Identification and validation of major-effect quantitative trait locus QMS-5B associated with male sterility in photo-thermo-sensitive genic male sterile wheat.

KEY MESSAGE: QMS-5B, a major QTL for photo-thermo-sensitive genic male sterility in wheat, was fine mapped in a 2.15 Mb region harboring a serine/threonine protein kinase gene TraesCS5B03G0887500, which was the most likely candidate gene. Genic male sterility is an essential trait in the utilization of heterosis and hybrid seed production for wheat. Currently, genic male sterile genes have been reported in wheat mutants, but the sterile genes controlling photo-thermo-sensitive genic male sterility in wheat have not been studied systematically. Here, 235 doubled haploid lines derived from a cross between photo-thermo-sensitive genic male sterile line BS462 and its restorer line CP279 were used to map male sterile gene by GenoBaits® Wheat 100 K Panel, bulked segregant exome sequencing (BSE-Seq) and wheat 660 K array. As a result, the major stable QTL on chromosome 5B, QMS-5B, was identified in all four environments, accounting for 7.3-36.4% of the phenotypic variances. Ulteriorly, QMS-5B was delimited to an approximate 2.15 Mb physical interval between KASP-5B5 and KASP-5B6 using kompetitive allele-specific PCR (KASP) markers. Within the interval, twenty-nine high-confidence genes were predicted according to Chinese Spring RefSeq v2.1. TraesCS5B03G0887500, encoding a serine/threonine protein kinase, was identified as the most likely candidate gene for QMS-5B based on weighted gene co-expression network analysis. Expression analysis confirmed that TraesCS5B03G0887500 was significantly differentially expressed in anthers of BS462 and CP279 at different stages under fertile and sterile environments. In addition, flanking KASP marker KASP-5B6 can effectively genotype male sterile lines and restorer lines, and can be used for molecular marker-assisted selection. This study provides insights into for exploring the mechanism of photo-thermo-sensitive genic male sterility in wheat.

Hsa_circRNA_0084043 promoting tumorigenesis in glioma through miR-577 sponging.

Circular RNAs (circRNAs) are important non-coding RNAs (ncRNAs) involved in the development of multiple human diseases, especially cancers. circRNA_0084043 is significantly involved in the progression of melanoma. However, whether circRNA_0084043 is associated with glioma remains unknown. In this study, the upregulation of circRNA_0084043 in glioma and the association between circRNA_0084043 and glioma grade were identified. Our results showed that circRNA_0084043 is significantly involved in the proliferative, migratory, and invasive capacities of glioma cells. The results obtained from starBase, luciferase reporter assays, RNA immunoprecipitation assays, and RNA pull-down assays demonstrated that circRNA_0084043 acts as a direct sponge for miR-577. TargetScan algorithm was used to identify potential miR-577 targets, it was found that sorting nexin 5 (SNX5) is a candidate bound to miR-577. Finally, cell experiments testified that circRNA_0084043 enhanced growth, migration and invasion of glioma through the regulation of miR-577-mediated SNX5. Taken together, we concluded that circRNA_0084043 in the miR-577/SNX5 axis can be used as a candidate target for glioma therapy.

Characterization and expression analysis of chalcone synthase gene family members suggested their roles in the male sterility of a wheat temperature-sensitive genic male sterile (TGMS) line.

