Cytoplasmic Male Sterility System Research Articles

Identification of stable restorers and high-yielding hybrids using diverse sorghum male sterile cytoplasmic sources and established pollen parents under different water regimes

Sorghum hybrids demonstrated increased productivity and helped offset the decreasing cultivated areas, particularly in Asia. The diversity in cytoplasmic male sterility systems, stability of restorers and high yield of sorghum is an important factor for achieving food security and sustainability. In sorghum, hybrid production has been limited to A1 cytoplasmic source to date, primarily due to limited number of restorers on other cytoplasmic sources. This work aimed at filling this gap through diversifying and assessing the producibility and stability of cytoplasmic backgrounds across environments, evaluating the stability of restorers across cytoplasms and environments, and evaluating the performance of the hybrid F1 products under different water regimes across cropping seasons. The effects of genotypes and interactions between the genotypes and the tested environments were shown visually using GGE biplot, which also categorized the genotypes based on performance per se and stability. Analysis of variance revealed significant (P< 0.001) differences between genotypes, environments and their interaction components for all the plant characteristics studied. The various GGE biplot patterns revealed that the restorers G4 (IS 33844-5), G5 (M 35-1-19), G8 (ICSV 15014), and G10 (ICSR 13009) were stable across environments and cytoplasms. We observed the following order of superiority in cytoplasmic restoration and seed producibility, A2 >A1>A3 >A4. The A2 cytoplasm can therefore reliably offer seed producibility advantage to commercial seed producers. Furthermore, the which-won–where model for hybrid grain yield revealed specific adaptations of hybrid genotypes G143, G195 and G215 to first mega environment (E1 and E3), whereas G167, G110, and G112 were adapted to the second mega-environment (E2 and E4). With a GGE biplot goodness of fit of 70% the best possible yield- and stability-based ranking of hybrid genotypes was G143, G215, G189, G200, G69, G162, G119, G54, and G81. The newly uncovered hybrid products should therefore be considered for downstream testing stages and candidates for release and commercial cultivation. Clearly, this work resulted in novel findings particularly in terms of seed producibility, universal restorers across the 4 cytoplasms and environments, and superior and stable hybrid products, all of which are expected to add value to conventional and heterosis breeding in the process of sorghum cultivar development.

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.

Identification, Elucidation and Deployment of a Cytoplasmic Male Sterility System for Hybrid Potato.

Recent advances in diploid F1 hybrid potato breeding rely on the production of inbred lines using the S-locus inhibitor (Sli) gene. As a result of this method, female parent lines are self-fertile and require emasculation before hybrid seed production. The resulting F1 hybrids are self-fertile as well and produce many undesirable berries in the field. Utilization of cytoplasmic male sterility would eliminate the need for emasculation, resulting in more efficient hybrid seed production and male sterile F1 hybrids. We observed plants that completely lacked anthers in an F2 population derived from an interspecific cross between diploid S. tuberosum and S. microdontum. We studied the antherless trait to determine its suitability for use in hybrid potato breeding. We mapped the causal locus to the short arm of Chromosome 6, developed KASP markers for the antherless (al) locus and introduced it into lines with T and A cytoplasm. We found that antherless type male sterility is not expressed in T and A cytoplasm, proving that it is a form of CMS. We hybridized male sterile al/al plants with P cytoplasm with pollen from al/al plants with T and A cytoplasm and we show that the resulting hybrids set significantly fewer berries in the field. Here, we show that the antherless CMS system can be readily deployed in diploid F1 hybrid potato breeding to improve hybridization efficiency and reduce berry set in the field.

