to the genetic regulatory networks (GRNs) between TFs and other genes or TFs by using GENIE3, we identified 3 TFs (ABI3/VP1, ARR-B and WRKY) mainly functioning in differential cold responses between two cultivars, and 3 key TFs (GRAS, AP2-EREBP and C2H2) primarily involved in cold responses. CONCLUSION: Collectively, our study provides valuable resources for transcriptome- based gene network studies of cold responses in tobacco. It helps to reveal how key cold responsive TFs or other genes are regulated through network. It also helps to identify the potential key cold responsive genes for the genetic manipulation of tobacco cultivars with enhanced cold tolerance in the future.

Genome-wide characterization of i-motifs and their potential roles in the stability and evolution of transposable elements in rice.

I-motifs (iMs) are non-canonical DNA secondary structures that fold from cytosine (C)-rich genomic DNA regions termed putative i-motif forming sequences (PiMFSs). The structure of iMs is stabilized by hemiprotonated C-C base pairs, and their functions are now suspected in key cellular processes in human cells such as genome stability and regulation of gene transcription. In plants, their biological relevance is still largely unknown. Here, we characterized PiMFSs with high potential for i-motif formation in the rice genome by developing and applying a protocol hinging on an iMab antibody-based immunoprecipitation (IP) coupled with high-throughput sequencing (seq), consequently termed iM-IP-seq. We found that PiMFSs had intrinsic subgenomic distributions, cis-regulatory functions and an intricate relationship with DNA methylation. We indeed found that the coordination of PiMFSs with DNA methylation may affect dynamics of transposable elements (TEs) among different cultivated Oryza subpopulations or during evolution of wild rice species. Collectively, our study provides first and unique insights into the biology of iMs in plants, with potential applications in plant biotechnology for improving important agronomic rice traits.

Single-Cell Transcriptome and Network Analyses Unveil Key Transcription Factors Regulating Mesophyll Cell Development in Maize.

BACKGROUND: Maize mesophyll (M) cells play important roles in various biological processes such as photosynthesis II and secondary metabolism. Functional differentiation occurs during M-cell development, but the underlying mechanisms for regulating M-cell development are largely unknown. RESULTS: We conducted single-cell RNA sequencing (scRNA-seq) to profile transcripts in maize leaves. We then identified coregulated modules by analyzing the resulting pseudo-time-series data through gene regulatory network analyses. WRKY, ERF, NAC, MYB and Heat stress transcription factor (HSF) families were highly expressed in the early stage, whereas CONSTANS (CO)-like (COL) and ERF families were highly expressed in the late stage of M-cell development. Construction of regulatory networks revealed that these transcript factor (TF) families, especially HSF and COL, were the major players in the early and later stages of M-cell development, respectively. Integration of scRNA expression matrix with TF ChIP-seq and Hi-C further revealed regulatory interactions between these TFs and their targets. HSF1 and COL8 were primarily expressed in the leaf bases and tips, respectively, and their targets were validated with protoplast-based ChIP-qPCR, with the binding sites of HSF1 being experimentally confirmed. CONCLUSIONS: Our study provides evidence that several TF families, with the involvement of epigenetic regulation, play vital roles in the regulation of M-cell development in maize.

A natural variation in Ribonuclease H-like gene underlies Rht8 to confer "Green Revolution" trait in wheat.

Rht8 is a gibberellin-sensitive Reduced height (Rht) locus that has been widely used in crop wheat semi-dwarfing breeding. In this study, the authors reported the map-based cloning of Rht8 candidate gene, and confirmed that loss of Ribonuclease H-Like 1 (RNHL-D1) is responsible for Rht8 semi-dwarfing effect.

A single nucleotide deletion in the third exon of FT-D1 increases the spikelet number and delays heading date in wheat (Triticum aestivum L.).

