Cytoplasmic Male Sterility Types Research Articles

Disruption of Energy Metabolism and Reactive Oxygen Species Homeostasis in Honglian Type-Cytoplasmic Male Sterility (HL-CMS) Rice Pollen

Honglian type-cytoplasmic male sterility (HL-CMS) is caused by the inter-communication between the nucleus and mitochondria. However, the mechanisms by which sterility genes regulate metabolic alterations and changes in mitochondrial morphology in the pollen of HL-CMS remains unclear. In this study, we compared the morphological differences between the pollen of the male sterile line YA and the near-isogenic line NIL-Rf6 using hematoxylin and eosin staining as well as 4ʹ,6-diamidino- 2-phenylindole (DAPI) staining. HL-CMS is characterized by gametophytic sterility, where the aborted pollen grains are empty and the tapetal layer remains intact. Transmission electron microscopy was employed to observe mitochondrial morphological changes at the microspore stage, revealing that significant mitochondrial alternations, characterized by the formation of 'large spherical mitochondria', occurred at the binucleate stage in the YA line. Additionally, metabolomics analysis revealed decreased levels of metabolites associated with the carbohydrate and flavonoid pathways. Notably, the decrease in flavonoids was found to contribute to an elevation in reactive oxygen species (ROS) levels. Therefore, we propose a model in which rice fertility is modulated by the levels of pollen carbohydrates and flavonoid metabolites, with impaired mitochondrial energy production and reduced flavonoid biosynthesis being the main causes of ROS accumulation and pollen abortion 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.

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.

Marker assisted selection reveal SNP deletion mutation in gene associated with cytoplasmic male sterility in Solanumvillosum

Male sterility (MS) is powerful tool for improvement of various traits of economic importance. MS has not been characterized in Solanum villosum, and molecular mechanism underlying mutation change have not yet been studied. In this study, Sanger sequence platform was employed to amplify the MS genic region using mt ATPase marker. The molecular mechanism governing sterility was studied, and the results detected the amplification region at 1150bp. The sequence query was 97 % identical with atp subunit 6 gene loci. Significance mutation detected in this study was deletion GG (delGG) and deletion C (delC) occurring between 536bp and 542bp position in atp6 gene transcript. The mutation score was 16 for delGG and 17 for delC, with a 90 % mutation confidence interval. The study observed that SNP changes due to base deletions in the atp6 gene region were the main cause of male sterility in the studied mutants due to anticipated frameshifts of the open reading frame (ORF) of the mitochondrial atp6 gene, this is regarded as cytoplasmic male sterility type (CMS). Deformed and low pollen count was morphological changes confirmed this study that associated with sterility. Disruption of the ATP synthesis in mitochondria, limits the energy supply for pollen grain formation and anticipated pollen sterility however, mitochondria nuclear gene interaction governing the CMS should be further studied in case of S.villosum. These findings shed-light on molecular mechanism underlying the CMS and can be utilized as a molecular marker for agronomic traits improvement of S.villosum.

ORF138 causes abnormal lipid metabolism in the tapetum leading to Ogu cytoplasmic male sterility in Brassica napus

Plant cytoplasmic male sterility is caused by mutations and rearrangements of mitochondrial genes. It serves as a significant way to utilize hybrid vigor to enhance crop yield. Ogu CMS is a natural cytoplasmic male sterility type discovered in radishes, being successfully transferred to rapeseed and cruciferous vegetables. However, current studies lack depth in exploring the molecular mechanisms of its male sterility. In this study, we confirmed orf138 is the causal gene for Ogu CMS through genetic transformation in Arabidopsis. Transcriptome analysis of aborted anthers in different stages suggested differentially expressed genes (DEGs) are mainly enriched in pathways such as glycerophospholipid metabolism and arginine and proline metabolism. It reveals that key genes involved in lipid metabolism pathways are significantly down-regulated in the sterile line (OguA), including BnaGPAT1, localized within the tapetum mitochondrial and endoplasmic reticulum. This could lead to changes in the metabolism of substances like acylglycerols within the tapetum, causing disruptions in lipid metabolism. This is consistent with morphological and subcellular structural changes in the tapetum and microspore cells as observed in the transmission electron microscopy. This abnormal lipid metabolism may trigger specific ROS accumulation in an oxidative stress response, ultimately leading to aborted microspore. Our study based on transcriptome has deepened our understanding of the molecular mechanisms in Ogu CMS.