Advances in Breeding for Oil Quality Enhancement in Indian Mustard (Brassica spp. L.): Achievements, Challenges, and Research Opportunities

Major objectives in oil crop improvement are enhancement of seed and oil yield, quality of oil according to its use, i.e. edible or industrial uses, breeding of varieties that fit in different cropping systems and breeding biotic and abiotic stress resistant/tolerant varieties. Despite traditional breeding approaches, including pure line breeding, yielding only modest gains in productivity, recent advancements in mustard breeding have led to significant breakthroughs in both productivity and oil quality. This review discusses the innovative breeding strategies that have contributed to these advancements, with a focus on hybrid development, oil quality enhancement, and biotechnological approaches. To enhance productivity, researchers at the University of Delhi have developed hybrid seed production techniques using transgenic Barnase-barstar systems and cytoplasmic male sterility (CMS) systems. These systems enable large-scale hybrid seed production, with field trials demonstrating significant yield heterosis ranging from 31% to 55% compared to national check varieties. In addition to productivity, improving oil and meal quality has been a key objective. By integrating genes from canola-quality mustard lines, breeders have achieved reductions in erucic acid and glucosinolates, enhancing the health profile and industrial applicability of mustard oil. A high-density linkage map developed using an F1 double haploid mapping population has facilitated the marker-assisted backcross breeding of desirable traits, enabling precise transfer of key quality traits. Transgenic approaches, such as antisense RNA technology, have led to the development of high-oleic, low-linoleic mustard lines with improved fatty acid profiles. These advancements reflect a strategic combination of conventional and biotechnological methods, demonstrating a clear pathway for boosting mustard yields while enhancing oil quality. Molecular markers reported for genetic diversity assessment, mapping and tagging genes/QTLs for different qualitative and quantitative traits and their use in marker-assisted selection have been presented. This progress not only addresses current challenges but also sets the stage for future research aimed at further optimizing productivity, oil quality, and resistance to pests and diseases in mustard cultivation.

Unveiling a half-century mystery of molecular bases for three-line hybrid rice breeding system.

Hybrid rice derived from Wild Abortive (WA) cytoplasmic male sterility type three-line systems is widely used in production. The fertility restoration gene Rf4 can reduce WA352c transcript levels in a dose-dependent manner. A two-copy haplotype of Rf4 (H1) is identified as the most valuable haplotype for future hybrid rice breeding.

A P-type pentatricopeptide repeat protein ZmRF5 promotes 5' region partial cleavages of atp6c transcripts to restore the fertility of CMS-C maize by recruiting a splicing factor.

A fast evolution within mitochondria genome(s) often generates discords between nuclear and mitochondria, which is manifested as cytoplasmic male sterility (CMS) and fertility restoration (Rf) system. The maize CMS-C trait is regulated by the chimeric mitochondrial gene, atp6c, and can be recovered by the restorer gene ZmRf5. Through positional cloning in this study, we identified the nuclear restorer gene, ZmRf5, which encodes a P-type pentatricopeptide repeat (PPR) family protein. The over-expression of ZmRf5 brought back the fertility to CMS-C plants, whereas its genomic editing by CRISPR/Cas9 induced abortive pollens in the restorer line. ZmRF5 is sorted to mitochondria, and recruited RS31A, a splicing factor, through MORF8 to form a cleaving/restoring complex, which promoted the cleaving of the CMS-associated transcripts atp6c by shifting the major cleavage site from 480th nt to 344 th nt for fast degradation, and preserved just right amount of atp6c RNA for protein translation, providing adequate ATP6C to assembly complex V, thus restoring male fertility. Interestingly, ATP6C in the sterile line CMo17A, with similar cytology and physiology changes to YU87-1A, was accumulated much less than it in NMo17B, exhibiting a contrary trend in the YU87-1 nuclear genome previously reported, and was restored to normal level in the presence of ZmRF5. Collectively these findings unveil a new molecular mechanism underlying fertility restoration by which ZmRF5 cooperates with MORF8 and RS31A to restore CMS-C fertility in maize, complemented and perfected the sterility mechanism, and enrich the perspectives on communications between nucleus and mitochondria.

