Male Sterility Research Articles

Genome-Wide Analysis of the Histone Modification Gene (HM) Family and Expression Investigation during Anther Development in Rice (Oryza sativa L.).

Histone modification plays a crucial role in chromatin remodeling and regulating gene expression, and participates in various biological processes, including plant development and responses to stress. Several gene families related to histone modification have been reported in various plant species. However, the identification of members and their functions in the rice (Oryza sativa L.) histone modification gene family (OsHM) at the whole-genome level remains unclear. In this study, a total of 130 OsHMs were identified through a genome-wide analysis. The OsHM gene family can be classified into 11 subfamilies based on a phylogenetic analysis. An analysis of the genes structures and conserved motifs indicates that members of each subfamily share specific conserved protein structures, suggesting their potential conserved functions. Molecular evolutionary analysis reveals that a significant number of OsHMs proteins originated from gene duplication events, particularly segmental duplications. Additionally, transcriptome analysis demonstrates that OsHMs are widely expressed in various tissues of rice and are responsive to multiple abiotic stresses. Fourteen OsHMs exhibit high expression in rice anthers and peaked at different pollen developmental stages. RT-qPCR results further elucidate the expression patterns of these 14 OsHMs during different developmental stages of anthers, highlighting their high expression during the meiosis and tetrad stages, as well as in the late stage of pollen development. Remarkably, OsSDG713 and OsSDG727 were further identified to be nucleus-localized. This study provides a fundamental framework for further exploring the gene functions of HMs in plants, particularly for researching their functions and potential applications in rice anthers' development and male sterility.

Quantitative proteomic analysis reveals hub proteins for high temperature-induced male sterility in bread wheat (Triticum aestivum L.).

High-temperature (HT) stress can induce male sterility in wheat; however, the underlying mechanisms remain poorly understood. This study examined proteomic alterations across three developmental stages between normal and HT-induced male-sterile (HT-ms) anthers in wheat. Utilizing tandem mass tags-based proteomics, we identified 2532 differentially abundant proteins (DAPs): 27 in the tetrad stage, 157 in the binuclear stage, and 2348 in the trinuclear stage. Analyses through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways indicated significant enrichment of these DAPs in seven pathways, namely phenylpropanoid biosynthesis, flavonoid biosynthesis, sphingolipid metabolism, MAPK signaling pathway, starch and sucrose metabolism, response to heat, and response to reactive oxygen species (ROS). Our results indicated the downregulation of DAPs associated with phenylpropanoid biosynthesis and starch and sucrose metabolism, which aligns with anther indehiscence and the lack of starch in HT-ms anthers. By contrast, DAPs in the ROS pathway were upregulated, which aligns with excessive ROS accumulation in HT-ms anthers. Additionally, we conducted protein-protein interaction analysis for the DAPs of these pathways, identifying 15 hub DAPs. The abundance of these hub proteins was confirmed through qRT-PCR, assessing mRNA expression levels of the corresponding transcripts. Collectively, these results offer insights into the molecular mechanisms underlying HT-induced male sterility in wheat at the proteomic level, providing a valuable resource for further research in plant sexual reproduction.

The BNB-GLID module regulates germline fate determination in Marchantia polymorpha.

Germline fate determination is a critical event in sexual reproduction. Unlike animals, plants specify the germline by reprogramming somatic cells at the late stages of their development. However, the genetic basis of germline fate determination and how it evolved during the land plant evolution are still poorly understood. Here, we report that the plant homeodomain finger protein GERMLINE IDENTITY DETERMINANT (GLID) is a key regulator of the germline specification in liverwort, Marchantia polymorpha. Loss of the MpGLID function causes failure of germline initiation, leading to the absence of sperm and egg cells. Remarkably, the overexpression of MpGLID in M. polymorpha induces the ectopic formation of cells with male germline cell features exclusively in male thalli. We further show that MpBONOBO (BNB), with an evolutionarily conserved function, can induce the formation of male germ cell-like cells through the activation of MpGLID by directly binding to its promoter. The Arabidopsis (Arabidopsis thaliana) MpGLID ortholog, MALE STERILITY1 (AtMS1), fails to replace the germline specification function of MpGLID in M. polymorpha, demonstrating that a derived function of MpGLID orthologs has been restricted to tapetum development in flowering plants. Collectively, our findings suggest the presence of the BNB-GLID module in complex ancestral land plants that has been retained in bryophytes, but rewired in flowering plants for male germline fate determination.

