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Abstract Background Two-line hybrid wheat technology system is one way to harness wheat heterosis both domestically and internationally. Seed vigor is a crucial parameter for assessing seed quality, as enhanced seed vigor can lead to yield increments of over 20% to a certain extent. MicroRNAs (miRNAs) were known to participate in the development and vigor of seed in plants, but its impact on seed vigor in two-line hybrid wheat remains poorly elucidated. Results The hybrid (BS1453/11GF5135) wheat exhibited superiority in seed vigor and anti-aging capacity, compared to its male parent (11GF5135, MP) and female parent (BS1453, FP). We identified four miRNAs associated with seed vigor, all of which are novel miRNAs. The majority of targets of miRNAs were related to ubiquitin ligases, kinases, sucrose synthases and hydrolases, involving in starch and sucrose metabolism, hydrolysis, catalysis, plant hormone signal transduction, and other pathways, which played crucial roles in seed development. Additionally, we also found miR531 was differentially expressed in both male parent and hybrid, and its target gene was a component of the E1 subunit of α-ketoate dehydrogenase complex, which interacted with dihydrolipoamide acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). Finally, We established a presumptive interaction model to speculate the relationship of miR531 and seed vigor. Conclusions This study analyzed the seed vigor of two-line hybrid wheat, and screened seed vigor-related miRNAs. Meanwhile speculated the genetic relationship of hybrid and parents, in terms of miRNAs. Consequently, the present study provides new insights into the miRNA-mediated gene and protein interaction network that regulates seed vigor. These findings hold significance for enhancing the yield and quality of two-line hybrid wheat, facilitating its future applications.

With the application of hybrid wheat, lodging is becoming one of the major factors limiting high yield in its production. However, few studies have focused on combining ability and heterosis analysis of stem-related traits. In this study, 24 crosses were made according to NCII genetic design, using the three (photo-sensitive male sterile lines)×eight (restorer lines) incomplete diallel crosses. The length of basal second internode (LBSI) and breaking strength of basal second internode (BSBSI) as well as other stem-related traits were used to perform the principal component analysis (PCA), combining ability and heterosis analysis. The PCA results showed that the variables could be classified into two main factors, which were named as the positive factor (factor 1) and the negative factor (factor 2), and accounted for 52.3 and 33.2%, respectively, of the total variance in different variables, combined with the analysis for index weight indicated that the factor 1-related traits play positive roles in lodging resistance formation of hybrids. Combining ability variance analysis indicated that its genetic performance was mainly dominated by additive gene effects, and the hybrid combinations with higher lodging resistance can be selected by using of 14GF6085 (R1), 14GF6343-2 (R4), 14GF6937 (R6), 14GF7433-1 (R7), and BS1086 (M3), which are with the features with lower general combining ability (GCA) effects of factor 2-related traits whereas higher GCA effects of factor 1-related traits. The heterosis analysis showed that the wide range of heterosis varied with the traits and combinations, and GCA or specific combining ability (SCA) effects of factor 1-related traits except wall thickness of basal second internode (WTBSI) were positively and closely related to the heterosis of lodging resistance. Generally, the correlation coefficients of heterosis to GCA effects of sterile lines (GCAm) of factor 1-related traits are significantly higher than that to GCA of restorer lines (GCAr) and SCA, combined with the higher GCAm variance values of factor 1-related traits compared to GCAr, the GCAm of factor 1-related traits should be particularly considered when breeding hybrid combinations. The heritability analysis showed that the narrow-sense heritability of the diameter of basal second internode (DBSI) and the center of gravity height (TCGH) were obviously lower (

Abstract 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.

Abstract 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.

Abstract 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.

The pollen fertility of photoperiod/temperature sensitive genic male sterile (P/TGMS) wheat is controlled by light and/or temperature. Circular RNA (circRNA) and long non-coding RNA (lncRNA) are known to participate in the development of anthers in plants, but their impact on male sterility in the P/TGMS line is not well understood. In this study, we carried out high-throughput sequencing to investigate the differential expression of lncRNAs and circRNAs and their biological functions in anthers of photo-thermosensitive genic male sterile (PTGMS) wheat line BS366-42L during the transition phase of male fertility under four different photoperiod and temperature treatments. Eight lncRNAs, 40 mRNAs and three circRNAs were screened out and thought as essential candidates that closely related to male sterility. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential functions of differentially expressed RNAs. The results indicated that carbohydrate-related metabolism was important for male sterility in the wheat PTGMS line BS366-42L. lncRNA/circRNA-mRNA-miRNA (ceRNA) integrate networks were constructed to reflect their complex inner association with male sterility. Our study provides a systematic perspective on the potential function of RNAs in male fertility in PTGMS lines of wheat.

Because of the yield increase of 3.5–15% compared to conventional wheat, hybrid wheat is considered to be one of the main ways to greatly improve the wheat yield in the future. In this study, we performed a principal component analysis (PCA) on two-line hybrids wheat and their parents using the grain weight (GW), the length of spike (LS), the kernel number of spike (KSN), and spike number (SPN) as variables. The results showed that the variables could be classified into three main factors, the weight factor (factor 1), the quantity factor 1 (factor 2) and the quantity factor 2 (factor 3), which accounted for 37.1, 22.6 and 18.5%, respectively of the total variance in different agronomic variables, suggesting that the GW is an important indicator for evaluating hybrid combinations, and the grain weight of restorer line (RGW) could be used as a reference for parents selection. The hybrid combination with a higher score in factor 1 direction and larger mid-parent heterosis (MPH) of the GW and its parents were used to carry out the analysis of grain filling, 1-aminocylopropane-1-carboxylicacid (ACC) and polyamine synthesis related genes. The results suggested that the GW of superior grain was significantly higher than that of inferior grains in BS1453×JS1 (H) and its parents. Both grain types showed a weight of H between BS1453 (M) and JS1(R), and a larger MPH, which may be caused by their differences in the active filling stage and the grain filling rate. The ADP-glucose pyrophosphorylase (AGPase), granule bound starch synthase I (GBSSI), starch synthase III (SSS), and starch branching enzyme-I (SBE-I) expression levels of H were intermediated between M and R, which might be closely related to MPH formation of the GW. Compared with R and H, the GW of M at maturity was the lowest. The expression levels of spermidine synthase 2 (Spd2), ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) had significantly positive correlations with the grain filling rate (r=0.77*, 0.51*, 0.59*), suggesting their major roles in the grain filling and heterosis formation. These provide a theoretical basis for improving the GW of photo-thermo-sensitive male sterile lines (PTSMSL) by changing the endogenous polyamine synthesis in commercial applications.