Chalcone synthase (CHS), also known as the plants-specific type III polyketide synthases (PKSs), catalyzes the first key step in the biosynthesis of plant flavonoids. Flavonoids are one of the most important secondary metabolites which participate in flower pigmentation and pollen fertility. Recent reports have demonstrated the role of the CHS family in plant pollen exine formation. This study focused on the potential roles of CHS in the pollen exine formation of wheat. In the present study, a genome-wide investigation of the CHS family was carried out, and 87 CHS genes were identified in wheat. TaCHS3, TaCHS10, and TaCHS13 are wheat orthologs of Arabidopsis LESS ADHESIVE POLLEN (LAP5); TaCHS58, TaCHS64, and TaCHS67 are wheat orthologs of AtLAP6. TaCHS3, TaCHS10, and TaCHS67 showed anther-specific patterns. The expression of TaCHS3, TaCHS10, and TaCHS67 was positively co-expressed with sporopollenin biosynthetic genes, including TaCYP703A2, TaCYP704B1, TaDRL1, TaTKPR2, and TaMS2. Coincidently, the expression of TaCHS3, TaCHS10, and TaCHS67, together with those sporopollenin biosynthetic genes, were repressed at the tetrads and uninucleate stages in the temperature-sensitive genic male-sterile (TGMS) line BS366 under sterile conditions. Wheat anther-specific CHS genes might participate in the exine formation of BS366 through co-expressing with sporopollenin biosynthetic genes, which will undoubtedly provide knowledge of the roles of CHS in wheat pollen development.

BRI1 EMS SUPPRESSOR1 genes regulate abiotic stress and anther development in wheat (Triticum aestivum L.).

BRI1 EMS SUPPRESSOR1 (BES1) family members are crucial downstream regulators that positively mediate brassinosteroid signaling, playing vital roles in the regulation of plant stress responses and anther development in Arabidopsis. Importantly, the expression profiles of wheat (Triticum aestivum L.) BES1 genes have not been analyzed comprehensively and systematically in response to abiotic stress or during anther development. In this study, we identified 23 BES1-like genes in common wheat, which were unevenly distributed on 17 out of 21 wheat chromosomes. Phylogenetic analysis clustered the BES1 genes into four major clades; moreover, TaBES1-3A2, TaBES1-3B2 and TaBES1-3D2 belonged to the same clade as Arabidopsis BES1/BZR1 HOMOLOG3 (BEH3) and BEH4, which participate in anther development. The expression levels of 23 wheat BES1 genes were assessed using real-time quantitative PCR under various abiotic stress conditions (drought, salt, heat, and cold), and we found that most TaBES1-like genes were downregulated under abiotic stress, particularly during drought stress. We therefore used drought-tolerant and drought-sensitive wheat cultivars to explore TaBES1 expression patterns under drought stress. TaBES1-3B2 and TaBES1-3D2 expression was high in drought-tolerant cultivars but substantially repressed in drought-sensitive cultivars, while TaBES1-6D presented an opposite pattern. Among genes preferentially expressed in anthers, TaBES1-3B2 and TaBES1-3D2 expression was substantially downregulated in thermosensitive genic male-sterile wheat lines compared to common wheat cultivar under sterile conditions, while we detected no obvious differences under fertile conditions. This result suggests that TaBES1-3B2 and TaBES1-3D2 might not only play roles in regulating drought tolerance, but also participate in low temperature-induced male sterility.

Efficacy of Femoral Neck System Fixation vs KHS Femoral Neck Dynamic Compression Locking Plate System in Femoral Neck Fracture.

Background: Femoral neck fracture is acknowledged as one of the common injuries in clinical orthopedics. Our study was aimed at investigating the efficacy of femoral neck fixation vs the KHS dynamic compression locking plate system in the treatment of femoral neck fracture. Methods: This was a prospective study. A toteal of 90 patients with femoral neck fracture who were admitted to The Third Hospital of Hebei Medical University in Shijiazhuang, China from August 2017 to March 2020 were enrolled in our study. The patients were randomly divided into the control group (45 patients allocated to intervention with the novel femoral neck dynamic compression locking plate system) and the study group (45 patients who underwent femoral neck system fixation). Intraoperative blood loss, surgery duration, fracture healing time and related complications in the 2 groups were monitored and evaluated. The recovery of hip joint function at different times in the 2 groups were closely monitored. Results: The 2 groups completed the surgery process, and the incision healed. All patients were followed up for 6 to 8 months, with an average follow-up time of 7.01 ± 0.21 months. Surgery duration, length of hospital stay and fracture healing time in the study group were significantly lower than in the control group (P < .05), while no significant difference was found in intraoperative blood loss between the 2 groups (P > .05). At 1 and 3 months after surgery, hip joint function in the study group was significantly higher than in the control group (P < .05), but 6 months after surgery, there was no significant difference between the 2 groups (P > .05). There were no complications in the study group, whereas 1 patient had a complication in the control group. The total incidence of complications in the study group was lower than in the control group, but the difference was not significant (P > .05). Conclusion: Femoral neck system fixation demonstrated superior efficacy to the KHS femoral neck dynamic compression locking plate system in femoral neck fracture, and is considered as a valid method for wide application.

