The characteristic analysis of TaTDF1 reveals its function related to male sterility in wheat (Triticum aestivum L.).

BACKGROUND: The male sterile lines are an important foundation for heterosis utilization in wheat (Triticum aestivum L.). Thereinto, pollen development is one of the indispensable processes of wheat reproductive development, and its fertility plays an important role in wheat heterosis utilization, and are usually influencing by genes. However, these key genes and their regulatory networks during pollen abortion are poorly understood in wheat. RESULTS: DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1) is a member of the R2R3-MYB family and has been shown to be essential for early tapetal layer development and pollen grain fertility in rice (Oryza sativa L.) and Arabidopsis thaliana. In order to clarify the function of TDF1 in wheat anthers development, we used OsTDF1 gene as a reference sequence and homologous cloned wheat TaTDF1 gene. TaTDF1 is localized in the nucleus. The average bolting time of Arabidopsis thaliana overexpressed strain (TaTDF1-OE) was 33 d, and its anther could be colored normally by Alexander staining solution, showing red. The dominant Mosaic suppression silence-line (TaTDF1-EAR) was blue-green in color, and the anthers were shrimpy and thin. The TaTDF1 interacting protein (TaMAP65) was confirmed using Yeast Two-Hybrid Assay (Y2H) and Bimolecular-Fluorescence Complementation (BiFC) experiments. The results showed that downregulated expression of TaTDF1 and TaMAP65 could cause anthers to be smaller and shrunken, leading to pollen abortion in TaTDF1 wheat plants induced by virus-induced gene-silencing technology. The expression pattern of TaTDF1 was influenced by TaMAP65. CONCLUSIONS: Thus, systematically revealing the regulatory mechanism of wheat TaTDF1 during anther and pollen grain development may provide new information on the molecular mechanism of pollen abortion in wheat.

A Systematic Review and Developmental Perspective on Origin of CMS Genes in Crops.

Cytoplasmic male sterility (CMS) arises from the incompatibility between the nucleus and cytoplasm as typical representatives of the chimeric structures in the mitochondrial genome (mitogenome), which has been extensively applied for hybrid seed production in various crops. The frequent occurrence of chimeric mitochondrial genes leading to CMS is consistent with the mitochondrial DNA (mtDNA) evolution. The sequence conservation resulting from faithfully maternal inheritance and the chimeric structure caused by frequent sequence recombination have been defined as two major features of the mitogenome. However, when and how these chimeric mitochondrial genes appear in the context of the highly conserved reproduction of mitochondria is an enigma. This review, therefore, presents the critical view of the research on CMS in plants to elucidate the mechanisms of this phenomenon. Generally, distant hybridization is the main mechanism to generate an original CMS source in natural populations and in breeding. Mitochondria and mitogenomes show pleomorphic and dynamic changes at key stages of the life cycle. The promitochondria in dry seeds develop into fully functioning mitochondria during seed imbibition, followed by massive mitochondria or mitogenome fusion and fission in the germination stage along with changes in the mtDNA structure and quantity. The mitogenome stability is controlled by nuclear loci, such as the nuclear gene Msh1. Its suppression leads to the rearrangement of mtDNA and the production of heritable CMS genes. An abundant recombination of mtDNA is also often found in distant hybrids and somatic/cybrid hybrids. Since mtDNA recombination is ubiquitous in distant hybridization, we put forward a hypothesis that the original CMS genes originated from mtDNA recombination during the germination of the hybrid seeds produced from distant hybridizations to solve the nucleo-cytoplasmic incompatibility resulting from the allogenic nuclear genome during seed germination.

Understanding the role of P-type ATPases in regulating pollen fertility and development in pigeonpea.