Correlation between the response of maize lines to CMS and their origin

Transferring maize hybrids to a sterile basis is the most important condition for their introduction into production. In this regard, it is important to study the behavior of the initial forms of hybrids, namely self-pollinated lines, in the sterile cytoplasm. The purpose of the current study was to estimate the response of new self-pollinated maize lines, depending on their origin, to the “M” and “C” types of CMS, to identify natural restoring agents and complete sterility fixatives. The study was carried out at the FSBSI “ARC “Donskoy” in 2021–2023. The subjects of the study were 20 new self-pollinated maize lines of various origins, 6 sources of sterility, 120 testcross maize hybrids. There was found that the response of maize lines depended on their origin. Lines belonging to the same group (by origin) usually had the same reaction to the Moldovan (“M”) and Bolivian (“C”) types of cytoplasmic male sterility (CMS). The closer the lines are related, the smaller the differences. The new self-pollinated lines of the first group ‘SL 171’, ‘SL 172’, ‘SL 174’, ‘SL 175’, ‘SL 176’ and ‘SL 177’ were characterized by complete consolidation of the type “M” of CMS (class 0). Lines of subgroup Ia were fixed, but subgroups Ib did not completely restore the type “C” of sterility. In the second group there were identified the complete ‘SL 181’, ‘SL 182’ (class 4, 5) and incomplete ‘SL 183’, ‘SL 184’ (class 2–5) reducing agents of the type “M” of sterility. There was determined that all lines of the second group perpetuated sterility of the Bolivian type (class 0). There have been identified the lines of the third group ‘SL 191’, ‘SL 192’, ‘SL 193’, ‘SL 194’, as full reducing agents of the type “M” of CMS, two of them ‘SL 191’ and ‘SL 193’ are also complete reducing agents of the type “C” of CMS (class 4, 5). All lines of the fourth group could be classified as complete fixatives (class 0, 1) of the studied types of CMS. The results obtained will improve the efficiency of work on transferring maize hybrids to a sterile basis.

Mapping of Rf20(t), a minor fertility restorer gene for rice wild abortive cytoplasmic male sterility in the maintainer line 'Zhenshan97B'.

Three-line hybrid rice has primarily been developed on wild abortive (WA)-type cytoplasmic male sterility (CMS) and has helped increase the yield of rice globally. The development of WA-type CMS lines and hybrids was expedited through the identification and mapping of the fertility restorer gene (Rf) in maintainers. This study observed fertile plants in WA-TianfengA/Zhenshan97B//TianfengB population, indicating that the maintainer line 'Zhenshan97B' should carry Rfs for WA-type CMS. Several advanced backcross populations were generated with the genetic background of the 'WA-TianfengA,' and the pollen fertility levels of the backcrossed individuals in BC3F1, BC4F1 and BC4F2 populations are governed by a new gene, Rf20(t), from 'Zhenshan97B.' Employing bulk segregant analysis of fertile and sterile pools from the BC4F1 population, Rf20(t) was genetically mapped to a candidate region on chromosome 10. Subsequently, Rf20(t) was located between RM24883 and RM24919 through recombination analysis of molecular markers using the BC4F2 population. Implementing a substitution mapping strategy, Rf20(t) was ultimately mapped to a 245-kb region between the molecular markers STS10-122 and STS10-126 and obtained the most likely candidate gene LOC_Os10g02650, which is predicted to encode pentatricopeptide repeat-containing (PPR) protein. These results enhance our understanding of the fertility restoration of WA-type CMS lines, facilitating the development of high-quality pairs of WA-type CMS and maintainer lines.