Temperature change regulates pollen fertility of a PTGMS rice line PA64S by modulating the ROS homeostasis and PCD within the tapetum.

Photoperiod and temperature-sensitive male sterility rice is an important line for two-line hybrid rice, and the changes in the cultivation temperature strictly control its pollen fertility. However, the mechanism by which temperature variation regulates pollen fertility is still unclear. This study obtained stable fertile PA64S(F) and sterile PA64S(S) rice from PA64S by controlling temperature changes. PA64S(F) shows a normal anther development and fertile pollen under low temperature (21°C), and PA64S(S) shows delayed degradation of the tapetum cells, leading to abnormal pollen wall formation and ubisch development under normal temperature (28°C). The accumulation of reactive oxygen species (ROS) positively correlates with the programmed cell death (PCD) process of tapetum cells. The delayed accumulation of ROS in the PA64S(S) tapetum at early stages leads to a delayed initiation of the PCD process. Importantly, we localized ascorbic acid (ASA) accumulation in the tapetum cells and determined that ASA is a major antioxidant for ROS homeostasis. ROS-inhibited accumulation plants (PA64S-ASA) demonstrated pollen sterility, higher ASA and lower ROS accumulation in the tapetum, and the absence of PCD processes in the tapetum cell. Abnormal changes in the tapetum of PA64S(S) rice disrupted metabolic pathways such as lipid metabolism, cutin and wax synthesis, sugar accumulation, and phenylpropane, affecting pollen wall formation and substance accumulation, suggesting that the timely accumulation of ROS is critical for male fertility. This study highlights the central role of ROS homeostasis in fertility alteration and also provides an avenue to address the effect of environmental temperature changes on pollen fertility in rice.

The mitochondrial genes orf113b and orf146 from Xinjiang wild rapeseed cause pollen abortion in alloplasmic male sterility

Nsa CMS, a type of cytoplasmic male sterility (CMS) in rapeseed, originates from the cross between Xinjiang wild rapeseed (Sinapis arvensis) and Xiangyou 15 (Brassica napus L.). Although this CMS variant shows promising applications, the factors contributing to its sterility and their underlying mechanisms remain unclear. To the best of our knowledge, we successfully assembled and analyzed the mitochondrial genome of 1258A (Nsa CMS) for the first time. This mitochondrial genome, spanning 263,010 bp, contains 91 genes, including 33 protein-coding and 36 orf genes. Our analysis identified a novel mitochondrial gene, orf113b, and a mutated, truncated gene, orf146, both likely linked to the sterility observed in 1258A. ORF113b and ORF146 were found to impede cell growth, disrupt gene expression associated with complexes I, III, and V of the mitochondrial oxidative phosphorylation pathway, and trigger reactive oxygen species (ROS) production. Additionally, transcriptome data analysis revealed key nuclear genes co-expressed with orf113b and orf146, suggesting that their aberrant expression may be influenced by retrograde signaling from the mitochondria. This signaling could lead to atypical programmed cell death (PCD) in the tapetum layer, resulting in pollen sterility. In conclusion, our study not only provides the first characterization of the Nsa CMS mitochondrial genome but also identifies orf113b and orf146 as crucial to pollen sterility. Furthermore, it suggests that ROS induced by these mitochondrial genes may play a central role in the abnormal regulation of nuclear genes essential for pollen development, offering new insights into the molecular mechanisms underlying Nsa CMS.