The P-type ATPase superfamily genes are the cation and phospholipid pumps that transport ions across the membranes by hydrolyzing ATP. They are involved in a diverse range of functions, including fundamental cellular events that occur during the growth of plants, especially in the reproductive organs. The present work has been undertaken to understand and characterize the P-type ATPases in the pigeonpea genome and their potential role in anther development and pollen fertility. A total of 59 P-type ATPases were predicted in the pigeonpea genome. The phylogenetic analysis classified the ATPases into five subfamilies: eleven P1B, eighteen P2A/B, fourteen P3A, fifteen P4, and one P5. Twenty-three pairs of P-type ATPases were tandemly duplicated, resulting in their expansion in the pigeonpea genome during evolution. The orthologs of the reported anther development-related genes were searched in the pigeonpea genome, and the expression profiling studies of specific genes via qRT-PCR in the pre- and post-meiotic anther stages of AKCMS11A (male sterile), AKCMS11B (maintainer) and AKPR303 (fertility restorer) lines of pigeonpea was done. Compared to the restorer and maintainer lines, the down-regulation of CcP-typeATPase22 in the post-meiotic anthers of the male sterile line might have played a role in pollen sterility. Furthermore, the strong expression of CcP-typeATPase2 in the post-meiotic anthers of restorer line and CcP-typeATPase46, CcP-typeATPase51, and CcP-typeATPase52 in the maintainer lines, respectively, compared to the male sterile line, clearly indicates their potential role in developing male reproductive organs in pigeonpea.

New Observations of the Effects of the Cytoplasm of Aegilops kotschyi Boiss. in Bread Wheat Triticum aestivum L.

The cytoplasm of Aegilops kotschyi is known for the induction of male sterility and haploidy in wheat. Both systems originally appeared rather simple, but manipulation of the standard chromosome constitution of the nuclear genome revealed additional interactions. This study shows that while there is little or no allelic variation at the main fertility restorer locus Rfmulti on chromosome arm 1BS, additional genes may also be involved in the nuclear-mitochondrial genome interactions, affecting not only male fertility but also the growth rate, from pollen competition for fertilization and early endosperm divisions all the way to seed size and plant maturity. Some of these effects appear to be of a sporophytic nature; others are gametophytic. Induction of parthenogenesis by a rye inducer in conjunction with the Ae. kotschyi cytoplasm is well known. However, here we show that the cytoplasmic-nuclear interactions affect all aspects of double fertilization: producing maternal haploids from unfertilized eggs, diploids from fertilized eggs or synergids, embryo-less kernels, and fertilized eggs without fertilization of the double nucleus in the embryo sack. It is unclear how frequent the inducers of parthenogenesis are, as variation, if any, is obscured by suppressors present in the wheat genome. Genetic dissection of a single wheat accession revealed five distinct loci affecting the rate of maternal haploid production: four acting as suppressors and one as an enhancer. Only when the suppressing haplotypes are confirmed may it be possible to the identify genetic variation of haploidy inducers, map their position(s), and determine their nature and the mode of action.

Functional analysis of the CaPIPLC5 gene in the regulation of the fertility restoration in pepper.

Cytoplasmic male sterility (CMS) is a very important factor to produce hybrid seeds, and the restoration of fertility involves the expression of many fertility-related genes. Our previous study showed that the expression of CaPIPLC5 was significantly up-regulated in pepper restorer accessions and minimally expressed in sterile accessions, speculating that CaPIPLC5 is related to the restoration of fertility. In this study, we further validated the function of CaPIPLC5 in the restoration of fertility. The results showed that CaPIPLC5 was specifically expressed in the anthers of the restorer accessions with the subcellular localization in the cytoplasm. Furthermore, the expression of CaPIPLC5 was significantly higher in restorer lines and restorer combinations than that in CMS lines and their maintainer lines. Silencing CaPIPLC5 led to the number of pollen decreased, pollen grains wrinkled, and the ratio of pollen germination reduced. In addition, the joint analysis of Yeast One-Hybrid (Y1H) and Dual-Luciferase (dual-LUC) assays suggested that transcription factors such as CaARF5, CabZIP24 and CaMYB-like1, interacted with the promoter regions of CaPIPLC5, which regulated the expression of CaPIPLC5. The present results provide new insights into the study of CaPIPLC5 involved in the restoration of fertility in pepper.