The spikelet number and heading date are two crucial and correlated traits for yield in wheat. Here, a quantitative trait locus (QTL) analysis was conducted in F8 recombinant inbred lines (RILs) derived from crossing two common wheats with different spikelet numbers. A total of 15 stable QTL influencing total spikelet number (TSN) and heading date (HD) were detected. Notably, FT-D1, a well-known flowering time gene in wheat, was located within the finely mapped interval of a major QTL on 7DS (QTsn/Hd.cau-7D). A causal indel of one G in the third exon of FT-D1 was significantly associated with total spikelet number and heading date. Consistently, CRISPR/Cas9 mutant lines with homozygous mutations in FT-D1 displayed an increase in total spikelet number and heading date when compared with wild type. Moreover, one simple and robust marker developed according to the polymorphic site of FT-D1 revealed that this one G indel had been preferentially selected to adapt to different environments. Collectively, these data provide further insights into the genetic basis of spikelet number and heading date, and the diagnostic marker of FT-D1 will be useful for marker-assisted pyramiding in wheat breeding.

Epigenomic features of DNA G-quadruplexes and their roles in regulating rice gene transcription.

A DNA G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure involved in many biological processes in mammals. The current knowledge on plant DNA G4s, however, is limited; whether and how DNA G4s impact gene expression in plants is still largely unknown. Here, we applied a protocol referred to as BG4-DNA-IP-seq followed by a comprehensive characterization of DNA G4s in rice (Oryza sativa L.); we next integrated dG4s (experimentally detectable G4s) with existing omics data and found that dG4s exhibited differential DNA methylation between transposable element (TE) and non-TE genes. dG4 regions displayed genic-dependent enrichment of epigenomic signatures; finally, we showed that these sites displayed a positive association with expression of DNA G4-containing genes when located at promoters, and a negative association when located in the gene body, suggesting localization-dependent promotional/repressive roles of DNA G4s in regulating gene transcription. This study reveals interrelations between DNA G4s and epigenomic signatures, as well as implicates DNA G4s in modulating gene transcription in rice. Our study provides valuable resources for the functional characterization or bioengineering of some of key DNA G4s in rice.

Transcriptomic and metabolic regulatory network characterization of drought responses in tobacco.

Drought stress usually causes huge economic losses for tobacco industries. Drought stress exhibits multifaceted impacts on tobacco systems through inducing changes at different levels, such as physiological and chemical changes, changes of gene transcription and metabolic changes. Understanding how plants respond and adapt to drought stress helps generate engineered plants with enhanced drought resistance. In this study, we conducted multiple time point-related physiological, biochemical,transcriptomic and metabolic assays using K326 and its derived mutant 28 (M28) with contrasting drought tolerance. Through integrative analyses of transcriptome and metabolome,we observed dramatic changes of gene expression and metabolic profiles between M28 and K326 before and after drought treatment. we found that some of DEGs function as key enzymes responsible for ABA biosynthesis and metabolic pathway, thereby mitigating impairment of drought stress through ABA signaling dependent pathways. Four DEGs were involved in nitrogen metabolism, leading to synthesis of glutamate (Glu) starting from NO-3 /NO-2 that serves as an indicator for stress responses. Importantly, through regulatory network analyses, we detected several drought induced TFs that regulate expression of genes responsible for ABA biosynthesis through network, indicating direct and indirect involvement of TFs in drought responses in tobacco. Thus, our study sheds some mechanistic insights into how plant responding to drought stress through transcriptomic and metabolic changes in tobacco. It also provides some key TF or non-TF gene candidates for engineering manipulation for breeding new tobacco varieties with enhanced drought tolerance.

Ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and Triticum petropavlovskyi with long outer glumes and grains.

Polish wheat (Triticum polonicum) is a unique tetraploid wheat species characterized by an elongated outer glume. The genetic control of the long-glume trait by a single semi-dominant locus, P1 (from Polish wheat), was established more than 100 years ago, but the underlying causal gene and molecular nature remain elusive. Here, we report the isolation of VRT-A2, encoding an SVP-clade MADS-box transcription factor, as the P1 candidate gene. Genetic evidence suggests that in T. polonicum, a naturally occurring sequence rearrangement in the intron-1 region of VRT-A2 leads to ectopic expression of VRT-A2 in floral organs where the long-glume phenotype appears. Interestingly, we found that the intron-1 region is a key ON/OFF molecular switch for VRT-A2 expression, not only because it recruits transcriptional repressors, but also because it confers intron-mediated transcriptional enhancement. Genotypic analyses using wheat accessions indicated that the P1 locus is likely derived from a single natural mutation in tetraploid wheat, which was subsequently inherited by hexaploid T. petropavlovskyi. Taken together, our findings highlight the promoter-proximal intron variation as a molecular basis for phenotypic differentiation, and thus species formation in Triticum plants.