From data to decisions: Exploring family planning services utilization in Karnataka and India through the prism of NFHS-4 and NFHS-5.

Family planning (FP) services are pivotal in assessing a country's healthcare efficacy. Despite India's strides in FP promotion, disparities persist in its utilization rates. This study analyzes Karnataka's FP trends by using National Family Health Survey (NFHS) rounds 4 and 5, comparing its indicators with national averages. A retrospective record review-based descriptive study design was employed. Data from NFHS-4 (2015-16) and NFHS-5 (2019-21) were analyzed using MS Excel 365. Key variables included age at marriage, total fertility rate (TFR), and various contraceptive methods. Karnataka exhibited a stable rate of early marriages for women and a decline among men aged 25-29 years. TFR in Karnataka slightly decreased, with increased contraceptive usage and significant growth in modern contraceptive methods. However, male sterilization rates remained low. There was an improvement in health workers' engagement with female non-users and in providing information on contraceptive side effects. While Karnataka has progressed in FP, challenges such as early marriages persist. Enhancing integration, leveraging technology, and empowering women are essential for comprehensive FP services in India.

Transformation of meiotic drive into hybrid sterility in Drosophila.

Hybrid male sterility is one of the fastest evolving intrinsic reproductive barriers between recently isolated populations. A leading explanation for the evolution of hybrid male sterility involves genomic conflicts with meiotic drivers in the male germline. There are, however, few examples directly linking meiotic drive to hybrid sterility. In this study, we report that the Sex-Ratio chromosome of Drosophila pseudoobscura, which causes X-chromosome drive within the USA subspecies, causes near-complete male sterility when it is moved into the genetic background of the Bogota subspecies. In addition, we show that this new form of sterility is genetically distinct from the sterility of F1 hybrid males in crosses between USA males and Bogota females. Our observations provide a tractable study system where noncryptic drive within species is transformed into strong hybrid sterility between very young subspecies.

Epididymis-specific RNase A family genes regulate fertility and small RNA processing.

Sperm small RNAs are implicated in intergenerational transmission of paternal environmental effects. Small RNAs generated by cleavage of tRNAs, known as tRNA fragments (tRFs), are an abundant class of RNAs in mature sperm, and can be modulated by environmental conditions. The ribonuclease(s) responsible for the biogenesis of tRFs in the male reproductive tract remains unknown. Angiogenin, a member of the Ribonuclease A superfamily (RNase A), cleaves tRNAs to generate tRFs in response to cellular stress. Four paralogs of Angiogenin, namely Rnase9, Rnase10, Rnase11, and Rnase12, are specifically expressed in the epididymis-a long, convoluted tubule where sperm mature and acquire fertility and motility. The biological functions of these genes remain largely unknown. Here, by generating mice deleted for all four genes (Rnase9-12-/-, termed "KO" for Knock Out), we report that these genes regulate fertility and RNA processing. KO mice showed complete male sterility. KO sperm fertilized oocytes in vitro but failed to efficiently fertilize oocytes in vivo, likely due to an inability of sperm to pass through the utero-tubular junction. Intriguingly, there were decreased levels of fragments of tRNAs (tRFs) and rRNAs (rRNA-derived small RNAs or rsRNAs) in the KO epididymis and epididymal luminal fluid, implying that Rnase9-12 regulate the biogenesis and/or stability of tRFs and rsRNAs. Importantly, KO sperm showed a dramatic decrease in the levels of tRFs, demonstrating a role of Rnase9-12 in regulating sperm RNA composition. Together, our results reveal an unexpected role of four epididymis-specific non-canonical RNase A family genes in fertility and RNA processing.