Structural variations of a new fertility restorer gene, Rf20, underlie the restoration of wild abortive-type cytoplasmic male sterility in rice.

The discovery of a wild abortive-type (WA) cytoplasmic male sterile (CMS) line and breeding its restorer line have led to the commercialization of three-line hybrid rice, contributing considerably to global food security. However, the molecular mechanisms underlying fertility abortion and the restoration of CMS-WA lines remain largely elusive. In this study, we cloned a restorer gene, Rf20, following a genome-wide association study analysis of the core parent lines of three-line hybrid rice. We found that Rf20 was present in all core parental lines, but different haplotypes and structural variants of its gene resulted in differences in Rf20 expression levels between sterile and restored lines. Rf20 could restore pollen fertility in the CMS-WA line and was found to be responsible for fertility restoration in some CMS lines under high temperatures. In addition, we found that Rf20 encodes a pentatricopeptide repeat protein that competes with WA352 for binding with COX11. This interaction enhances COX11's function as a scavenger of reactive oxygen species, which in turn restores pollen fertility. Collectively, our study suggests a new action mode for pentatricopeptide repeat proteins in the fertility restoration of CMS lines, providing an essential theoretical basis for breeding robust restorer lines and for overcoming high temperature-induced fertility recovery of some CMS lines.

Comparative transcriptome analysis reveals the impact of daily temperature difference on male sterility in photo-thermo-sensitive male sterile wheat.

BACKGROUND: Photo-thermo-sensitive male sterility (PTMS), which refers to the male sterility triggered by variations in photoperiod and temperature, is a crucial element in the wheat two-line hybrid system. The development of safe production and efficient propagation for male sterile lines holds utmost importance in two-line hybrid wheat. Under the stable photoperiod condition, PTMS is mainly induced by high or low temperatures in wheat, but the effect of daily temperature difference (DTD) on the fertility conversion of PTMS lines has not been reported. Here, three BS type PTMS lines including BS108, BS138, and BS366, as well as a control wheat variety J411 were used to analyze the correlation between fertility and DTD using differentially sowing tests, photo-thermo-control experiments, and transcriptome sequencing. RESULTS: The differentially sowing tests suggested that the optimal sowing time for safe seed production of the three PTMS lines was from October 5th to 25th in Dengzhou, China. Under the condition of 12 h 12 °C, the PTMS lines were greatly affected by DTD and exhibited complete male sterility at a temperature difference of 15 °C. Furthermore, under different temperature difference conditions, a total of 20,677 differentially expressed genes (DEGs) were obtained using RNA sequencing. Moreover, through weighted gene co-expression network analysis (WGCNA) and KEGG enrichment analysis, the identified DEGs had a close association with "starch and sucrose metabolism", "phenylpropanoid biosynthesis", "MAPK signaling pathway-plant", "flavonoid biosynthesis", and "cutin, and suberine and wax biosynthesis". qRT-PCR analysis showed the expression levels of core genes related to KEGG pathways significantly decreased at a temperature difference of 15 ° C. Finally, we constructed a transcriptome mediated network of temperature difference affecting male sterility. CONCLUSIONS: The findings provide important theoretical insights into the correlation between temperature difference and male sterility, providing guidance for the identification and selection of more secure and effective PTMS lines.

Pentatricopeptide repeat 153 (PPR153) restores maize C-type cytoplasmic male sterility in conjunction with RF4.