Targeted knockout of a conserved plant mitochondrial gene by genome editing

Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I. By generating a large number of TALEN-mediated mitochondrial deletion lines, we isolated, in addition to mutants with rearranged genomes, homochondriomic mutants harbouring clean nad9 deletions. Characterization of the knockout plants revealed impaired complex I biogenesis, male sterility and defects in leaf and flower development. We show that these defects can be restored by expressing a functional Nad9 protein from the nuclear genome, thus creating a synthetic cytoplasmic male sterility system. Our data (1) demonstrate the feasibility of using genome editing to study mitochondrial gene functions by reverse genetics, (2) highlight the role of complex I in plant development and (3) provide proof-of-concept for the construction of synthetic cytoplasmic male sterility systems for hybrid breeding by genome editing.

Male sterility in plants: an overview of advancements from natural CMS to genetically manipulated systems for hybrid seed production.

The male sterility system in plants has traditionally been utilized for hybrid seed production. In last three decades, genetic manipulation for male sterility has revolutionized this area of research related to hybrid seed production technology. Here, we have surveyed some of the natural cytoplasmic male sterility (CMS) systems that existed/ were developed in different crop plants for developing male sterility-fertility restoration systems used in hybrid seed production and highlighted some of the recent biotechnological advancements in the development of genetically engineered systems that occurred in this area. We have indicated the possible future directions toward the development of engineered male sterility systems. Cytoplasmic male sterility (CMS) is an important trait that is naturally prevalent in many plant species, which has been used in the development of hybrid varieties. This is associated with the use of appropriate genes for fertility restoration provided by the restorer line that restores fertility on the corresponding CMS line. The development of hybrids based on a CMS system has been demonstrated in several different crops. However, there are examples of species, which do not have usable cytoplasmic male sterility and fertility restoration systems (Cytoplasmic Genetic Male Sterility Systems-CGMS) for hybrid variety development. In such plants, it is necessary to develop usable male sterile lines through genetic engineering with the use of heterologous expression of suitable genes that control the development of male gametophyte and fertile male gamete formation. They can also be developed through gene editing using the recently developed CRISPR-Cas technology to knock out suitable genes that are responsible for the development of male gametes. The present review aims at providing an insight into the development of various technologies for successful production of hybrid varieties and is intended to provide only essential information on male sterility systems starting from naturally occurring ones to the genetically engineered systems obtained through different means.

Identification of Good Restorer Lines through Molecular Confirmation of Rf3 and Rf4 Genes in Rice Hybrids

Pollen fertility can be restored by nuclear-encoded genes known as fertility restorer (Rf) genes. The initial stage in creating high-yielding heterotic hybrids involves the identification of restorers capable of effectively restoring the fertility of CMS lines. Hybrid rice breeding programs have proven effective in achieving these goals by utilizing cytoplasmic male sterility (CMS) systems in combination with restorer genes. Among these, Rf3 and Rf4 genes play critical roles in restoring male fertility and are essential components of successful hybrid rice breeding. In this study, we aimed to investigate the molecular conformation of Rf3 and Rf4 genes in a set of restorer lines and hybrids in rice. DNA was isolated from 10 restorer lines and 30 rice hybrids, two checks and KMR3R were used for comparison for positive alleles. Two functional markers, RM SF21-5 for Rf3 and RMS-PRR-9-1 for Rf4, were employed to identify the allelic status of the fertility restorer genes. Results revealed that among the 42 rice entries screened, five rice hybrids lacked both Rf3 and Rf4 genes and Four rice restorer lines and four hybrids were found to possess both Rf3 and Rf4 genes, making them valuable for hybrid breeding programs. Conversely, five hybrids lacked restorer genes and were unsuitable for such breeding programs. These findings establish Rf3and Rf4 as major fertility restoring genes in rice, consistently contributing to complete fertility restoration. The genotyping results provide valuable insights for selecting appropriate parental lines and restorers in hybrid rice breeding programs. The presence or absence of these fertility-restoring genes plays a crucial role in developing high-yielding and productive rice varieties through effective breeding strategies.