Male condition and seminal fluid affect female host-marking behavior in the Mexican fruit fly

Mating and the transfer of seminal fluid components including male accessory glands (MAGs) proteins can affect oviposition behavior in insects. After oviposition, some species of fruit flies deposit a host-marking pheromone (HMP) on the fruit that discourages oviposition by other females of the same or different species or genus and reduces competition between larvae. However, we know very little about how mating, receiving seminal fluid, or male condition can affect female host marking behavior. Here, we tested how the physiological state of females (mated or unmated), the receipt of seminal fluid, and the condition of the male (wild or sterile) affect oviposition and host-marking behavior (HMB) in Anastrepha ludens (Diptera: Tephritidae). We also determined the efficiency of the host-marking pheromone from mated or unmated females in deterring oviposition. In a further examination of how seminal fluid may be affecting HMB we assessed if there were differences in the size of wild or sterile MAGs and the protein quantity transferred during mating. Our results indicate that receiving seminal fluid increased egg laying and increased time invested in host-marking (HM). Unmated females laid fewer eggs than mated females but invested the same amount of time in depositing host-marking pheromone, which had similar effectiveness in deterring oviposition as that of mated females. Females that mated with sterile males laid the same number of eggs as females that mated with wild males but spent less time depositing host-marking pheromone, which suggests that females detect the condition of the male and invest less in marking hosts. Finally, sterile males had larger accessory glands and transferred more MAGs proteins during mating compared to wild males. Seminal proteins could be manipulating HM behavior and female investment into their current reproductive effort. We are only beginning to understand how male condition and seminal fluid can affect female physiology and maternal investment in HMP.

In-Depth Understanding of Cytoplasmic Male Sterility by Metabolomics in Spring Stem Mustard (Brassica juncea var. tumida Tsen et Lee)

Male sterility (MS) caused by aborted pollen is a vital germplasm resource. In this study, metabolomic, transcriptomic, and proteomic analyses were performed to investigate the molecular regulatory mechanism of cytoplasmic male sterility (CMS) in the flower buds of the male sterile line 09-05A and its maintainer line, 09-05B, of Brassica juncea. Our metabolomic analysis revealed that 41 metabolites involved in pollen development and male fertility formation were differentially accumulated between 09-05A and 09-05B at the bi-nucleate stage of B. juncea. Integrated omics indicated that the expression of key genes and proteins in the amino acid and phenylpropanoid metabolic pathways was remarkably downregulated in the flower buds of 09-05A. Furthermore, the abnormal expression of key transcription factor (TF) genes related to tapetum development and pollen wall formation may affect pollen development in the 09-05A CMS line. The results indicated that the downregulated expression level of critical genes and proteins in amino acid metabolism; phenylpropanoid synthesis; and TF genes, such as shikimate kinase, phenylalanine ammonia-lyase, and MYB103, may have led to impaired tapetum and pollen wall development, thereby causing pollen abortion in 09-05A. This study provides new insights into the metabolic and molecular regulatory mechanisms underlying the formation of CMS in B. juncea and lays a foundation for detailed studies on the identity and characteristics of MS-related genes.

Genome-Wide Identification and Male Sterility-Related Expression Analysis of Papain-like Cysteine Protease Gene Family in Capsicum annuum

PLCPs (papain-like cysteine proteases) are one of the most abundant groups of cysteine proteases and play vital roles in multiple processes. The pepper (Capsicum annuum) is an important Solanaceae vegetable crop; its commercial hybrid seeds are widely used in production. Male sterility is a valuable trait for hybrid seed production. However, the function of PLCPs and the underlying mechanisms of male sterility in peppers remain unclear. In this study, we comprehensively identified the PLCP gene family in peppers, identifying 31 CaPLCPs. A phylogenetic analysis classified 31 members into eight clades. These CaPLCPs were unevenly distributed across eight chromosomes, and five segmental duplicated pairs were observed. The promoter cis-acting element analysis indicated that CaPLCP promoters contained abundant hormone-responsive and stress-responsive cis-elements, suggesting that CaPLCPs may play important roles in responding to abiotic stress, such as drought and low temperatures, as well as in plant immunity. The qRT-PCR analysis demonstrated that the expression levels of CaPLCP1, CaPLCP5, CaPLCP11, CaPLCP12, CaPLCP13, CaPLCP17, CaPLCP19, and CaPLCP21 were significantly reduced in the flowers of MS (male sterile pepper) at least at one stage, indicating their potential roles as regulatory factors in pepper male sterility. These findings provide important insights into the functional analysis of the PLCP gene family in peppers and other species, laying a crucial foundation for understanding the mechanisms of male sterility in peppers.

