Cytoplasmic Male Sterility Genes Research Articles

The mitochondrial orf117Sha gene desynchronizes pollen development and causes pollen abortion in Arabidopsis Sha cytoplasmic male sterility.

Cytoplasmic male sterility (CMS) is of major agronomical relevance in hybrid breeding. In gametophytic CMS, abortion of pollen is determined by the grain genotype, while in sporophytic CMS, it is determined by the mother plant genotype. While several CMS mechanisms have been dissected at the molecular level, gametophytic CMS has not been straightforwardly accessible. We used the gametophytic Sha-CMS in Arabidopsis to characterize the cause and process of pollen abortion by implementing in vivo biosensing in single pollen and mitoTALEN mutagenesis. We obtained conclusive evidence that orf117Sha is the CMS-causing gene, despite distinct characteristics from other CMS genes. We measured the in vivo cytosolic ATP content in single pollen, followed pollen development, and analyzed pollen mitochondrial volume in two genotypes that differed only by the presence of the orf117Sha locus. Our results showed that the Sha-CMS is not triggered by ATP deficiency. Instead, we observed desynchronization of a pollen developmental program. Pollen death occurred independently in pollen grains at diverse stages and was preceded by mitochondrial swelling. We conclude that pollen death is grain-autonomous in Sha-CMS and propose that mitochondrial permeability transition, which was previously described as a hallmark of developmental and environmental-triggered cell death programs, precedes pollen death in Sha-CMS.

Mitochondrial phylogeny and distribution of cytoplasmic male sterility-associated genes in Beta vulgaris.

Cytoplasmic male sterility (CMS) is a mitochondrial-encoded trait that confers reproductive defects in males but not in females or any vegetative function. Why CMS is so often found in plants should be investigated from the viewpoint of mitochondrial phylogeny. Beta vulgaris, including the wild subspecies maritima and cultivated subspecies vulgaris (e.g., sugar beet), is known to be mitochondrially polymorphic, from which multiple CMS mitochondria have been found, but their evolutionary relationship has been obscure. We first refined the B. vulgaris reference mitochondrial genome to conduct a more accurate phylogenetic study. We identified mitochondrial single-nucleotide polymorphic sites from 600 B. vulgaris accessions. Principal component analysis, hierarchical clustering analysis, and creation of a phylogenetic tree consistently suggested that B. vulgaris mitochondria can be classified into several groups whose geographical distribution tends to be biased toward either the Atlantic or Mediterranean coasts. We examined the distribution of CMS-associated mitochondrial genes from Owen, E- and G-type CMS mitochondria. About one-third of cultivated beets had Owen-type CMS, which reflects the prevalence of using Owen-type CMS in hybrid breeding. Occurrence frequencies for each of the three CMS genes in wild beet were less than 4%. CMS genes were tightly associated with specific mitochondrial groups that are phylogenetically distinct, suggesting their independent origin. However, homologous sequences of the Owen type CMS gene occurred in several different mitochondrial groups, for which an intricate explanation is necessary. Whereas the origin of cultivated beet had been presumed to be Greece, we found an absence of Owen-type mitochondria in Greek accessions.