The top Bulgarian contributions to sunflower breeding

Abstract The sunflower (Helianthus annuus L.) is a fundamental oil-yielding culture for Bulgaria and the world and thus, special attention is paid to it. Various selection methods have been used over the years and as a result of successful breeding programs new sunflower forms, lines (В/А and R) and hybrids (oil and confectionery type) were created in Bulgaria. Some of these new forms have new plant architectonic, different vegetation periods, seeds with different sizes and coloration, high combining ability, resistance to diseases, the parasite Broomrape and some herbicides, high seed oil and fatty acid content, genes for restoration of fertility (Rf genes), and different types of cytoplasmic male sterility (CMS).

Floral morphology of 'Anand' rapid cycling Brassica oleracea .

Abstract The 'Anand' type of cytoplasmic male sterility (CMS) was introduced to Brassica oleracea through protoplast fusion (Cardi and Earle, 1997), providing a source of male sterility that is of value in producing F1 hybrid varieties of this species without the cold sensitivity of 'Ogura' cytoplasm. In this species, as in others in which it has been introduced, 'Anand' mitochondria are associated with flower abnormalities. The best lines have been used by commercial breeders. This paper describes 'Anand' flower abnormalities in further detail. Rapid cycling brassica with 'Anand' cytoplasm may have promise as a model system for research on floral patterning. Materials and Methods. Three lines of rapid cycling B. oleracea carrying the 'Anand' cytoplasm and one wild type were grown. The flower morphology of 'Anand' lines was classified as good in F25-F-1A, fair in F25-F-6B, and poor in F23-F-1B (Cardi and Earle, 1997). Seedlings were started in an indoor growth room, but were maintained in a naturally lit greenhouse at 20° night, 25° day during flowering. Five flowers on 5 plants of each line were examined. Results. Morphological abnormalities affected all floral parts (Table 1). There was substantial variation among flowers on individual plants and among lines but little within lines. Conclusions. "Good" flowers have large petals that push open the sepals before the carpel elongates. Female fertility could be reduced due to occlusion of the stigma or physical damage caused by the stigma pressing against the end of a sepal and kinking the style. Female fertility in some flowers was unlikely because the carpel suture failed to fuse. In some cases the edge of the split carpel was sepal-like, consistent with the absence of SEPALLATA expression. The stigma on a sepal represents the converse. Stamens were most strongly affected, with outer stamens most reduced. This whorl is the one most frequently suppressed in flowering plants (Masters, 1869). The number of stamens was reduced to varying degrees. First to be lost was one or both of the pair outside the petals. Then the outer pair inside the petals was affected. One or both of these stamens could either be lost or doubled to give 3 to 5 stamens. Again a positional cue for stamens appears to be weakened. Anther development was often limited, the expected and desired consequence of the Anand genotype. Some undehisced " good " anthers contained pollen. There is variation even among the lines initially described as " good ". Further selection among these may help identify those that are most promising for further use in hybrid production.

Adaptation and High Yield Performance of Honglian Type Hybrid Rice in Pakistan with Desirable Agricultural Traits