Multi-Omics analysis reveals the important role of indole-3-acetic acid homeostasis in male fertility of cotton

Auxin (Indole-3-acetic acid, IAA) is essential for anther development and male fertility, but little is known about its homeostasis in developing anthers due to its complex and diverse synthesis pathways. Here, we employed transcriptomic, targeted metabolomic, and amino acid analyses on a wild type (WT) cotton (Gossypium hirsutum) line SD98–6 and its nearly-isogenic male sterile (MS) line SD98–6A, to delve into the relationship between IAA and anther fertility. In WT anthers, IAA homeostasis was achieved mainly through self-synthesis, conjugate hydrolysis, and polar transport, and accumulation of IAA exhibited an inverted "U" pattern with the highest content observed at the tetrad stage. In MS anthers, no IAA accumulation was observed after the meiosis stage and the IAA content remained constantly low, mainly due to reduction of the substrates (phenylalanine and tryptophan) for IAA synthesis, and significantly decreased expression of the genes involved in IAA synthesis. Meanwhile, carbohydrate metabolism pathways directly contributing to IAA synthesis and synthesis of amino acids were malfunctional in MS anthers based on the expression profile of the metabolic genes and content measurement. These results provide valuable insights into the IAA homeostasis in cotton anthers and imply that genes related to IAA homeostasis are potential targets for development of IAA-sensitive male sterile cotton lines.

Mutations in nuclear genes encoding mitochondrial ribosome proteins restore pollen fertility in S male-sterile maize.

The interaction of plant mitochondrial and nuclear genetic systems is exemplified by mitochondria-encoded cytoplasmic male sterility (CMS) under the control of nuclear restorer-of-fertility genes. The S type of CMS in maize is characterized by a pollen collapse phenotype and a unique paradigm for fertility restoration in which numerous nuclear restorer-of-fertility lethal mutations rescue pollen function but condition homozygous-lethal seed phenotypes. Two nonallelic restorer mutations recovered from Mutator transposon-active lines were investigated to determine the mechanisms of pollen fertility restoration and seed lethality. Mu Illumina sequencing of transposon-flanking regions identified insertion alleles of nuclear genes encoding mitochondrial ribosomal proteins RPL6 and RPL14 as candidate restorer-of-fertility lethal mutations. Both candidates were associated with lowered abundance of mitochondria-encoded proteins in developing maize pollen, and the rpl14 mutant candidate was confirmed by independent insertion alleles. While the restored pollen functioned despite reduced accumulation of mitochondrial respiratory proteins, normal-cytoplasm plants heterozygous for the mutant alleles showed a significant pollen transmission bias in favor of the nonmutant Rpl6 and Rpl14 alleles. CMS-S fertility restoration affords a unique forward genetic approach to investigate the mitochondrial requirements for, and contributions to, pollen and seed development.