Microspore-expressed SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen development in wheat.

Sporopollenin is one of the most structurally sophisticated and chemically recalcitrant biopolymers. In higher plants, sporopollenin is the dominant component of exine, the outer wall of pollen grains, and contains covalently linked phenolics that protect the male gametes from harsh environments. Although much has been learned about the biosynthesis of sporopollenin precursors in the tapetum, the nutritive cell layer surrounding developing microspores, little is known about how the biopolymer is assembled on the microspore surface. We identified SCULP1 (SKS clade universal in pollen) as a seed plant conserved clade of the multicopper oxidase family. We showed that SCULP1 in common wheat (Triticum aestivum) is specifically expressed in the microspore when sporopollenin assembly takes place, localized to the developing exine, and binds p-coumaric acid in vitro. Through genetic, biochemical, and 3D reconstruction analyses, we demonstrated that SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen viability. Moreover, we found that SCULP1 accumulation is compromised in thermosensitive genic male sterile wheat lines and its expression partially restored exine integrity and male fertility. These findings identified a key microspore protein in autonomous sporopollenin polymer assembly, thereby laying the foundation for elucidating and engineering sporopollenin biosynthesis.

Characterization and evolutionary analysis of phosphate starvation response genes in wheat and other major gramineous plants.

Developing cultivars with improved Pi use efficiency is essential for the sustainability of agriculture as well as the environment. Phosphate starvation response (PHR) regulators have not yet been systematically studied in wheat. This study provides the detailed characteristics of PHRs in hexaploid wheat as well as other major gramineous plants at the genome-wide level. The identified PHR proteins were divided into six subfamilies through phylogeny analysis, and a total of 63 paralogous TaPHR pairs were designated as arising from duplication events, with strong purifying selection. The promoters of TaPHRs were identified as stations for many transcription factors. Protein-protein interaction network and gene ontology enrichment analysis indicated a core biological process of cellular response to phosphate starvation. The three-dimensional structures of core PHR proteins showed a high phylogenetic relationship, but amino acid deletions in core protein domains may cause functional differentiation between rice and wheat. TaPHR3 could interact with TaSPX1 and TaSPX5 proteins, which is regarded as a novel interaction mode. Under different Pi gradient treatments, TaPHRs showed low inducible expression patterns among all subfamilies. Our study is the first to comprehensively clarify the basic properties of TaPHR proteins and might accumulate basic data for improving grain yield and environmental homeostasis.

Comparative Transcriptome Analysis Reveals Key Insights into Fertility Conversion in the Thermo-Sensitive Cytoplasmic Male Sterile Wheat.

Thermo-sensitive cytoplasmic male sterility (TCMS) plays a crucial role in hybrid production and hybrid breeding; however, there are few studies on molecular mechanisms related to anther abortion in the wheat TCMS line. In this study, FA99, a new wheat thermo-sensitive cytoplasmic male sterility line, was investigated. Fertility conversion analysis showed that FA99 was mainly controlled by temperature, and the temperature-sensitive stage was pollen mother cell formation to a uninucleate stage. Further phenotypic identification and paraffin section showed that FA99 was characterized by indehiscent anthers and aborted pollen in a sterile environment and tapetum was degraded prematurely during the tetrad period, which was the critical abortion period of FA99. The contents of O2-, H2O2, MDA and POD were significantly changed in FA99 under a sterile environment by the determination of physiological indexes. Furthermore, through transcriptome analysis, 252 differentially expressed genes were identified, including 218 downregulated and 34 upregulated genes. Based on KOG function classification, GO enrichment and KEGG pathways analysis, it was evident that significant transcriptomic changes in FA99 under different fertility environments, and the major differences were "phenylalanine metabolism", "phenylpropanoid biosynthesis", "cutin, suberine and wax biosynthesis", "phenylalanine, tyrosine and tryptophan biosynthesis" and "citrate cycle (TCA cycle)". Finally, we proposed an intriguing transcriptome-mediated pollen abortion and male sterility network for FA99. These findings provided data on the molecular mechanism of fertility conversion in thermo-sensitive cytoplasmic male sterility wheat.