Honglian type cytoplasmic male sterility (CMS) is one of the three known major CMS types of rice (Oryza sativa L.) commercially used in hybrid rice seed production. Hybrid rice generated by the Honglian type CMS is a special group of hybrid rice, having distinct agricultural characteristics. The main objective of the study was to screen out the Honglian hybrid rice adapted for growing in Pakistan based on desirable traits. Different Honglian-type hybrid rice varietieswere tested locally in different locations in Pakistan based on various desirabletraits. Three Honglian types of hybrids (HP1, HP2, HP3) performed well, had better agricultural traits and showed high yield potential over the check variety. Different qualitative and quantitative traits were studied to conclude the advantages of these varieties for Pakistani local adaptation evaluations. Forty-eight SSR markers were used to study the genetic diversities of the hybrids. Nine selected polymorphic SSR markers (RM-219, RM-236, RM-274, RM-253, RM-424, RM-567, RM-258, RM-481, RM-493) showed genetic variations among Honglian hybrid rice varieties through PCR analysis. In 2019 and 2020, the increment of the yield potential of HP1, HP2 and HP3 was better (+43.90%, +35.44%, +37.13% and +30.91%, +33.37%, +33.62%, respectively, in both years)than the check variety KSK-133. All the desirable traits were analyzed through Principal Component Analysis (PCA). The principal components with more than one eigenvalue showed more variability. The average variability of 74.78% was observed among genotypes and their desirable traits in both years. National Uniform Yield Trial (NUYT) and Distinctness, Uniformity, Stability (DUS) trials are being conducted under the supervision of National Coordinated Rice (NCR) and Federal Seed Certification and Registration Department (FSCRD), Government of Pakistan. In the 2020 trial, the average yield of 104 rice varieties/hybrids was 8608 kg/ha; HP1, HP2 and HP3 (8709 kg/ha, 8833 kg/ha, and 9338 kg/ha, respectively) were all higher than the average yield, and HP3 yield was higher than over check varieties (D-121, Guard-53). In the 2021 trial, the average yield of 137 varieties was 7616 kg/ha; the HP1 yield (7863 kg/ha) was higher than the average overcheck varieties/hybrids. Various qualitative and quantitative traits showed desirable genetic diversity among the rice hybrids. It was also observed that, under higher temperatures, the seeds setting rate of Honglian-type hybrid rice was stable, which is the guarantee for stable yield and rice production in Pakistan. Moreover, it was considerably better, suggesting that Honglian-type hybrid rice varieties can be grown in Pakistan because they are less risky under climate change, especially the global warming challenges.

Identification and fine mapping of a fertility restorer gene for wild abortive cytoplasmic male sterility in the elite indica rice non-restorer line 9311

The wild abortive (WA)-type cytoplasmic male sterility (CMS) derived from the wild rice species Oryza rufipogon Griff. is used widely in three-line indica hybrids. The identification and mapping of restorer of fertility (Rf) genes aided in the development of WA-type hybrids. Here we report that testcross F1 plants from the WA-type CMS line and 9311 exhibited stainable pollen grains with no seed set, indicating that 9311 carries minor-effect Rfs for WA-type CMS. We developed an advanced backcross population consisting of plants harboring small regions of donor chromosomal segments from 9311 in the WA-TianfengA genetic background with moderate seed setting rates. Genetic analysis showed that the pollen fertility levels of the backcross individuals are governed by a single gene from 9311 that we named Rf19(t). By use of the RICE 40 K gene chip, three introduced segments were identified in the fertile lines, and a candidate region spanning 4.37–8.29 Mb on chromosome 1 was identified for Rf19(t). Finally, Rf19(t) was fine-mapped to a region of 90 kb between the DNA marker loci STS1-163 and STS1-183, in which eight ORFs were predicted. Also, using relative expression analyses, comparative sequence analyses and functional domain analyses, we identified LOC_Os01g10530 as the most likely candidate gene for Rf19(t). Furthermore, Rf19(t) was found to function in fertility restoration, most probably by regulating the degradation of mRNA transcribed from the mitochondrial gene WA352. These results increase our knowledge of fertility restoration in WA-type CMS lines and will facilitate the development of high-quality pairs of WA-type CMS and maintainer lines.