HSP70 and APX1 play important roles in cotton male fertility by mediating ROS homeostasis

Male sterility is used in the production of hybrid seeds and can improve the breeding efficiency of cotton hybrids. Reactive oxygen species is closely associated with the tapetum and pollen development, but their relationship in cotton male fertility remains unclear. In this study, we comprehensively compared the cytology and proteome of the anthers from an Upland cotton (Gossypium hirsutum) material, Shida 98 (WT), and its nearly-isogenic male sterile line Shida 98A (MS). Cytology indicated delayed PCD in the tapetum and defects in microspores in MS anthers. And further studies revealed disruption of ROS homeostasis. Proteomic analysis identified proteins with differential abundance mainly being related to redox homeostasis, protein folding, and apoptotic signaling pathways. GhAPX1 interacted with GhHSP70 and played a crucial role in the development of cotton anthers. Exogenous application of HSP70 inhibitor increased H2O2 content and decreased the activity of APX1 and pollen viability. The GhAPX1 mutants generated by CRISPR/Cas9-mediated gene editing exhibited premature degradation of the tapetum, significant decrease in pollen viability, and significant increase in H2O2 content. Altogether, our results imply HSP70 and APX1 being the key players jointly regulating male fertility by mediating ROS homeostasis. These results provide insights into the proteins associated with male fertility.

Histone demethylase KDM2A recruits HCFC1 and E2F1 to orchestrate male germ cell meiotic entry and progression

In mammals, the transition from mitosis to meiosis facilitates the successful production of gametes. However, the regulatory mechanisms that control meiotic initiation remain unclear, particularly in the context of complex histone modifications. Herein, we show that KDM2A, acting as a lysine demethylase targeting H3K36me3 in male germ cells, plays an essential role in modulating meiotic entry and progression. Conditional deletion of Kdm2a in mouse pre-meiotic germ cells results in complete male sterility, with spermatogenesis ultimately arrested at the zygotene stage of meiosis. KDM2A deficiency disrupts H3K36me2/3 deposition in c-KIT+ germ cells, characterized by a reduction in H3K36me2 but a dramatic increase in H3K36me3. Furthermore, KDM2A recruits the transcription factor E2F1 and its co-factor HCFC1 to the promoters of key genes required for meiosis entry and progression, such as Stra8, Meiosin, Spo11, and Sycp1. Collectively, our study unveils an essential role for KDM2A in mediating H3K36me2/3 deposition and controlling the programmed gene expression necessary for the transition from mitosis to meiosis during spermatogenesis.

Insights into cellular crosstalk regulating cytoplasmic male sterility and fertility restoration.

Cytoplasmic male sterility has been a popular genetic tool in development of hybrids. The molecular mechanism behind maternal sterility varies from crop to crop. An understanding of underlying mechanism can help in development of new functional CMS gene in crops which lack effective and stable CMS systems. In crops where seed or fruit is the commercial product, fertility must be recovered in F1 hybrids so that higher yield gains can be realized. This necessitates the presence of fertility restorer gene (Rf) in nucleus of male parent to overcome the effect of sterile cytoplasm. Fertility restoring genes have been identified in crops like wheat, maize, sunflower, rice, pepper, sugar beet, pigeon pea etc. But in crops like eggplant, bell pepper, barley etc. unstable fertility restorers hamper the use of Cytoplasmic genic male sterility (CGMS) system. Stability of CGMS system is influenced by environment, genetic background or interaction of these factors. This review thus aims to understand the genetic mechanisms controlling mitochondrial-nuclear interactions required to design strong and stable restorers without any pleiotropic effects in F1 hybrids.

Wild rice: unlocking the future of rice breeding.

Germplasm resources serve as the foundations of advancements in breeding and are crucial for maintaining food security. Wild rice species of the genus Oryza include rich sources of genetic diversity and high adaptability, making them a substantial resource for rice breeding. The discovery of wild-type cytoplasmic male sterility resources enabled the achievement of the 'three lines' goal in hybrid rice, significantly increasing rice yields. The application of resistance alleles from wild rice enables rice production to withstand losses caused by stress. Reduced genetic diversity due to rice breeding poses a significant limitation to further advances and can be alleviated through a systematic use of wild genetic resources that integrate geographic, climatic and environmental data of the original habitat, along with extensive germplasm collection and identification using advanced methods. Leveraging technological advancements in plant genomics, the understanding of genetic mechanisms and the application of artificial intelligence and gene editing can further enhance the efficiency and accuracy of this process. These advancements facilitate rapid isolation and functional studies of genes, and precise genome manipulation. This review systematically summarizes the utilization of superior genes and germplasm resources derived from wild rice sources, while also exploring the collection, conservation, identification and utilization of further wild rice germplasm resources. A focus on genome sequencing and biotechnology developments is leading to new breeding and biotechnology opportunities. These new opportunities will not only promote the development of rice varieties that exhibit high yields, superior stress resistance and high quality but also expand the genetic diversity among rice cultivars.