Progress in identifying male sterility genes and utilizing heterosis in wheat

<p indent="0mm">Male sterility is a valuable trait for plant breeding and heterosis utilization. Identifying and characterizing male-sterile genes in crops have improved our understanding of the molecular mechanisms controlling anther and pollen grain development, and enabled the development and effective use of many biotechnology-based male sterile systems in hybrid crop breeding. Research progress on male-sterile genes in wheat (<italic>Triticum</italic> spp.) lags that in rice (<italic>Oryza sativa</italic>) and maize (<italic>Zea mays</italic>) because of the polyploid nature and complex genome of wheat; therefore, only a few wheat male-sterile genes have been identified or cloned. Due to the limited progress in understanding the basis of male sterility, a low-cost and efficient wheat hybridization system has not been developed. In this review, we summarize the progress in characterizing genetic male sterility (GMS), cytoplasmic male sterility (CMS), and environment-sensitive GMS (EGMS) in wheat. At least five GMS genes were reported in wheat via forward genetic approaches, of which the dominant gene <italic>Ms2</italic> and the recessive genes <italic>ms1</italic> and <italic>ms5</italic> have been cloned. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) gene editing technology was used to characterize the role of four wheat homologs of maize <italic>Ms26</italic> and <italic>Ms45</italic>, rice <italic>NO POLLEN1 </italic>(<italic>OsNP1</italic>), and <italic>Arabidopsis thaliana</italic> <italic>DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 </italic>(<italic>TDF1</italic>) in male fertility. Over 70 different types of wheat CMS systems have been reported; in most cases, CMS is caused by mitochondrial genome rearrangements, and can be reversed with nuclear restorer of fertility (<italic>Rf</italic>) genes. Current approaches to identify wheat <italic>Rf</italic> genes for fertility restoration focus on T-type (<italic>Triticum timopheevii</italic>) and K-type (<italic>Aegilops</italic><italic>kotschyi</italic> Boiss) CMS lines. At least 15 wheat <italic>Rf</italic> genes have been identified, but only two,<italic> Rf1</italic> and <italic>Rf3</italic>, were cloned by genetic mapping and comparative sequence analysis. These two restorer genes and their mode of action in CMS provide new opportunities for wheat hybrid breeding. The candidate CMS restorer genes <italic>Rfk1</italic> and <italic>Rf</italic> ^{<italic>multi</italic>} were also predicted using comparative genomics approaches. EGMS may involve epistatic regulation of non-coding RNAs that restore fertility under specific conditions, and thus provides a special class of germplasm for hybrid production in several crops; however, only a few wheat EGMS genes have been reported and none of them have been cloned. Next, we summarize the wheat hybridization systems that rely on male-sterile genes, such as the “three-line system” based on CMS genes, the “two-line system” based on EGMS genes, and the next generation of hybrid seed production based on nuclear GMS genes. Recent efforts in hybrid wheat breeding have produced hybrid varieties with desirable attributes, which have been marketed in Europe and China. Finally, we discuss future perspectives and challenges in the research and application of next-generation wheat hybrid production technology. This review provides a reference for researching and utilizing male-sterile genes in <italic>Triticeae</italic> crops.

Fine mapping of Rf2, a minor Restorer-of-fertility (Rf) gene for cytoplasmic male sterility in chili pepper G164 (Capsicum annuum L.).

Genome re-sequencing and recombination analyses identified Capana06g000193 as a strong candidate for the minor male fertility restoration locus Rf2 in chili pepper G164 harboring two dominant male fertility restoration genes. Male fertility restoration genes of chili pepper restorer line G164 (Capsicum annuum L.) were studied using molecular marker genotypes of an F2 population (7G) of G164 crossed with the cytoplasmic male sterility line 77013A. The ratio of sterile to fertile single plants in the F2 population was 1:15. This result indicates that chili pepper G164 has two dominant restoration genes, which we designated as Rf1 and Rf2. An individual plant recessive for Rf1 and heterozygous for Rf2, 7G-112 (rf1rf1Rf2rf2), was identified by molecular marker selection and genetic analysis, and a single Rf2 gene-segregating population with a 3:1 ratio of fertile to sterile plants was developed from the self-pollination of male fertile individuals of 77013A and 7G-112 hybrid progeny. Bulk segregant analysis of fertile and sterile pools from the segregating populations was used to genetically map Rf2 to a 3.1-Mb region on chromosome 6. Rf2 was further narrowed to a 179.3-kb interval through recombination analysis of molecular markers and obtained the most likely candidate gene, Capana06g000193.

The cotton mitochondrial chimeric gene orf610a causes male sterility by disturbing the dynamic balance of ATP synthesis and ROS burst

Plant cytoplasmic male sterility (CMS) is maternally inherited and often manifested as aborted pollen development, but the molecular basis of abortion remains to be identified. To facilitate an investigation of CMS in cotton, the complete sequence of cotton mitochondrial (mt) genome for CMS-D2 line ZBA was determined. The mt genome was assembled as a single circular molecule with 634,036 bp in length. A total of 194 ORFs, 36 protein‐coding genes, six rRNAs, and 24 tRNAs were identified. Several chimeric genes encoding hypothetical proteins with transmembrane domains were identified. Among them, a previously unknown chimeric gene, orf610a, which is composed of atp1 and a 485-bp downstream sequence of unknown nature, was identified. RT-PCR and qRT-PCR validation indicated that orf610a was expressed specifically in a sterile line. Ectopic expression of orf610a in yeast resulted in excessive accumulation of reactive oxygen species and reduction in ATP content, in addition to inhibition of cellular growth. Transgenic A. thaliana overexpressing orf610a fused with a mitochondrial targeting peptide displayed partial male sterility. Interaction between ORF610a and the nuclear-encoded protein RD22 indicated an association between ORF610a and pollen abortion. Positive feedback during transcriptional regulation between nuclear regulatory factors and the mt CMS gene may account for the male sterility of ZBA.