Teacher's Emotional Display Affects Students' Perceptions of Teacher's Competence, Feelings, and Productivity in Online Small-Group Discussions.

Teacher's emotions have been shown to be highly important in the quality and effectiveness of teaching and learning. There is a recognized need to examine the essential role of teacher's emotions in students' academic achievement. However, the influence of teacher's displays of emotions on students' outcomes in small-group interaction activities, especially in the online environment, has received little attention in prior research. The aim of the present study was to explore the relationship between teacher's different emotional displays and students' perceptions of the teacher's competence, as well as students' collaborative feelings and productivity in online small-group discussions. Using a three-level between-subjects design, 74 participants were randomly divided into four-member groups comprising a teacher and three other participants. All the groups were asked to discuss an open-ended realistic problem using online software, during which the teacher's display of emotions varied (positive vs. negative vs. neutral). The participants' self-reported questionnaire data (perception of the teacher's competence, students' feeling of pleasure, collaborative satisfaction, and willingness to continue collaborating) and productivity (number of effective ideas expressed within a given time) were measured to compare the participants who were exposed to different emotional displays. As expected, the results showed that the participants who received the teacher's positive emotional display reported that they experienced higher levels of pleasure during the task. However, in contrast to our expectations, those under the negative emotional display condition showed a significantly higher level of productivity in the group task. In addition, compared to emotional display, the participants' perceptions of the teacher's competence were rated significantly higher under the neutral condition, and they reported higher levels of collaborative satisfaction and greater willingness to continue collaborating with their group. The findings have the potential benefit of informing educational practice on whether teachers should display their emotions in a small-group discussion or how they should display emotions following adjustment for the relative aim of the teaching activities.

Profiling of H3K4me3 and H3K27me3 and Their Roles in Gene Subfunctionalization in Allotetraploid Cotton.

Cotton is an excellent model for studying crop polyploidization and domestication. Chromatin profiling helps to reveal how histone modifications are involved in controlling differential gene expression between A and D subgenomes in allotetraploid cotton. However, the detailed profiling and functional characterization of broad H3K4me3 and H3K27me3 are still understudied in cotton. In this study, we conducted H3K4me3- and H3K27me3-related ChIP-seq followed by comprehensively characterizing their roles in regulating gene transcription in cotton. We found that H3K4me3 and H3K27me3 exhibited active and repressive roles in regulating the expression of genes between A and D subgenomes, respectively. More importantly, H3K4me3 exhibited enrichment level-, position-, and distance-related impacts on expression levels of related genes. Distinct GO term enrichment occurred between A/D-specific and homeologous genes with broad H3K4me3 enrichment in promoters and gene bodies, suggesting that broad H3K4me3-marked genes might have some unique biological functions between A and D subgenome. An anticorrelation between H3K27me3 enrichment and expression levels of homeologous genes was more pronounced in the A subgenome relative to the D subgenome, reflecting distinct enrichment of H3K27me3 in homeologous genes between A and D subgenome. In addition, H3K4me3 and H3K27me3 marks can indirectly influence gene expression through regulatory networks with TF mediation. Thus, our study provides detailed insights into functions of H3K4me3 and H3K27me3 in regulating differential gene expression and subfunctionalization of homeologous genes, therefore serving as a driving force for polyploidization and domestication in cotton.

Pleiotropic QTL influencing spikelet number and heading date in common wheat (Triticum aestivum L.).