The 4R QTLs pollen fertility restoration in triticale with CMS Triticum timopheevii

AbstractThe Triticum timopheevii Zhuk. (Tt) cytoplasm is the most exploited cytoplasmic male sterility (CMS) type used in triticale in commercial seed production of hybrid varieties. The present study established a recombinant inbred line (RIL) mapping population to identify genetic loci controlling fertility restoration in triticale with CMS Tt. The trait was evaluated via phenotyping the BC1F1 materials originating from a backcross of maternal line MS 112(15)‐2‐1 (on CMS Tt) and RILs. A genetic map spanning 2293 cM over 21 linkage groups was constructed using 1,170 single nucleotide polymorphisms and silico diversity array technology (DArT) markers generated through DArTseq genotyping. Two quantitative trait loci (QTL) controlling fertility restoration were localized at the 4RL chromosome. They were stable during two vegetative seasons and, depending on the year, explained 17.8 to 37.5 (QRft‐4R.1) and 17.8 to 27.1% (QRft‐4R.2) of phenotypic variance. Bioinformatic analysis was performed to predict genes potentially responsible for the trait. Four markers linked to the trait were successfully converted into a polymerase chain reaction‐based assay to select the pollen fertility QTLs easily. The exact position of QTLs detected previously in the rye and here in the triticale suggests that the same loci might control the trait.

Developmentally regulated mitochondrial biogenesis and cell death competence in maize pollen

BackgroundCytoplasmic male sterility (CMS) is a maternally inherited failure to produce functional pollen that most commonly results from expression of novel, chimeric mitochondrial genes. In Zea mays, cytoplasmic male sterility type S (CMS-S) is characterized by the collapse of immature, bi-cellular pollen. Molecular and cellular features of developing CMS-S and normal (N) cytoplasm pollen were compared to determine the role of mitochondria in these differing developmental fates.ResultsTerminal deoxynucleotidyl transferase dUTP nick end labeling revealed both chromatin and nuclear fragmentation in the collapsed CMS-S pollen, demonstrating a programmed cell death (PCD) event sharing morphological features with mitochondria-signaled apoptosis in animals. Maize plants expressing mitochondria-targeted green fluorescent protein (GFP) demonstrated dynamic changes in mitochondrial morphology and association with actin filaments through the course of N-cytoplasm pollen development, whereas mitochondrial targeting of GFP was lost and actin filaments were disorganized in developing CMS-S pollen. Immunoblotting revealed significant developmental regulation of mitochondrial biogenesis in both CMS-S and N mito-types. Nuclear and mitochondrial genome encoded components of the cytochrome respiratory pathway and ATP synthase were of low abundance at the microspore stage, but microspores accumulated abundant nuclear-encoded alternative oxidase (AOX). Cytochrome pathway and ATP synthase components accumulated whereas AOX levels declined during the maturation of N bi-cellular pollen. Increased abundance of cytochrome pathway components and declining AOX also characterized collapsed CMS-S pollen. The accumulation and robust RNA editing of mitochondrial transcripts implicated translational or post-translational control for the developmentally regulated accumulation of mitochondria-encoded proteins in both mito-types.ConclusionsCMS-S pollen collapse is a PCD event coincident with developmentally programmed mitochondrial events including the accumulation of mitochondrial respiratory proteins and declining protection against mitochondrial generation of reactive oxygen species.

Characterization, inheritance of male sterility and development of male sterile and maintainer lines in ridge gourd (Luffa acutangula (Roxb.) L.)

Two male sterile mutants IIHRRG-12MS (long fruited) and IIHRRG-28MS (medium long fruited) were identified from the ridge gourd germplasm IIHR-12 and IIHR-28 respectively at ICAR-IIHR, Bengaluru. These two male-sterile (ms) sources were characterized by the production of rudimentary male flowers in the racemes in contrast to the bright yellow flowers with fertile pollen and healthy anthers in male fertile, monoecious plants. Using these ms lines the inheritance of male sterility was worked out, which is cytoplasmic genic male sterility (CGMS) type, with single dominant gene either in homozygous or heterozygous condition restoring male fertility in the presence of sterile cytoplasm. In order to develop F1 hybrids using male sterility, several male sterile and maintainer lines were developed in different genetic back grounds such as green/dark green fruit colour and short/medium long/long fruit length.