Detection of seed purity of hybrid wheat using reflectance and transmittance hyperspectral imaging technology.

Chemical hybridization and genic male sterility systems are two main methods of hybrid wheat production; however, complete sterility of female wheat plants cannot be guaranteed owing to the influence of the growth stage and weather. Consequently, hybrid wheat seeds are inevitably mixed with few parent seeds, especially female seeds. Therefore, seed purity is a key factor in the popularization of hybrid wheat. However, traditional seed purity detection and variety identification methods are time-consuming, laborious, and destructive. Therefore, to establish a non-destructive classification method for hybrid and female parent seeds, three hybrid wheat varieties (Jingmai 9, Jingmai 11, and Jingmai 183) and their parent seeds were sampled. The transmittance and reflectance spectra of all seeds were collected via hyperspectral imaging technology, and a classification model was established using partial least squares-discriminant analysis (PLS-DA) combined with various preprocessing methods. The transmittance spectrum significantly improved the classification of hybrids and female parents compared to that obtained using reflectance spectrum. Specifically, using transmittance spectrum combined with a characteristic wavelength-screening algorithm, the Detrend-CARS-PLS-DA model was established, and the accuracy rates in the testing sets of Jingmai 9, Jingmai 11, and Jingmai 183 were 95.69%, 98.25%, and 97.25%, respectively. In conclusion, transmittance hyperspectral imaging combined with a machine learning algorithm can effectively distinguish female parent seeds from hybrid seeds. These results provide a reference for rapid seed purity detection in the hybrid production process. Owing to the non-destructive and rapid nature of hyperspectral imaging, the detection of hybrid wheat seed purity can be improved by online sorting in the future.

Comparative Transcriptome Analysis Reveals Hormone Signal Transduction and Sucrose Metabolism Related Genes Involved in the Regulation of Anther Dehiscence in Photo-Thermo-Sensitive Genic Male Sterile Wheat.

Anther dehiscence is an important process to release pollen and then is a critical event in pollination. In the wheat photo-thermo-sensitive genic male sterility (PTGMS) line, pollen cannot release from anther since the anther cannot dehisce during anther dehiscence stage in a sterile condition. In this study, we carried out RNA-sequencing to analyze the transcriptome of one wheat PTGMS line BS366 during anther dehiscence under fertile and sterile conditions to explore the mechanism. We identified 6306 differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) and KEGG analysis showed that DEGs were mainly related to "hormone signal transduction pathway" and "starch and sucrose metabolism". We identified 35 and 23 DEGs related hormone signal transduction and sucrose metabolism, respectively. Compared with conventional wheat Jing411, there were some changes in the contents of hormones, including JA, IAA, BR, ABA and GA3, and sucrose, during three anther dehiscence stages in the sterile condition in BS366. We performed qRT-PCR to verify the expression levels of some critical DEGs of the hormone signaling pathway and the starch and sucrose metabolism pathway. The results showed disparate expression patterns of the critical DEGs of the hormone signaling pathway and the starch and sucrose metabolism pathway in different conditions, suggesting these genes may be involved in the regulation of the anther dehiscence in BS366. Finally, we conducted a hypothesis model to reveal the regulation pathway of hormones and sucrose on anther dehiscence. The information provided new clues to the molecular mechanisms of anther dehiscence in wheat and improved wheat hybrid breeding.