KEY MESSAGE: Three pleiotropic QTL regions associated with spikelet number and heading date were identified, with FT-A1 considered the candidate gene for QTspn/Hd.cau-7A. Spikelet number traits and heading date (HD) play key roles in yield improvement of wheat and its wide adaptation to different environments. Here, we used a Recombinant Inbred Lines population derived from a cross between Yi5029 (5029) and Nongda4332 (4332) to construct a high-density genetic linkage map and identify quantitative trait loci (QTL) associated with total spikelet number per spike (TSPN), fertile spikelet number per spike (FSPN), sterile spikelet number per spike (SSPN) and HD. A total of 22 environmentally stable QTL for TSPN, FSPN, SSPN and HD were identified. Notably, three pleiotropic QTL regions for TSPN and HD were detected on chromosomes 2A, 7A and 7D. The QTL associated with TSPN and HD on chromosome 7AS was designated QTspn/Hd.cau-7A. Furthermore, the candidate gene FT-A1 located in the region of QTspn/Hd.cau-7A had a single-nucleotide polymorphism (T-G) within the third exon, which might be the cause of diversity in spikelet number and HD between the two parents. Additionally, we developed a semi-thermal asymmetric reverse PCR (STARP) marker to analyze the geographical distribution and evolution of FT-A1 (T or G) alleles. This study contributes to our understanding of the molecular mechanisms of the four traits (TSPN, FSPN, SSPN and HD) and provides further insights into the genetic relationship between spikelet number traits and HD in wheat.

A Single Amino Acid Substitution in STKc_GSK3 Kinase Conferring Semispherical Grains and Its Implications for the Origin of Triticum sphaerococcum.

Six subspecies of hexaploid wheat (Triticum aestivum) have been identified, but the origin of Indian dwarf wheat (Triticum sphaerococcum), the only subspecies with round grains, is currently unknown. Here, we isolated the grain-shape gene Tasg-D1 in T sphaerococcum via positional cloning. Tasg-D1 encodes a Ser/Thr protein kinase glycogen synthase kinase3 (STKc_GSK3) that negatively regulates brassinosteroid signaling. Expression of TaSG-D1 and the mutant form Tasg-D1 in Arabidopsis (Arabidopsis thaliana) suggested that a single amino acid substitution in the Thr-283-Arg-284-Glu-285-Glu-286 domain of TaSG-D1 enhances protein stability in response to brassinosteroids, likely leading to formation of round grains in wheat. This gain-of-function mutation has pleiotropic effects on plant architecture and exhibits incomplete dominance. Haplotype analysis of 898 wheat accessions indicated that the origin of T sphaerococcum in ancient India involved at least two independent mutations of TaSG-D1 Our results demonstrate that modest genetic changes in a single gene can induce dramatic phenotypic changes.

Dissection of genetic factors underlying grain size and fine mapping of QTgw.cau-7D in common wheat (Triticum aestivum L.).

KEY MESSAGE: Thirty environmentally stable QTL controlling grain size and/or plant height were identified, among which QTgw.cau-7D was delimited into the physical interval of approximately 4.4 Mb. Grain size and plant height (PHT) are important agronomic traits in wheat breeding. To dissect the genetic basis of these traits, we conducted a quantitative trait locus (QTL) analysis using recombinant inbred lines (RILs). In total, 30 environmentally stable QTL for thousand grain weight (TGW), grain length (GL), grain width (GW) and PHT were detected. Notably, one major pleiotropic QTL on chromosome arm 3DS explained the highest phenotypic variance for TGW, GL and PHT, and two stable QTL (QGw.cau-4B and QGw.cau-7D) on chromosome arms 4BS and 7DS contributed greater effects for GW. Furthermore, the stable QTL controlling grain size (QTgw.cau-7D and QGw.cau-7D) were delimited into the physical interval of approximately 4.4 Mb harboring 56 annotated genes. The elite NILs of QTgw.cau-7D increased TGW by 12.79-21.75% and GW by 4.10-8.47% across all three environments. Collectively, these results provide further insight into the genetic basis of TGW, GL, GW and PHT, and the fine-mapped QTgw.cau-7D will be an attractive target for positional cloning and marker-assisted selection in wheat breeding programs.