Responses of nitrogen accumulation and translocation in five cytoplasmic hybrid rice cultivars

To study the difference among cytoplasm at the different nitrogen conditions, a research experiment was conducted using five different cytoplasmic male sterile (CMS) hybrid rice with nitrogen levels at N0, N1, N2, and N3; the nitrogen application rates were 0, 90, 180, and 270 kg/ha, from 2018 to 2019. Results showed that among tested cultivars of CMS hybrid rice, JW (J803A × Chenghui727) showed the highest yield in both years for the low nitrogen and high nitrogen treatments. The dry matter accumulation and translation of JW type in nutritive organs were higher than that of others during the low nitrogen level (N1). We concluded that the nutrient translocation within plants organs and dry biomass accumulation were highly dependent on CMS type and nitrogen application. This research indicates that selecting a rice cultivar with greater efficiency of nitrogen is favourable for raising the number of grains per panicle, grain yield, and nitrogen use efficiency. JW cytoplasm displayed great efficiency in low nitrogen, which is a potential cytoplasmic resource.

SSR-маркеры, ассоциированные с геном-восстановителем ЦМС 9Е сорго

9E type of cytoplasmic male sterility (CMS) in sorghum belongs to environmentally-regulated types of male sterility in which the restoration of fertility is regulated, along with the genotype, by plant water availability and air humidity on the eve and during the flowering period. For studying the genetic control and understanding the mechanisms of epigenetic processes that regulate the restoration of fertility in this CMS type, the identification of fertility restoring genes (Rf-9E) is extremely important. To solve this problem, we carried out the work on identification of molecular markers associated with the Rf-9E genes. For this purpose, we used the plants of the BC1 population [(9E Pishchevoe 614 × 84/19) × Pishchevoe 614] segregated for fertility restoration. In total, 55 SSR markers were used, mapping all 10 chromosomes of the sorghum genome. It was found that SSR markers located on chromosome 2 of the sorghum genome, sam60498 (23911554…23913675 bp) and sam37585 (33055782…33056867 bp), demonstrate a significant association with the restoration of male fertility in the studied population. In silico analysis of the reference genome of S. bicolor (BTx623) showed that near this region, which includes more than 9 million bp, there are a pericentromeric heterochromatin region, and two PPR genes, 002G142700.1 and 002G144300.1 located at 23465796…23468350 bp and 23810047…23812824bp, respectively. Given that the vast majority of cloned Rf-genes in various plant species, including sorghum, contain sequences of PPR genes, the SSR markers identified in this study can indeed be the molecular markers of 9ECMS fertility-restoring genes. A hypothesis on the relationship between the localization of CMS 9E fertility-restoring genes near heterochromatin and the “moisture-dependent” nature of male fertility restoration in this CMS type is discussed.

The main restorer Rf3 of maize S type cytoplasmic male sterility encodes a PPR protein that functions in reduction of the transcripts of orf355

Male sterile lines, especially cytoplasmic male sterile (CMS) lines, are extensively used in commercial hybrid seed production. Therefore a better understanding of the genetic basis of fertility restoration for CMS is important for the utility of heterosis in crops. In general, restorer of fertility (Rf) is controlled by one or two major genomic genes, and most of the Rf genes encode proteins containing P type pentatricopeptide repeat (PPR) motifs (Wang et al., 2006; Hu et al., 2012; Tang et al., 2014; Liu et al., 2016).

The effects of Rf5 and Rf6 on fertility restoration in Honglian-type cytoplasmic male sterile (CMS) lines of japonica rice (Oryza sativa L. ssp. japonica).

The online version contains supplementary material available at 10.1007/s11032-021-01256-7.