The Central Circadian Clock Protein TaCCA1 Regulates Seedling Growth and Spike Development in Wheat (Triticum aestivum L.).

The biological functions of the circadian clock on growth and development have been well elucidated in model plants, while its regulatory roles in crop species, especially the roles on yield-related traits, are poorly understood. In this study, we characterized the core clock gene CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) homoeologs in wheat and studied their biological functions in seedling growth and spike development. TaCCA1 homoeologs exhibit typical diurnal expression patterns, which are positively regulated by rhythmic histone modifications including histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 9 acetylation (H3K9Ac), and histone H3 lysine 36 trimethylation (H3K36me3). TaCCA1s are preferentially located in the nucleus and tend to form both homo- and heterodimers. TaCCA1 overexpression (TaCCA1-OE) transgenic wheat plants show disrupted circadian rhythmicity coupling with reduced chlorophyll and starch content, as well as biomass at seedling stage, also decreased spike length, grain number per spike, and grain size at the ripening stage. Further studies using DNA affinity purification followed by deep sequencing [DNA affinity purification and sequencing (DAP-seq)] indicated that TaCCA1 preferentially binds to sequences similarly to "evening elements" (EE) motif in the wheat genome, particularly genes associated with photosynthesis, carbon utilization, and auxin homeostasis, and decreased transcriptional levels of these target genes are observed in TaCCA1-OE transgenic wheat plants. Collectively, our study provides novel insights into a circadian-mediated mechanism of gene regulation to coordinate photosynthetic and metabolic activities in wheat, which is important for optimal plant growth and crop yield formation.

Integrated Analysis of Microarray, Small RNA, and Degradome Datasets Uncovers the Role of MicroRNAs in Temperature-Sensitive Genic Male Sterility in Wheat.

Temperature-sensitive genic male sterile (TGMS) line Beijing Sterility 366 (BS366) has been utilized in hybrid breeding for a long time, but the molecular mechanism underlying male sterility remains unclear. Expression arrays, small RNA, and degradome sequencing were used in this study to explore the potential role of miRNA in the cold-induced male sterility of BS366. Microspore observation showed defective cell plates in dyads and tetrads and shrunken microspores at the vacuolated stage. Differential regulation of Golgi vesicle transport, phragmoplast formation, sporopollenin biosynthesis, pollen exine formation, and lipid metabolism were observed between cold and control conditions. Pollen development was significantly represented in the 352 antagonistic miRNA-target pairs in the integrated analysis of miRNA and mRNA profiles. The specific cleavage of ARF17 and TIR1 by miR160 and miR393 were found in the cold-treated BS366 degradome, respectively. Thus, the cold-mediated miRNAs impaired cell plate formation through repression of Golgi vesicle transport and phragmoplast formation. The repressed expression of ARF17 and TIR1 impaired pollen exine formation. The results of this study will contribute to our understanding of the roles of miRNAs in male sterility in wheat.

Comprehensive molecular evaluation of the histone methyltransferase gene family and their important roles in two-line hybrid wheat.

BACKGROUND: Histone methylation usually plays important roles in plant development through post-translational regulation and may provide a new visual field for heterosis. The histone methyltransferase gene family has been identified in various plants, but its members and functions in hybrid wheat related in heterosis is poorly studied. RESULTS: In this study, 175 histone methyltransferase (HMT) genes were identified in wheat, including 152 histone lysine methyltransferase (HKMT) genes and 23 protein arginine methyltransferase (PRMT) genes. Gene structure analysis, physicochemical properties and subcellular localization predictions of the proteins, exhibited the adequate complexity of this gene family. As an allohexaploid species, the number of the genes (seven HKMTs orthologous groups and four PRMTs orthologous groups) in wheat were about three times than those in diploids and showed certain degrees of conservation, while only a small number of subfamilies such as ASH-like and Su-(var) subfamilies have expanded their members. Transcriptome analysis showed that HMT genes were mainly expressed in the reproductive organs. Expression analysis showed that some TaHMT genes with different trends in various hybrid combinations may be regulated by lncRNAs with similar expression trends. Pearson correlation analysis of the expression of TaHMT genes and two yield traits indicated that four DEGs may participate in the yield heterosis of two-line hybrid wheat. ChIP-qPCR results showed that the histone modifications (H3K4me3, H3K36me3 and H3K9ac) enriched in promoter regions of three TaCCA1 genes which are homologous to Arabidopsis heterosis-related CCA1/LHY genes. The higher expression levels of TaCCA1 in F1 than its parents are positive with these histone modifications. These results showed that histone modifications may play important roles in wheat heterosis. CONCLUSIONS: Our study identified characteristics of the histone methyltransferase gene family and enhances the understanding of the evolution and function of these members in allohexaploid wheat. The causes of heterosis of two-line hybrid wheat were partially explained from the perspective of histone modifications.