Intraspecific variations of the cytoplasmic male sterility genes orf108 and orf117 in Brassica maurorum and Moricandia arvensis, and the specificity of the mRNA processing.

The mitochondrial gene orf108, which is co-transcribed with atp1 and causes cytoplasmic male sterility in Brassica crops, is widely distributed across wild species and genera of Brassicaceae. However, to date, intraspecific variations in the presence of orf108 have not yet been studied, and the mechanisms underlying the wide distribution of the gene remain unclear. We analyzed the presence and sequence variations of orf108 in two wild species, Brassica maurorum and Moricandia arvensis. After polymerase chain reaction amplification of the 5' region of atp1 and the coding sequence of orf108, we determined the DNA sequences. Brassica maurorum and M. arvensis showed variations in the presence of orf108 or orf117 (orf108V117) both between and within accessions and were not fixed to the mitochondrial type with the male sterile genes. Sequencing of the amplicons showed that B. maurorum had orf108V117 instead of orf108. Sequencing also indicated mitochondrial heteroplasmy in the two species; in particular, in B. maurorum, one plant possessed both orf108 and orf108V117 sequences. These results suggest that substoichiometric shifting of mitochondrial genomes leads to the acquisition or loss of orf108. Furthermore, fertility restorer genes of the two species were involved in the processing of the mRNA of male sterility genes at different sites.

Current Perspectives on Introgression Breeding in Food Legumes.

Food legumes are important for defeating malnutrition and sustaining agri-food systems globally. Breeding efforts in legume crops have been largely confined to the exploitation of genetic variation available within the primary genepool, resulting in narrow genetic base. Introgression as a breeding scheme has been remarkably successful for an array of inheritance and molecular studies in food legumes. Crop wild relatives (CWRs), landraces, and exotic germplasm offer great potential for introgression of novel variation not only to widen the genetic base of the elite genepool for continuous incremental gains over breeding cycles but also to discover the cryptic genetic variation hitherto unexpressed. CWRs also harbor positive quantitative trait loci (QTLs) for improving agronomic traits. However, for transferring polygenic traits, “specialized population concept” has been advocated for transferring QTLs from CWR into elite backgrounds. Recently, introgression breeding has been successful in developing improved cultivars in chickpea (Cicer arietinum), pigeonpea (Cajanus cajan), peanut (Arachis hypogaea), lentil (Lens culinaris), mungbean (Vigna radiata), urdbean (Vigna mungo), and common bean (Phaseolus vulgaris). Successful examples indicated that the usable genetic variation could be exploited by unleashing new gene recombination and hidden variability even in late filial generations. In mungbean alone, distant hybridization has been deployed to develop seven improved commercial cultivars, whereas in urdbean, three such cultivars have been reported. Similarly, in chickpea, three superior cultivars have been developed from crosses between C. arietinum and Cicer reticulatum. Pigeonpea has benefited the most where different cytoplasmic male sterility genes have been transferred from CWRs, whereas a number of disease-resistant germplasm have also been developed in Phaseolus. As vertical gene transfer has resulted in most of the useful gene introgressions of practical importance in food legumes, the horizontal gene transfer through transgenic technology, somatic hybridization, and, more recently, intragenesis also offer promise. The gains through introgression breeding are significant and underline the need of bringing it in the purview of mainstream breeding while deploying tools and techniques to increase the recombination rate in wide crosses and reduce the linkage drag. The resurgence of interest in introgression breeding needs to be capitalized for development of commercial food legume cultivars.