Unraveling the Genetic Basis of Fertility Restoration for Cytoplasmic Male Sterile Line WNJ01A Originated From Brassica juncea in Brassica napus.

Crosses that lead to heterosis have been widely used in the rapeseed (Brassica napus L.) industry. Cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) systems represent one of the most useful tools for rapeseed production. Several CMS types and their restorer lines have been identified in rapeseed, but there are few studies on the mechanisms underlying fertility restoration. Here, we performed morphological observation, map-based cloning, and transcriptomic analysis of the F2 population developed by crossing the CMS line WNJ01A with its restorer line Hui01. Paraffin-embedded sections showed that the sporogenous cell stage was the critical pollen degeneration period, with major sporogenous cells displaying loose and irregular arrangement in sterile anthers. Most mitochondrial electron transport chain (mtETC) complex genes were upregulated in fertile compared to sterile buds. Using bulked segregant analysis (BSA)-seq to analyze mixed DNA pools from sterile and fertile F2 buds, respectively, we identified a 6.25 Mb candidate interval where Rfw is located. Using map-based cloning experiments combined with bacterial artificial chromosome (BAC) clone sequencing, the candidate interval was reduced to 99.75 kb and two pentatricopeptide repeat (PPR) genes were found among 28 predicted genes in this interval. Transcriptome sequencing showed that there were 1679 DEGs (1023 upregulated and 656 downregulated) in fertile compared to sterile F2 buds. The upregulated differentially expressed genes (DEGs) were enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) lysine degradation pathway and phenylalanine metabolism, and the downregulated DEGs were enriched in cutin, suberine, and wax biosynthesis. Furthermore, 44 DEGs were involved in pollen and anther development, such as tapetum, microspores, and pollen wall development. All of them were upregulated except a few such as POE1 genes (which encode Pollen Ole e I allergen and extensin family proteins). There were 261 specifically expressed DEGs (9 and 252 in sterile and fertile buds, respectively). Regarding the fertile bud-specific upregulated DEGs, the ubiquitin–proteasome pathway was enriched. The top four hub genes in the protein–protein interaction network (BnaA09g56400D, BnaA10g18210D, BnaA10g18220D, and BnaC09g41740D) encode RAD23d proteins, which deliver ubiquitinated substrates to the 26S proteasome. These findings provide evidence on the pathways regulated by Rfw and improve our understanding of fertility restoration.

Genomic Scan of Male Fertility Restoration Genes in a 'Gülzow' Type Hybrid Breeding System of Rye (Secale cereale L.).

Efficient and stable restoration of male fertility (Rf) is a prerequisite for large-scale hybrid seed production but remains an inherent issue in the predominant fertility control system of rye (Secale cereale L.). The ‘Gülzow’ (G)-type cytoplasmic male sterility (CMS) system in hybrid rye breeding exhibits a superior Rf. While having received little scientific attention, one major G-type Rf gene has been identified on 4RL (Rfg1) and two minor genes on 3R (Rfg2) and 6R (Rfg3) chromosomes. Here, we report a comprehensive investigation of the genetics underlying restoration of male fertility in a large G-type CMS breeding system using recent advents in rye genomic resources. This includes: (I) genome-wide association studies (GWAS) on G-type germplasm; (II) GWAS on a biparental mapping population; and (III) an RNA sequence study to investigate the expression of genes residing in Rf-associated regions in G-type rye hybrids. Our findings provide compelling evidence of a novel major G-type non-PPR Rf gene on the 3RL chromosome belonging to the mitochondrial transcription termination factor gene family. We provisionally denote the identified novel Rf gene on 3RL RfNOS1. The discovery made in this study is distinct from known P- and C-type systems in rye as well as recognized CMS systems in barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). We believe this study constitutes a stepping stone towards understanding the restoration of male fertility in the G-type CMS system and potential resources for addressing the inherent issues of the P-type system.