Genome Wide Identification and Characterization of Wheat GH9 Genes Reveals Their Roles in Pollen Development and Anther Dehiscence.

Glycoside hydrolase family 9 (GH9) is a key member of the hydrolase family in the process of cellulose synthesis and hydrolysis, playing important roles in plant growth and development. In this study, we investigated the phenotypic characteristics and gene expression involved in pollen fertility conversion and anther dehiscence from a genomewide level. In total, 74 wheat GH9 genes (TaGH9s) were identified, which were classified into Class A, Class B and Class C and unevenly distributed on chromosomes. We also investigated the gene duplication and reveled that fragments and tandem repeats contributed to the amplification of TaGH9s. TaGH9s had abundant hormone-responsive elements and light-responsive elements, involving JA-ABA crosstalk to regulate anther development. Ten TaGH9s, which highly expressed stamen tissue, were selected to further validate their function in pollen fertility conversion and anther dehiscence. Based on the cell phenotype and the results of the scanning electron microscope at the anther dehiscence period, we found that seven TaGH9s may target miRNAs, including some known miRNAs (miR164 and miR398), regulate the level of cellulose by light and phytohormone and play important roles in pollen fertility and anther dehiscence. Finally, we proposed a hypothesis model to reveal the regulation pathway of TaGH9 on fertility conversion and anther dehiscence. Our study provides valuable insights into the GH9 family in explaining the male sterility mechanism of the wheat photo-thermo-sensitive genetic male sterile (PTGMS) line and generates useful male sterile resources for improving wheat hybrid breeding.

Systematic Analysis and Identification of Drought-Responsive Genes of the CAMTA Gene Family in Wheat (Triticum aestivum L.).

The calmodulin-binding transcription activator (CAMTA) is a Ca2+/CaM-mediated transcription factor (TF) that modulates plant stress responses and development. Although the investigations of CAMTAs in various organisms revealed a broad range of functions from sensory mechanisms to physiological activities in crops, little is known about the CAMTA family in wheat (Triticum aestivum L.). Here, we systematically analyzed phylogeny, gene expansion, conserved motifs, gene structure, cis-elements, chromosomal localization, and expression patterns of CAMTA genes in wheat. We described and confirmed, via molecular evolution and functional verification analyses, two new members of the family, TaCAMTA5-B.1 and TaCAMTA5-B.2. In addition, we determined that the expression of most TaCAMTA genes responded to several abiotic stresses (drought, salt, heat, and cold) and ABA during the seedling stage, but it was mainly induced by drought stress. Our study provides considerable information about the changes in gene expression in wheat under stress, notably that drought stress-related gene expression in TaCAMTA1b-B.1 transgenic lines was significantly upregulated under drought stress. In addition to providing a comprehensive view of CAMTA genes in wheat, our results indicate that TaCAMTA1b-B.1 has a potential role in the drought stress response induced by a water deficit at the seedling stage.

Global survey of alternative splicing and gene modules associated with fertility regulation in a thermosensitive genic male sterile wheat.