Comparative Transcriptome Profiling of CMS-D2 and CMS-D8 Systems Characterizes Fertility Restoration Genes Network in Upland Cotton

Resolving the genetic basis of fertility restoration for cytoplasmic male sterility (CMS) can improve the efficiency of three-line hybrid breeding. However, the genetic determinants of male fertility restoration in cotton are still largely unknown. This study comprehensively compared the full-length transcripts of CMS-D2 and CMS-D8 systems to identify potential genes linked with fertility restorer genes Rf1 or Rf2. Target comparative analysis revealed a higher percentage of differential genes in each restorer line as compared to their corresponding sterile and maintainer lines. An array of genes with specific expression in the restorer line of CMS-D2 had functional annotations related to floral development and pathway enrichments in various secondary metabolites, while specifically expressed genes in the CMS-D8 restorer line showed functional annotations related to anther development and pathway enrichment in the biosynthesis of secondary metabolites. Further analysis identified potentially key genes located in the target region of fertility restorer genes Rf1 or Rf2. In particular, Ghir_D05G032450 can be the candidate gene related to restorer gene Rf1, and Ghir_D05G035690 can be the candidate gene associated with restorer gene Rf2. Further gene expression validation with qRT-PCR confirmed the accuracy of our results. Our findings provide useful insights into decoding the potential regulatory network that retrieves pollen fertility in cotton and will help to further reveal the differences in the genetic basis of fertility restoration for two CMS systems.

Genetic Variability of Floral and Agronomic Characteristics that Influence Outcrossing Rate Percentage of Cytoplasmic Male Sterile Rice

Developing high-yielding hybrid rice varieties is much needed to ensure food security to meet the requirement of the increasing population across the world. Cytoplasmic male genetic sterility system is a valuable technology for the exploitation of heterosis and production of high-yielding hybrid rice with better grain quality. Large-scale commercial exploitation of hybrid vigor in rice largely depends on the availability of good CMS (Cytoplasmic Male Sterile) lines. A total of eighteen traits of five CMS lines of rice were evaluated to study the variability and correlation between floral and other yield-contributing traits with outcrossing rate using Randomized Complete Block Design with three replications. High significant variation was observed among the genotypes for all the studied traits. High GCV (Genotypic Coefficient of Variance) and PCV (phenotypic Coefficient of Variance) coupled with high heritability and high genetic advance in percent of mean were observed for number of panicles per plant, filled grain, flag leaf breadth, stigma length and breadth, filament length and outcrossing rate which indicated the broad genetic base, less environmental influence, and these traits are under the control of additive gene action. character association and path analysis indicated that number of grains per panicle, filled grains per panicle, stigma length, and breadth possessed both positive significant correlation and positive direct effects on outcrossing rate therefore, selection for the traits mentioned above will certainly bring in the improvement of the outcrossing percentage of the lines, which in turn enhances the seed quantity of the hybrid.

Comparative study on abortion characteristics of Nsa CMS and Pol CMS and analysis of long non-coding RNAs related to pollen abortion in Brassica napus.

Cytoplasmic male sterile system (CMS) is one of the important methods for the utilization of heterosisin Brassica napus. The involvement of long non-coding RNAs (lncRNAs) in anther and pollen development in B.napus has been recognized, but there is little data on the involvement of lncRNAs in pollen abortion in different types of rapeseed CMS. The present study compared the cytological, physiological and biochemical characteristics of Nsa CMS (1258A) and Pol CMS (P5A) during pollen abortion, and high-throughput sequencing of flower buds of different sizes before and after pollen abortion. The results showed that insufficient energy supply was an important physiological basis for 1258A and P5A pollen abortion, and 1258A had excessive ROS (reactive oxygen species) accumulation in the stage of pollen abortion. Functional analysis showed that Starch and sucrose metabolism and Sulfur metabolism were significantly enriched before and after pollen abortion in 1258A and P5A, and a large number of genes were down-regulated. In 1258A, 227 lncRNAs had cis-targeting regulation, and 240 cis-target genes of the lncRNAs were identified. In P5A, 116 lncRNAs had cis-targeting regulation, and 101 cis-target genes of the lncRNAs were identified. There were five lncRNAs cis-target genes in 1258A and P5A during pollen abortion, and LOC106445716 encodes β-D-glucopyranosyl abscisate β-glucosidase and could regulate pollen abortion. Taken together, this study, provides a new perspective for lncRNAs to participate in the regulation of Nsa CMS and Pol CMS pollen abortion.