Reconsidering the Lessons Learned from the 1970 Southern Corn Leaf Blight Epidemic.

The southern corn leaf blight epidemic of 1970 caused estimated losses of about 16% for the U.S. corn crop, equivalent to about $8 billion in current terms. The epidemic was caused by the prevalence of Texas male sterile cytoplasm (cms-T), used to produce most of the hybrid corn seed planted that year, combined with the emergence of a novel race of the fungus Cochliobolus heterostrophus that was exquisitely virulent on cms-T corn. Remarkably, the epidemic lasted just a single year. This episode has often been portrayed in the literature and textbooks over the last 50 years as a catastrophic mistake perpetrated by corn breeders and seed companies of the time who did not understand or account for the dangers of crop genetic uniformity. In this perspective article, we aim to present an alternative interpretation of these events. First, we contend that, rather than being caused by a grievous error on the part of the corn breeding and seed industry, this epidemic was a particularly unfortunate, unusual, and unlucky consequence of a technological advancement intended to improve the efficiency of corn seed production for America's farmers. Second, we tell the story of the resolution of the epidemic as an example of timely, meticulously applied research in the public sector for the public good.

Morphological and cytological assessments reveal pollen degradation causes pollen abortion in cotton cytoplasmic male sterility lines

BackgroundUnderstanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources. However, only a few cytoplasmic male sterility (CMS) lines of cotton have been produced due to several challenges, like inadequate variation of agronomic traits, incomplete sterility, weak resilience of restorer lines, and difficulty in combining strong dominance. Therefore, the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding.ResultsTwo F2 segregating populations of cotton were constructed from cytoplasmic male sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal). Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants. Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants. However, sterile plants exhibited smaller floral organs, shortened filament lengths, and anther atrophy on the flowering day in comparison with the fertile plants. When performed scanning electron microscopy (SEM), the two F2 populations revealed morphological variations in the anther epidermis. Cellular analysis showed no significant differences in pollen development before pollen maturation. Interestingly, between the pollen maturation and flowering stages, the tapetum layer of sterile plants degenerated prematurely, resulting in abnormal pollen grains and gradual pollen degradation.ConclusionThe results of this study suggest that fertility-restoring genes are controlled by a single dominant gene. Sterile plants exhibit distinctive floral morphology, which is characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs between pollen maturity and flowering, indicating that premature tapetum degradation may be the primary cause of pollen abortion. Overall, our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations, mechanisms of sterility, and the role of sterile and restorer genes.

Improvement of Flowering Stage in Japonica Rice Variety Jiahe212 by Using CRISPR/Cas9 System.

The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome-editing vector containing five genes related to three traits, Hd2, Ghd7, and DTH8 (flowering-stage genes), along with the recessive rice blast resistance gene Pi21 and the aromatic gene BADH2. This vector was introduced into the high-quality japonica rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T0 plants with various effective mutations. Among the 17 mutant T1 lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T2 plants, we identified seven, six, and two fragrant glutinous plants devoid of transgenic components. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for indica-japonica interspecific three-line hybrid rice. In summary, our research lays the foundation for the creation of short-growth-period CMS (cytoplasmic male sterility, CMS) lines, and also provides materials and a theoretical basis for indica-japonica interspecific hybrid rice breeding with wider adaptability.