Maize (Zea mays L.) C-type cytoplasmic male sterility (CMS-C) is a highly used CMS system for maize commercial hybrid seed production. Rf4 is the major dominant restorer gene for CMS-C. Inbreds were recently discovered which contain the restoring Rf4 allele yet are unable to restore fertility due to the lack of an additional gene required for Rf4's restoration. To find this additional gene, QTL mapping and positional cloning were performed using an inbred that contained Rf4 but was incapable of restoring CMS-C. The QTL was mapped to a 738-kb interval on chromosome 2, which contains a Pentatricopeptide Repeat (PPR) gene cluster. Allele content comparisons of the inbreds identified three potential candidate genes responsible for fertility restoration in CMS-C. Complementation via transformation of these three candidate genes showed that PPR153 (Zm00001eb114660) is required for Rf4 to restore fertility to tassels. The PPR153 sequence is present in B73 genome, but it is not capable of restoring CMS-C without Rf4. Analysis using NAM lines revealed that Rf4 requires the presence of PPR153 to restore CMS-C in diverse germplasms. This research uncovers a major CMS-C genetic restoration pathway and can be used for identifying inbreds suitable for maize hybrid production with CMS-C cytoplasm.

Lipid and sugar metabolism play an essential role in pollen development and male sterility: a case analysis in Brassica napus.

AIMS: The genic male sterility (GMS) system is an important strategy for generating heterosis in plants. To better understand the essential role of lipid and sugar metabolism and to identify additional candidates for pollen development and male sterility, transcriptome and metabolome analysis of a GMS line of 1205AB in B. napus was used as a case study. DATA RESOURCES GENERATED: To characterize the GMS system, the transcriptome and metabolome profiles were generated for 24 samples and 48 samples of 1205AB in B. napus, respectively. Transcriptome analysis yielded a total of 156.52 Gb of clean data and revealed the expression levels of 109,541 genes and 8,501 novel genes. In addition, a total of 1,353 metabolites were detected in the metabolomic analysis, including 784 in positive ion mode and 569 in negative ion mode. KEY RESULTS: A total of 15,635 differentially expressed genes (DEGs) and 83 differential metabolites (DMs) were identified from different comparison groups, most of which were involved in lipid and sugar metabolism. The combination of transcriptome and metabolome analysis revealed 49 orthologous GMS genes related to lipid metabolism and 46 orthologous GMS genes related to sugar metabolism, as well as 45 novel genes. UTILITY OF THE RESOURCE: The transcriptome and metabolome profiles and their analysis provide useful reference data for the future discovery of additional GMS genes and the development of more robust male sterility breeding systems for use in the production of plant hybrids.

Expediting Next-Generation Hybrid Technology in Recalcitrant Maize Inbreds through In Vivo Targeted Activity of CRISPR/Cas9.

The Manipulated Genic Male Sterile Maintainer (MGM) system, a next-generation hybrid seed technology, enables efficient production of sortable seeds from genic male sterile (GMS) lines. However, implementing robust MGM systems in commercial maize inbred lines requires stable transformation, a genotype-specific and laborious process. This study aimed to integrate MGM technology into the commercial maize inbred line Z372, developing both GMS and MGM lines. We utilized the MGM line ZC01-3A-7, which contains the MS26ΔE5 editor T-DNA and MGM T-DNA, previously established in the highly transformable ZC01 recipient plants. Through a combination of crossing and backcrossing with Z372, we targeted the fertility gene Ms26 within the Z372 genome for mutation using the in vivo CRISPR/Cas9 activity within the MS26ΔE5 editor T-DNA construct. This approach facilitated precise editing of the Ms26 locus, minimizing linkage drag associated with the Ms26 mutation. Whole-genome SNP analysis achieved a 98.74% recovery rate for GMS and 96.32% for MGM in the BC2F2 generation. Importantly, the Z372-GMS line with the ms26ΔE5 mutation is non-transgenic, avoiding linkage drag and demonstrating production readiness. This study represents a significant advancement in maize breeding, enabling the rapid generation of GMS and MGM lines for efficient hybrid seed production.

Integrated transcriptomic and metabolomic analysis provides insight into the pollen development of CMS-D1 rice.