The Alpha Subunit of Mitochondrial Processing Peptidase Participated in Fertility Restoration in Honglian-CMS Rice

The cytoplasmic male sterility (CMS) and nuclear-controlled fertility restoration system is a favorable tool for the utilization of heterosis in plant hybrid breeding. Many restorer-of-fertility (Rf) genes have been characterized in various species over the decades, but more detailed work is needed to investigate the fertility restoration mechanism. Here, we identified an alpha subunit of mitochondrial processing peptidase (MPPA) that is involved in the fertility restoration process in Honglian-CMS rice. MPPA is a mitochondrial localized protein and interacted with the RF6 protein encoded by the Rf6. MPPA indirectly interacted with hexokinase 6, namely another partner of RF6, to form a protein complex with the same molecular weight as the mitochondrial F1F0-ATP synthase in processing the CMS transcript. Loss-of-function of MPPA resulted in a defect in pollen fertility, the mppa+/- heterozygotes showed semi-sterility phenotype and the accumulation of CMS-associated protein ORFH79, showing restrained processing of the CMS-associated atp6-OrfH79 in the mutant plant. Taken together, these results threw new light on the process of fertility restoration by investigating the RF6 fertility restoration complex. They also reveal the connections between signal peptide cleavage and the fertility restoration process in Honglian-CMS rice.

Integrated transcriptomic and proteomic analysis of a cytoplasmic male sterility line and associated maintainer line in soybean.

Heterosis is a critical phenomenon in crop improvement. Cytoplasmic male sterility (CMS) and Restorer gene (Rf) systems are essential components for heterosis-based breeding. However, the molecular mechanism underlying CMS remains largely unclear in soybean. We integrated a morphological investigation with comparative analyses of transcriptomic and proteomic changes in pollen from the CMS line W931A and its maintainer line, W931B, at the uninucleate microspore (UM) and binucleate pollen (BP) stages. Compared to W931B, which had healthy, oval pollen grains, W931A showed shrunken or degraded pollen grains with an irregularly thickened endothelium and decreased starch accumulation. Transcriptomic comparisons revealed a total of 865 differentially expressed genes (DEGs) in W931A over the two stages. These genes were primarily associated with pentose and glucuronate interconversions, sphingolipid metabolism, and glycerolipid metabolism. Proteomic analysis revealed 343 differentially expressed proteins (DEPs), which were mainly involved in carbon metabolism, glycolysis/gluconeogenesis, and nitrogen metabolism. Consistently, Gene Ontology (GO) biological process terms related to pollen development were enriched among DEGs at the UM and BP stages. Notably, four genes with demonstrated roles in pollen development were differentially expressed, including AGAMOUS-LIKE 104, PROTEIN-TYROSINE-PHOSPHATASE 1, and PHOSPHOLIPASE A2. A total of 53 genes and the corresponding proteins were differentially expressed in W931A at both the UM and BP stages, and many of these were pectinesterases, polygalacturonases, peroxidases, and ATPases. The results of this study suggest that pollen development in W931A is likely regulated through suppression of the identified DEGs and DEPs. These findings increase our understanding of the molecular mechanism underlying CMS in soybean, aiding future research into soybean fertility and promoting the efficient use of heterosis for soybean improvement.

Abrin inactivating mitochondrial ribosome induced anther abortion in cotton CMS line LD6A by damaging mitochondrial membrane structure

Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we aimed to elucidate the molecular pathways, regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) lines were analyzed for physiological and proteomic changes during the development process. Transmission electron micrographs of anther tapetum showed a relatively indistinct inner ridge of CMS than of LD6B mitochondria at tetrad stage. Furthermore, at pollen mother cell and tetrad stages, CMS anthers accumulated a significantly higher reactive oxygen species than the maintainer line. At the protein level, CMS anthers induced significantly higher Abrin (XP_016705148.2), lower tricarboxylic acid cycle enzyme (malate dehydrogenase, malic enzyme, isocitrate dehydrogenase), and histone acetyltransferase expression than its maintainer line. Suggesting that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein Abrin in CMS line damaged mitochondrial membrane. This inhibited energy supply from the non-degraded tapetum, and consequently induced pollen sterility in LD6A. These data provide new insights into CMS mechanism in cotton crops that can be used for developing new CMS germplasm resources.