Thermosensitive genic male sterile (TGMS) wheat lines are the core of two-line hybrid systems. Understanding the mechanism that regulates male sterility in TGMS wheat lines is helpful for promoting wheat breeding. Several studies have obtained information regarding the mechanisms associated with male sterility at the transcriptional level, but it is not clear how the post-transcriptional process of alternative splicing might contribute to controlling male sterility. In this study, we performed genome-wide analyses of alternative splicing during the meiosis stage in TGMS line BS366 using PacBio and RNA-Seq hybrid sequencing. Cytological observations indicated that cytoskeleton assembly in pollen cells, calcium deposition in pollen and tapetal cells, and vesicle transport in tapetal cells were deficient in BS366. According to our cytological findings, 49 differentially spliced genes were isolated. Moreover, 25 long non-coding RNA targets and three bHLH transcription factors were identified. Weighted gene co-expression network analysis detected four candidate differentially spliced genes that had strong co-relation with the seed setting percentage, which is the direct representation of male sterility in BS366. In this study, we obtained comprehensive data regarding the alternative splicing-mediated regulation of male sterility in TGMS wheat. The candidates identified may provide the molecular basis for an improved understanding of male sterility.

Comparative transcriptome and DNA methylation analysis in temperature-sensitive genic male sterile wheat BS366.

BACKGROUND: Known as the prerequisite component for the heterosis breeding system, the male sterile line determines the hybrid yield and seed purity. Therefore, a deep understanding of the mechanism and gene network that leads to male sterility is crucial. BS366, a temperature-sensitive genic male sterile (TGMS) line, is male sterile under cold conditions (12 °C with 12 h of daylight) but fertile under normal temperature (20 °C with 12 h of daylight). RESULTS: During meiosis, BS366 was defective in forming tetrads and dyads due to the abnormal cell plate. During pollen development, unusual vacuolated pollen that could not accumulate starch grains at the binucleate stage was also observed. Transcriptome analysis revealed that genes involved in the meiotic process, such as sister chromatid segregation and microtubule-based movement, were repressed, while genes involved in DNA and histone methylation were induced in BS366 under cold conditions. MethylRAD was used for reduced DNA methylation sequencing of BS366 spikes under both cold and control conditions. The differentially methylated sites (DMSs) located in the gene region were mainly involved in carbohydrate and fatty acid metabolism, lipid metabolism, and transport. Differentially expressed and methylated genes were mainly involved in cell division. CONCLUSIONS: These results indicated that the methylation of genes involved in carbon metabolism or fatty acid metabolism might contribute to male sterility in BS366 spikes, providing novel insight into the molecular mechanism of wheat male sterility.

Comprehensive analysis of formin gene family highlights candidate genes related to pollen cytoskeleton and male fertility in wheat (Triticum aestivum L.).

BACKGROUND: Formin, a highly conserved multi-domain protein, interacts with microfilaments and microtubules. Although specifically expressed formin genes in anthers are potentially significant in research on male sterility and hybrid wheat breeding, similar reports in wheat, especially in thermo-sensitive genic male sterile (TGMS) wheat, remain elusive. RESULTS: Herein, we systematically characterized the formin genes in TGMS wheat line BS366 named TaFormins (TaFHs) and predicted their functions in inducing stress response. In total, 25 TaFH genes were uncovered, majorly localized in 2A, 2B, and 2D chromosomes. According to the neighbor-joining (NJ) method, all TaFH proteins from wheat and other plants clustered in 6 sub-groups (A-F). The modeled 3D structures of TaFH1-A/B, TaFH2-A/B, TaFH3-A/B and TaFH3-B/D were validated. And different numbers of stress and hormone-responsive regulatory elements in their 1500 base pair promoter regions were contained in the TaFH genes copies. TaFHs had specific temporal and spatial expression characteristics, whereby TaFH1, TaFH4, and TaFH5 were expressed highly in the stamen of BS366. Besides, the accumulation of TaFHs was remarkably lower in a low-temperature sterile condition (Nanyang) than fertile condition (Beijing), particularly at the early stamen development stage. The pollen cytoskeleton of BS366 was abnormal in the three stages under sterile and fertile environments. Furthermore, under different stress levels, TaFHs expression could be induced by drought, salt, abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), indole-3-acetic acid (IAA), polyethylene glycol (PEG), and low temperature. Some miRNAs, including miR167, miR1120, and miR172, interacts with TaFH genes; thus, we constructed an interaction network between microRNAs, TaFHs, phytohormone responses, and distribution of cytoskeleton to reveal the regulatory association between upstream genes of TaFH family members and sterile. CONCLUSIONS: Collectively, this comprehensive analysis provides novel insights into TaFHs and miRNA resources for wheat breeding. These findings are, therefore, valuable in understanding the mechanism of TGMS fertility conversion in wheat.