BACKGROUND: Cytoplasmic male sterility (CMS) has greatly improved the utilization of heterosis in crops due to the absence of functional male gametophyte. The newly developed sporophytic D1 type CMS (CMS-D1) rice exhibits unique characteristics compared to the well-known sporophytic CMS-WA line, making it a valuable resource for rice breeding. RESULTS: In this research, a novel CMS-D1 line named Xingye A (XYA) was established, characterized by small, transparent, and shriveled anthers. Histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays conducted on anthers from XYA and its maintainer line XYB revealed that male sterility in XYA is a result of delayed degradation of tapetal cells and abnormal programmed cell death (PCD) of microspores. Transcriptome analysis of young panicles revealed that differentially expressed genes (DEGs) in XYA, compared to XYB, were significantly enriched in processes related to chromatin structure and nucleosomes during the microspore mother cell (MMC) stage. Conversely, processes associated with sporopollenin biosynthesis, pollen exine formation, chitinase activity, and pollen wall assembly were enriched during the meiosis stage. Metabolome analysis identified 176 specific differentially accumulated metabolites (DAMs) during the meiosis stage, enriched in pathways such as α-linoleic acid metabolism, flavone and flavonol biosynthesis, and linolenic acid metabolism. Integration of transcriptomic and metabolomic data underscored the jasmonic acid (JA) biosynthesis pathway was significant enriched in XYA during the meiosis stage compared to XYB. Furthermore, levels of JA, MeJA, OPC4, OPDA, and JA-Ile were all higher in XYA than in XYB at the meiosis stage. CONCLUSIONS: These findings emphasize the involvement of the JA biosynthetic pathway in pollen development in the CMS-D1 line, providing a foundation for further exploration of the molecular mechanisms involved in CMS-D1 sterility.

Cloning and functional verification of the male sterile gene BrQRT3 in Chinese cabbage.

Chinese cabbage is a cross-pollinated crop with significant heterosis, and male sterile lines are an important way to produce hybrid seeds. In this study, a male sterile mutant msm0795 was identified in an EMS-mutagenized population of Chinese cabbage. Cytological observations revealed that the microspores failed to separate after the tetrad stage, and thus developed into abnormal pollen grains, resulting in anther abortion. MutMap combined with Kompetitive Allele Specific PCR genotyping showed that BraA01g011280.3.5 C was identified as the candidate gene, which encodes polygalacturonase QRT3 and plays a direct role in the degradation of pollen mother cell wall during microspore development, named BrQRT3. Subcellular localization and expression analyses demonstrated that BrQRT3 was localized in the cell membrane and was ubiquitously expressed in roots, stems, leaves, flower buds, and flowers, but the expression of BrQRT3 was gradually suppressed with the anther development. Ectopic expression confirmed that over-expression of BrQRT3 in qrt3 background Arabidopsis mutant can rescue the pollen defects caused by loss of AtQRT3 function. It is the first time to achieve a male sterile mutant caused by the mutation of BrQRT3 in Chinese cabbage. These findings contribute to elucidate the mechanism of BrQRT3 in regulating stamen development of Chinese cabbage.

GhTKPR1_8 functions to inhibit anther dehiscence and reduce pollen viability in cotton.

Sporopollenin, as the main component of the pollen exine, is a highly resistant polymer that provides structural integrity under unfavourable environmental conditions. Tetraketone α-pyrone reductase 1 (TKPR1) is essential for sporopollenin formation, catalyzing the reduction of tetraketone carbonyl to hydroxylated α-pyrone. The functional role of TKPR1 in male sterility has been reported in flowering plants such as maize, rice, and Arabidopsis. However, the molecular cloning and functional characterization of TKPR1 in cotton remain unaddressed. In this study, we identified 68 TKPR1s from four cotton species, categorized into three clades. Transcriptomics and RT-qPCR demonstrated that GhTKPR1_8 exhibited typical expression patterns in the tetrad stage of the anther. GhTKPR1_8 was localized to the endoplasmic reticulum. Moreover, ABORTED MICROSPORES (GhAMS) transcriptionally activated GhTKPR1_8 as indicated by luciferase complementation tests. GhTKPR1_8-knockdown inhibited anther dehiscence and reduced pollen viability in cotton. Additionally, overexpression of GhTKPR1_8 in the attkpr1 mutant restored its male sterile phenotype. This study offers novel insights into the investigation of TKPR1 in cotton while providing genetic resources for studying male sterility.