CMS systems in rapeseed and their use in the breeding of domestic hybrids

Development of heterotic hybrids is the most efficient approach to solve the problem of increasing the yield of rapeseed (Brassica napus L.), a leading oilseed crop. The cytoplasmic male sterility (CMS), widely used in rapeseed hybrid seed production, makes it possible to control hybridization between female and male lines. A review of publications on the nature of CMS systems in rapeseed and their utilization in breeding is presented. In rapeseed there are more than 10 known CMS systems of alloplasmic and homoplasmic origin. The male sterility character proved to be determined by chimeric mitochondrial genes, characterized by the presence of novel transcribed open reading frames (orf). Mitochondrial CMS genes associated with nap, pol, ogu and Nsa sterility types as well as nuclear Rf genes for pollen fertility restoration were identified. Molecular marker systems for identification of CMS-inducing and male fertility restoring genes were developed. The ogu, pol, MSL and inap CMS systems are commonly used for producing industrial rapeseed hybrids. The State Register of the Russian Federation for 2019 contains rapeseed hybrids of only foreign origin. Main achievements in domestic rapeseed hybrid production are highlighted. Research and breeding institutions developed new source material for rapeseed hete rotic hybrid breeding in various regions of the country. The sterility and fertility restoration sources were received from Canadian and French institutions as well as from domestic working collections. The yield structure traits did not deteriorate after transferring hybrid maternal lines to the sterile cytoplasm, while the glucosinolate content increased when pollen fertility restoring genes were transferred into paternal lines. Dihaploid (androclinium) lines and in vitro culture of unfertilized ovules were used to accelerate the breeding process. Experimental hybrids were developed using pol and ogu CMS.

Genome-Wide Correlation of 36 Agronomic Traits in the 287 Pepper (Capsicum) Accessions Obtained from the SLAF-seq-Based GWAS.

There are many agronomic traits of pepper (Capsicum L.) with abundant phenotypes that can benefit pepper growth. Using specific-locus amplified fragment sequencing (SLAF-seq), a genome-wide association study (GWAS) of 36 agronomic traits was carried out for 287 representative pepper accessions. To ensure the accuracy and reliability of the GWAS results, we analyzed the genetic diversity, distribution of labels (SLAF tags and single nucleotide polymorphisms (SNPs)) and population differentiation and determined the optimal statistical model. In our study, 1487 SNPs were highly significantly associated with 26 agronomic traits, and 2126 candidate genes were detected in the 100-kb region up- and down-stream near these SNPs. Furthermore, 13 major association peaks were identified for 11 key agronomic traits. Then we examined the correlations among the 36 agronomic traits and analyzed SNP distribution and found 37 SNP polymerization regions (total size: 264.69 Mbp) that could be selected areas in pepper breeding. We found that the stronger the correlation between the two traits, the greater the possibility of them being in more than one polymerization region, suggesting that they may be linked or that one pleiotropic gene controls them. These results provide a theoretical foundation for future multi-trait pyramid breeding of pepper. Finally, we found that the GWAS signals were highly consistent with those from the nuclear restorer-of-fertility (Rf) gene for cytoplasmic male sterility (CMS), verifying their reliability. We further identified Capana06g002967 and Capana06g002969 as Rf candidate genes by functional annotation and expression analysis, which provided a reference for the study of cytoplasmic male sterility in Capsicum.

Advances in understanding the molecular mechanisms of cytoplasmic male sterility and restoration in rice.

Cytoplasmic male sterility (CMS) in plants is a male reproductive defect determined by mitochondrial genes and inherited maternally. CMS can be suppressed by nuclear restorer of fertility (Rf) genes. Therefore, CMS/Rf systems provide a classic model for the study of mitochondrial-nuclear interactions in plants. Moreover, CMS/Rf systems are economical, effective tools for the production of hybrid seeds. For example, CMS/Rf systems have been applied in over forty countries to breed hybrid rice (Oryza sativa L.) with improved yields due to hybrid vigor. The production of hybrid rice mainly depends on three types of CMS systems, namely Wild-Abortive type CMS (CMS-WA), Hong-Lian type CMS (CMS-HL) and Boro II type CMS (CMS-BT). Understanding the molecular mechanisms underlying these CMS/Rf systems will help us to understand mitochondrial-nuclear interactions, and accelerate the utilization of heterosis for improvement in yield. In the past decades, research benefitting from the availability of the high-quality, annotated mitochondrial and nuclear genome sequences of rice has isolated many CMS genes, identified the cognate nuclear Rf genes and studied the molecular mechanisms underlying CMS and restoration in rice. Here, we focus on recent advances in studies of the three major CMS/Rf systems in rice and discuss the key issues facing basic research and application of CMS/Rf systems in the future.