Development of mitochondrial simple sequence repeat markers to simultaneously distinguish cytoplasmic male sterile sources in cotton.

Deleterious effects on anther development andmain economy traits caused by sterile genes or cytoplasms are one of the important genetic characteristics of cytoplasmic male sterility (CMS) systems in cotton, which severely hinder the large-scale application of "three-line" hybrids in production. Therefore, distinct characterization of each cytoplasmic type is mandatory to improve the breeding efficiency of cotton hybrids. In this study, four isonuclear-alloplasmic cotton male sterile lines withG. hirsutum (CMS-(AD)1), G. barbadense (CMS-(AD)2), G. harknessii (CMS-D2), and G. trilobum (CMS-D8) cytoplasms were first created by multiple backcrosses with common genotype Shikang126. Then, 64 pairs of mitochondrial simple sequence repeat (mtSSR) markers were designed to explore the mitochondrial DNA diversities among four isonuclear-alloplasmic cotton male sterile lines, and a total of nine pairs of polymorphic mtSSR molecular markers were successfully developed. Polymorphism analysis indicated that mtSSR59 marker correlated to the atp1 gene could effectively divide the CMS-D2, CMS-(AD)1, and CMS-(AD)2 in one category while the CMS-D8 in another category. Further cytological observation and determination of ATP contents also confirmed the accurate classification of CMS-D2 and CMS-D8 lines. Moreover, the mtSSR59 marker was successfully applied in the marker-assisted selection (MAS) for breeding new male sterile lines and precise differentiation or purity identification of different CMS-based "three-line" and conventional cotton hybrids. This study provides new technical measures for classifying various cytoplasmic sterile lines, and our results will significantly improve the efficiency of there-line hybrid breeding in cotton.

Genetics of fertility restoration in A2‐based cytoplasmic genetic male sterility system in pigeonpea (Cajanus cajan)

AbstractThe three short duration cytoplasmic genetic male sterility (CGMS) hybrids developed using A2 (Cajanus scarabeoides) cytoplasm‐based male sterile lines (CORG 990047A, CORG 990052A and CORG 7A) and the restorer inbred AK 261322 and their segregating populations (F2 and BC1F1) were subjected to the study of inheritance of fertility restoration in pigeonpea. The fertility restoration was studied based on three different criteria, namely, anther colour, pollen grain fertility and pollen grain morphology and staining. The F2 and BC1F1 populations of the three crosses, namely, CORG 990047A × AK 261322, CORG 990052A × AK 261322 and CORG 7A × AK 261322, segregated in the ratio of 3:1 and 1:1, for anther colour (yellow:pale yellow), pollen grain fertility (fertile:sterile) and for pollen grain morphology and staining. The above study confirmed that the trait fertility restoration was controlled by single dominant gene. This finding can be utilized for the identification of potential restorers, which can be further used in the development of CGMS‐based hybrids in pigeonpea.

Induction of Male Sterility by Targeted Mutation of a Restorer-of-Fertility Gene with CRISPR/Cas9-Mediated Genome Editing in Brassica napus L.