Identification and characterization of the CRK gene family in the wheat genome and analysis of their expression profile in response to high temperature-induced male sterility.

Cysteine-rich receptor-like kinases (CRKs) play many important roles during plant development, including defense responses under both biotic and abiotic stress, reactive oxygen species (ROS) homeostasis, callose deposition and programmed cell death (PCD). However, there are few studies on the involvement of the CRK family in male sterility due to heat stress in wheat (Triticum aestivum L.). In this study, a genome-wide characterization of the CRK family was performed to investigate the structural and functional attributes of the wheat CRKs in anther sterility caused by heat stress. A total of 95 CRK genes were unevenly distributed on 18 chromosomes, with the most genes distributed on chromosome 2B. Paralogous homologous genes with Ka/Ks ratios less than 1 may have undergone strong purifying selection during evolution and are more functionally conserved. The collinearity analysis results of CRK genes showed that wheat and Arabidopsis (A. thaliana), foxtail millet, Brachypodium distachyon (B. distachyon), and rice have three, 12, 15, and 11 pairs of orthologous genes, respectively. In addition, the results of the network interactions of genes and miRNAs showed that five miRNAs were in the hub of the interactions map, namely tae-miR9657b-5p, tae-miR9780, tae-miR9676-5p, tae-miR164, and tae-miR531. Furthermore, qRT-PCR validation of the six TaCRK genes showed that they play key roles in the development of the mononuclear stage anthers, as all six genes were expressed at highly significant levels in heat-stressed male sterile mononuclear stage anthers compared to normal anthers. We hypothesized that the TaCRK gene is significant in the process of high-temperature-induced sterility in wheat based on the combination of anther phenotypes, paraffin sections, and qRT-PCR data. These results improve our understanding of their relationship.

The Dark Side of the pollen: BSA-seq identified genomic regions linked to male sterility in globe artichoke.

Globe artichoke (Cynara cardunculus var. scolymus; 2n = 2x = 34) is a food crop consumed for its immature flower heads. Traditionally, globe artichoke varietal types are vegetatively propagated. However, seed propagation makes it possible to treat the crop as annual, increasing field uniformity and reducing farmers costs, as well as pathogens diffusion. Despite globe artichoke's significant agricultural value and the critical role of heterosis in the development of superior varieties, the production of hybrids remains challenging without a reliable system for large-scale industrial seed production. Male sterility (MS) presents a promising avenue for overcoming these challenges by simplifying the hybridization process and enabling cost-effective seed production. However, within the Cynara genus, genic male sterility has been linked to three recessive loci in globe artichoke, with no definitive genetic mechanism elucidated to date. A 250 offsprings F2 population, derived from a cross between a MS globe artichoke and a male fertile (MF) cultivated cardoon (C. cardunculus var. altilis) and fitting a monogenic segregation model (3:1), was analyzed through BSA-seq, aiming at the identification of genomic regions/genes affecting male sterility. Four QTL regions were identified on chromosomes 4, 12, and 14. By analyzing the sequence around the highest pick on chromosome 14, a cytochrome P450 (CYP703A2) was identified, carrying a deleterious substitution (R/Q) fixed in the male sterile parent. A single dCAPS marker was developed around this SNP, allowing the discrimination between MS and MF genotypes within the population, suitable for applications in plant breeding programs. A 3D model of the protein was generated by homology modeling, revealing that the mutated amino acid is part of a highly conserved motif crucial for protein folding.

Characterization and validation of TaAGL66, a gene related to fertility conversion of wheat in the presence of Aegilops kotschyi cytoplasm.