Genome-wide identification and transcriptional characterization of DNA methyltransferases conferring temperature-sensitive male sterility in wheat.

BACKGROUND: DNA methyltransferase (DMT) genes contribute to plant stress responses and development by de novo establishment and subsequent maintenance of DNA methylation during replication. The photoperiod and/or temperature-sensitive genic male sterile (P/TGMS) lines play an important role in hybrid seed production of wheat. However, only a few studies have reported on the effect of DMT genes on temperature-sensitive male sterility of wheat. Although DMT genes have been investigated in some plant species, the identification and analysis of DMT genes in wheat (Triticum aestivum L.) based on genome-wide levels have not been reported. RESULTS: In this study, a detailed overview of phylogeny of 52 wheat DMT (TaDMT) genes was presented. Homoeolog retention for TaDMT genes was significantly above the average retention rate for whole-wheat genes, indicating the functional importance of many DMT homoeologs. We found that the strikingly high number of TaDMT genes resulted mainly from the significant expansion of the TaDRM subfamily. Intriguingly, all 5 paralogs belonged to the wheat DRM subfamily, and we speculated that tandem duplications might play a crucial role in the TaDRM subfamily expansion. Through the transcriptional analysis of TaDMT genes in a TGMS line BS366 and its hybrids with the other six fertile lines under sterile and fertile conditions, we concluded that TaCMT-D2, TaMET1-B1, and TaDRM-U6 might be involved in male sterility in BS366. Furthermore, a correlation analysis showed that TaMET1-B1 might negatively regulate the expression of TaRAFTIN1A, an important gene for pollen development, so we speculated regarding an epigenetic regulatory mechanism underlying the male sterility of BS366 via the interaction between TaMET1-B1 and TaRAFTIN1A. CONCLUSIONS: Our findings presented a detailed phylogenic overview of the DMT genes and could provide novel insights into the effects of DMT genes on TGMS wheat.

Genome-Wide Characterization of OFP Family Genes in Wheat (Triticum aestivum L.) Reveals That TaOPF29a-A Promotes Drought Tolerance.

OVATE family proteins (OFPs) are plant-specific transcription factors that play important roles in plant development. Although common wheat (Triticum aestivum L.) is a major staple food worldwide, OFPs have not been systematically analyzed in this important crop. Here, we performed a genome-wide survey of OFP genes in wheat and identified 100 genes belonging to 34 homoeologous groups. Arabidopsis thaliana, rice (Oryza sativa), and wheat OFP genes were divided into four subgroups based on their phylogenetic relationships. Structural analysis indicated that only four TaOFPs contain introns. We mapped the TaOFP genes onto the wheat chromosomes and determined that TaOFP17 was duplicated in this crop. A survey of cis-acting elements along the promoter regions of TaOFP genes suggested that subfunctionalization of homoeologous genes might have occurred during evolution. The TaOFPs were highly expressed in wheat, with tissue- or organ-specific expression patterns. In addition, these genes were induced by various hormone and stress treatments. For instance, TaOPF29a-A was highly expressed in roots in response to drought stress. Wheat plants overexpressing TaOPF29a-A had longer roots and higher dry weights than nontransgenic plants under drought conditions, suggesting that this gene improves drought tolerance. Our findings provide a starting point for further functional analysis of this important transcription factor family and highlight the potential of using TaOPF29a-A to genetically engineer drought-tolerant crops.