Abstract Background MYC transcriptional factors are members of the bHLH (basic helix-loop-helix) superfamily, and play important roles in plant growth and development. Recent studies have revealed that some MYCs are involved in the crosstalk between Jasmonic acid regulatory pathway and light signaling in Arabidopsis, but such kinds of studies are rare in wheat, especially in photo-thermo-sensitive genic male sterile (PTGMS) wheat line. Results 27 non-redundant MYC gene copies, which belonged to 11 TaMYC genes, were identified in the whole genome of wheat (Chinese Spring). These gene copies were distributed on 13 different chromosomes, respectively. Based on the results of phylogenetic analysis, 27 TaMYC gene copies were clustered into group I, group III, and group IV. The identified TaMYC genes copies contained different numbers of light, stress, and hormone-responsive regulatory elements in their 1500 base pair promoter regions. Besides, we found that TaMYC3 was expressed highly in stem, TaMYC5 and TaMYC9 were expressed specially in glume, and the rest of TaMYC genes were expressed in all tissues (root, stem, leaf, pistil, stamen, and glume) of the PTGMS line BS366. Moreover, we found that TaMYC3, TaMYC7, TaMYC9, and TaMYC10 were highly sensitive to methyl jasmonate (MeJA), and other TaMYC genes responded at different levels. Furthermore, we confirmed the expression profiles of TaMYC family members under different light quality and plant hormone stimuli, and abiotic stresses. Finally, we predicted the wheat microRNAs that could interact with TaMYC family members, and built up a network to show their integrative relationships. Conclusions This study analyzed the size and composition of the MYC gene family in wheat, and investigated stress-responsive and light quality induced expression profiles of each TaMYC gene in the PTGMS wheat line BS366. In conclusion, we obtained lots of important information of TaMYC family, and the results of this study was supposed to contribute novel insights and gene and microRNA resources for wheat breeding, especially for the improvement of PTGMS wheat lines.

Abstract Background COI (CORONATINE INSENSITIVE), an F-box component of the Skp1-Cullin-F-box protein (SCFCOI1) ubiquitin E3 ligase, plays important roles in the regulation of plant growth and development. Recent studies have shown that COIs are involved in pollen fertility. In this study, we identified and characterized COI genes in the wheat genome and analyzed expression patterns under abiotic stress. Results A total of 18 COI candidate sequences for 8 members of COI gene family were isolated in wheat (Triticum aestivum L.). Phylogenetic and structural analyses showed that these COI genes could be divided into seven distinct subfamilies. The COI genes showed high expression in stamens and glumes. The qRT-PCR results revealed that wheat COIs were involved in several abiotic stress responses and anther/glume dehiscence in the photoperiod-temperature sensitive genic male sterile (PTGMS) wheat line BS366. Conclusions The structural characteristics and expression patterns of the COI gene family in wheat as well as the stress-responsive and differential tissue-specific expression profiles of each TaCOI gene were examined in PTGMS wheat line BS366. In addition, we examined SA- and MeJA-induced gene expression in the wheat anther and glume to investigate the role of COI in the JA signaling pathway, involved in the regulation of abnormal anther dehiscence in the PTGMS wheat line. The results of this study contribute novel and detailed information about the TaCOI gene family in wheat and could be used as a benchmark for future studies of the molecular mechanisms of PTGMS in other crops.

MicroRNAs (miRNAs) are endogenous small RNAs which play important negative regulatory roles at both the transcriptional and post-transcriptional levels in plants. Wheat is the most commonly cultivated plant species worldwide. In this study, RNA-seq analysis was used to examine the expression profiles of miRNA in the spikelets of photo-thermosenisitive genic male sterile (PTGMS) wheat line BS366 during male fertility transition. Through mapping on their corresponding precursors, 917–7,762 novel miRNAs were found in six libraries. Six novel miRNAs were selected for examination of their secondary structures and confirmation by stem-loop RT-PCR. In a differential expression analysis, 20, 22, and 58 known miRNAs exhibited significant differential expression between developmental stages 1 (secondary sporogenous cells had formed), 2 (all cells layers were present and mitosis had ceased), and 3 (meiotic division stage), respectively, of fertile and sterile plants. Some of these differential expressed miRNAs, such as tae-miR156, tae-miR164, tae-miR171, and tae-miR172, were shown to be associated with their targets. These targets were previously reported to be related to pollen development and/or male sterility, indicating that these miRNAs and their targets may be involved in the regulation of male fertility transition in the PTGMS wheat line BS366. Furthermore, target genes of miRNA cleavage sites were validated by degradome sequencing. In this study, a possible signal model for the miRNA-mediated signaling pathway during the process of male fertility transition in the PTGMS wheat line BS366 was developed. This study provides a new perspective for understanding the roles of miRNAs in male fertility in PTGMS lines of wheat.