MAIN CONCLUSION: TaAGL66, a MADS-box transcription factor highly expressed in fertile anthers of KTM3315A, regulates anther and/or pollen development, as well as male fertility in wheat with Aegilops kotschyi cytoplasm. Male sterility, as a string of sophisticated biological processes in higher plants, is commonly regulated by transcription factors (TFs). Among them, MADS-box TFs are mainly participated in the processes of floral organ formation and pollen development, which are tightly related to male sterility, but they have been little studied in the reproductive development in wheat. In our study, TaAGL66, a gene that was specifically expressed in spikes and highly expressed in fertile anthers, was identified by RNA sequencing and the expression profiles data of these genes, and qRT-PCR analyses, which was localized to the nucleus. Silencing of TaAGL66 under fertility condition in KTM3315A, a thermo-sensitive male sterile line with Ae. kotschyi cytoplasm, displayed severe fertility reduction, abnormal anther dehiscence, defective pollen development, decreased viability, and low seed-setting. It can be concluded that TaAGL66 plays an important role in wheat pollen development in the presence of Ae. kotschyi cytoplasm, providing new insights into the utilization of male sterility.

Identification and fine mapping of Brmmd1 gene controlling recessive genic male sterility in Brassica rapa L.

Recessive genic male sterility (RGMS) provides an effective approach for the commercial exploitation of heterosis, especially in Brassica crops. Although some artificial RGMS mutants have been reported in B. rapa, no causal genes derived from these natural mutants have been identified so far. In this study, a spontaneous RGMS mutant Bcajh97-01A derived from the 'Aijiaohuang' line traced back to the 1980 s was identified. Genetic analysis revealed that the RGMS trait was controlled by a single locus in the Bcajh97-01A/B system. Bulk segregant analysis (BSA) in combination with linkage analysis was employed to delimit the causal gene to an approximate 129 kb interval on chromosome A02. The integrated information of transcriptional levels and the predicted genes in the target region indicated that the Brmmd1 (BraA02g017420) encoding a PHD-containing nuclear protein was the most likely candidate gene. A 374 bp miniature inverted-repeat transposable element (MITE) was inserted into the first exon to prematurely stop the Brmmd1 gene translation, thus blocking the normal expression of this gene at the tetrad stage in the Bcajh97-01A. Additionally, a co-segregating structure variation (SV) marker was developed to rapidly screen the RGMS progenies from Bcajh97-01A/B system. Our findings reveal that BraA02g017420 is the causal gene responsible for the RGMS trait. This study lays a foundation for marker-assisted selection and further molecular mechanism exploration of pollen development in B. rapa.

Transcriptome analysis reveals the potential lncRNA-mRNA modules involved in genetic male sterility and fertility of Chinese cabbage (brassica rapa L. ssp. pekinensis).

BACKGROUND: Long non-coding RNAs (lncRNAs) play a crucial role in regulating gene expression vital for the growth and development of plants. Despite this, the role of lncRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) pollen development and male fertility remains poorly understood. RESULTS: In this study, we characterized a recessive genic male sterile mutant (366-2 S), where the delayed degradation of tapetum and the failure of tetrad separation primarily led to the inability to form single microspores, resulting in male sterility. To analyze the role of lncRNAs in pollen development, we conducted a comparative lncRNA sequencing using anthers from the male sterile mutant line (366-2 S) and the wild-type male fertile line (366-2 F). We identified 385 differentially expressed lncRNAs between the 366-2 F and 366-2 S lines, with 172 of them potentially associated with target genes. To further understand the alterations in mRNA expression and explore potential lncRNA-target genes (mRNAs), we performed comparative mRNA transcriptome analysis in the anthers of 366-2 S and 366-2 F at two stages. We identified 1,176 differentially expressed mRNAs. Remarkably, GO analysis revealed significant enrichment in five GO terms, most notably involving mRNAs annotated as pectinesterase and polygalacturonase, which play roles in cell wall degradation. The considerable downregulation of these genes might contribute to the delayed degradation of tapetum in 366-2 S. Furthermore, we identified 15 lncRNA-mRNA modules through Venn diagram analysis. Among them, MSTRG.9997-BraA04g004630.3 C (ß-1,3-glucanase) is associated with callose degradation and tetrad separation. Additionally, MSTRG.5212-BraA02g040020.3 C (pectinesterase) and MSTRG.13,532-BraA05g030320.3 C (pectinesterase) are associated with cell wall degradation of the tapetum, indicating that these three candidate lncRNA-mRNA modules potentially regulate pollen development. CONCLUSION: This study lays the foundation for understanding the roles of lncRNAs in pollen development and for elucidating their molecular mechanisms in regulating male sterility in Chinese cabbage.