Brassica napus L. (canola, oil seed rape) is one of the world's most important oil seed crops. In the last four decades, the discovery of cytoplasmic male-sterility (CMS) systems and the restoration of fertility (Rf) genes in B. napus has improved the crop traits by heterosis. The homologs of Rf genes, known as the restoration of fertility-like (RFL) genes, have also gained importance because of their similarities with Rf genes. Such as a high non-synonymous/synonymous codon replacement ratio (dN/dS), autonomous gene duplications, and a possible engrossment in fertility restoration. B. napus contains 53 RFL genes on chromosomes A9 and C8. Our research aims to study the function of BnaRFL11 in fertility restoration using the CRISPR/Cas9 genome editing technique. A total of 88/108 (81.48%) T0 lines, and for T1, 110/145 (75%) lines carried T-DNA insertions. Stable mutations were detected in the T0 and T1 generations, with an average allelic mutation transmission rate of 81%. We used CRISPR-P software to detect off-target 50 plants sequenced from the T0 generation that showed no off-target mutation, signifying that if the designed sgRNA is specific for the target, the off-target effects are negligible. We also concluded that the mutagenic competence of the designed sgRNAs mediated by U6-26 and U6-29 ranged widely from 31% to 96%. The phenotypic analysis of bnarfl11 revealed defects in the floral structure, leaf size, branch number, and seed production. We discovered a significant difference between the sterile line and fertile line flower development after using a stereomicroscope and scanning electron microscope. The pollen visibility test showed that the pollen grain had utterly degenerated. The cytological observations of homozygous mutant plants showed an anther abortion stage similar to nap-CMS, with a Orf222, Orf139, Ap3, and nad5c gene upregulation. The bnarfl11 shows vegetative defects, including fewer branches and a reduced leaf size, suggesting that PPR-encoding genes are essential for the plants' vegetative and reproductive growth. Our results demonstrated that BnaRFL11 has a possible role in fertility restoration. The current study's findings suggest that CRISPR/Cas9 mutations may divulge the functions of genes in polyploid species and provide agronomically desirable traits through a targeted mutation.

Transcript Complexity and New Insights of Restorer Line in CMS-D8 Cotton Through Full-Length Transcriptomic Analysis.

Hybrid utilization has proficiently increased crop production worldwide. The cytoplasmic male sterility (CMS) system has emerged as an efficient tool for commercial hybrid cotton seed production. The restorer line with dominant Rf2 gene can restore the fertility of the CMS-D8 sterile line. However, the molecular mechanism of fertility restoration remains unclear in CMS-D8 cotton that limits wider utilization of three-line hybrid breeding. In our study, the Pacific Biosciences (PacBio) Iso-Seq technology was applied to understand fertility restoration mechanism of CMS-D8 cotton. In total, 228,106 full-length non-chimeric transcriptome sequences were obtained from anthers of developing flowering buds. The analysis results identified 3,174 novel isoforms, 2,597 novel gene loci, 652 long non-coding RNAs predicted from novel isoforms, 7,234 alternative splicing events, 114 fusion transcripts, and 1,667 genes with alternative polyadenylation. Specially, two novel genes associated with restoration function, Ghir_D05.742.1 and m64033_190821_201011/21103726/ccs were identified and showed significant higher levels of expression in restorer line than sterile and maintainer lines. Our comparative full-length transcriptome analysis provides new insights into the molecular function of Rf2 fertility restorer gene. The results of this study offer a platform for fertility restoration candidate gene discovery in CMS-D8 cotton.

Comparative analysis of mitochondrial genomes provides insights into the mechanisms underlying an S-type cytoplasmic male sterility (CMS) system in wheat (Triticum aestivum L.)

Cytoplasmic male sterility (CMS) has been widely used in crop cross breeding. There has been much research on wheat CMS. However, the correlation between S-type CMS and mitochondrial genome remains elusive. Herein, we sequenced the mitochondrial genome of wheat CMS line and compared it with the maintainer line. The results showed that the mitochondrial genome of CMS line encoded 26 tRNAs, 8 rRNAs, and 35 protein-coding genes, and the cob encoding complex III in which the protein coding gene is mutated. This protein is known to affect reactive oxygen (ROS) production. The analysis of ROS metabolism in developing anthers showed that the deficiency of antioxidants and antioxidant enzymes in the sterile system aggravated membrane lipid oxidation, resulting in ROS accumulation, and influencing the anther development. Herein, cob is considered as a candidate causative gene sequence for CMS.