The vesicle trafficking gene, OsRab7, is critical for pollen development and male fertility in cytoplasmic male-sterility rice.

Rice cytoplasmic male sterility (CMS) provides an exceptional model for studying genetic interaction within plant nuclei given its inheritable trait of non-functional male gametophyte. Gaining a comprehensive understanding of the genes and pathways associated with the CMS mechanism is imperative for improving the vigor of hybrid rice agronomically, such as its productivity. Here, we observed a significant decrease in the expression of a gene named OsRab7 in the anther of the CMS line (SJA) compared to the maintainer line (SJB). OsRab7 is responsible for vesicle trafficking and loss function of OsRab7 significantly reduced pollen fertility and setting rate relative to the wild type. Meanwhile, over-expression of OsRab7 enhanced pollen fertility in the SJA line while a decrease in its expression in the SJB line led to the reduced pollen fertility. Premature tapetum and abnormal development of microspores were observed in the rab7 mutant. The expression of critical genes involved in tapetum development (OsMYB103, OsPTC1, OsEAT1 and OsAP25) and pollen development (OsMSP1, OsDTM1 and OsC4) decreased significantly in the anther of rab7 mutant. Reduced activities of the pDR5::GUS marker in the young panicle and anther of the rab7 mutant were also observed. Furthermore, the mRNA levels of genes involved in auxin biosynthesis (YUCCAs), auxin transport (PINs), auxin response factors (ARFs), and members of the IAA family (IAAs) were all downregulated in the rab7 mutant, indicating its impact on auxin signaling and distribution. In summary, these findings underscore the importance of OsRab7 in rice pollen development and its potential link to cytoplasmic male sterility.

Functional study of the soybean stamen-preferentially expressed gene GmFLA22a in regulating male fertility.

China has a high dependence on soybean imports, yield increase at a faster rate is an urgent problem that need to be solved at present. The application of heterosis is one of the effective ways to significantly increase crop yield. In recent years, the development of an intelligent male sterility system based on recessive nuclear sterile genes has provided a potential solution for rapidly harnessing the heterosis in soybean. However, research on male sterility genes in soybean has been lagged behind. Based on transcriptome data of soybean floral organs in our research group, a soybean stamen-preferentially expressed gene GmFLA22a was identified. It encodes a fasciclin-like arabinogalactan protein with the FAS1 domain, and subcellular localization studies revealed that it may play roles in the endoplasmic reticulum. Take advantage of the gene editing technology, the Gmfla22a mutant was generated in this study. However, there was a significant reduction in the seed-setting rate in the mutant plants at the reproductive growth stage. The pollen viability and germination rate of Gmfla22a mutant plants showed no apparent abnormalities. Histological staining demonstrated that the release of pollen grains in the mutant plants was delayed and incomplete, which may due to the locule wall thickening in the anther development. This could be the reason of the reduced seed-setting rate in Gmfla22a mutants. In summary, our study has preliminarily revealed that GmFLA22a may be involved in regulating soybean male fertility. It provides crucial genetic materials for further uncovering its molecular function and gene resources and theoretical basis for the utilization of heterosis in soybean.