Origins of rice cytoplasmic male sterility genes.

Cytoplasmic male sterility (CMS) factors have long been known in some wild plants, and also in some domesticated species, where they are used to produce plants to be used as maternal parents, for example to breed hybrids that display hybrid vigor. Their origins have been mystifying, and now a study recently published in Cell Research helps understand how one widely-used rice CMS factor evolved.

Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes.

New gene origination is a major source of genomic innovations that confer phenotypic changes and biological diversity. Generation of new mitochondrial genes in plants may cause cytoplasmic male sterility (CMS), which can promote outcrossing and increase fitness. However, how mitochondrial genes originate and evolve in structure and function remains unclear. The rice Wild Abortive type of CMS is conferred by the mitochondrial gene WA352c (previously named WA352) and has been widely exploited in hybrid rice breeding. Here, we reconstruct the evolutionary trajectory of WA352c by the identification and analyses of 11 mitochondrial genomic recombinant structures related to WA352c in wild and cultivated rice. We deduce that these structures arose through multiple rearrangements among conserved mitochondrial sequences in the mitochondrial genome of the wild rice Oryza rufipogon, coupled with substoichiometric shifting and sequence variation. We identify two expressed but nonfunctional protogenes among these structures, and show that they could evolve into functional CMS genes via sequence variations that could relieve the self-inhibitory potential of the proteins. These sequence changes would endow the proteins the ability to interact with the nucleus-encoded mitochondrial protein COX11, resulting in premature programmed cell death in the anther tapetum and male sterility. Furthermore, we show that the sequences that encode the COX11-interaction domains in these WA352c-related genes have experienced purifying selection during evolution. We propose a model for the formation and evolution of new CMS genes via a "multi-recombination/protogene formation/functionalization" mechanism involving gradual variations in the structure, sequence, copy number, and function.

The Evolutionary Dynamics of Gynodioecy inLobelia

Premise of research. Plant mitochondria are expected to show no within-species haplotype diversity. However, sex is determined in some plants by an interaction between mitochondrial cytoplasmic male sterility (CMS) genes and nuclear restorers, resulting in gynodioecy. A question arises as to whether CMS genes are transient or under balancing selection. With balancing selection, rare CMS genes should be favored, potentially resulting in the maintenance of multiple mitochondrial haplotypes. Given that populations with multiple CMS genes are expected to have higher proportions of females, we predicted that the percentage of females in a population would be positively correlated with mitochondrial haplotype diversity.Methodology. We compared sequence diversity in regions of the mitochondrial cob and cox1 genes for populations of multiple species of Lobelia, which includes hermaphroditic and gynodioecious members.Pivotal results. Sequence variation was low, with haplotypes across five North American species va...

Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

Somatic hybridization is performed to obtain significant cytoplasmic male sterility (CMS) lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-1A, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-1B, and the abortive process of SaNa-1A were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-1A was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-1A aborted in the pollen mother cell (PMC) stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-1A is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

A codominant molecular marker in linkage disequilibrium with a restorer-of-fertility gene (Ms) and its application in reevaluation of inheritance of fertility restoration in onions

To reveal the linkage relationship between the Ms locus, a restorer-of-fertility gene for cytoplasmic male-sterility (CMS) caused by CMS-S cytoplasm in onion (Allium cepa L.) and previously reported molecular markers linked to the Ms locus, 11 recombinants selected from 4,273 segregating plants originating from the cross between male-sterile maternal and male-fertile paternal lines were analyzed. Results showed that genotypes of a codominant marker, jnurf12, were perfectly matched with the male-fertility phenotypes in all recombinants, but that this marker was not applicable in diverse breeding lines due to multiple band patterns. For the development of more reliable markers, a 12-bp indel was identified from the sequences which were obtained by genome walking, and was used to develop a simple PCR marker which was designated jnurf13. When 104 diverse breeding lines containing CMS-S cytoplasm were analyzed with the jnurf13 marker, male-fertility phenotypes of all breeding lines were perfectly matched with marker genotypes. To our surprise, phenotypes of 153 breeding lines containing CMS-T-like cytoplasm were also matched with genotypes of the jnurf13 marker which was linked to the Ms locus for the CMS-S system. Furthermore, phenotypes of four F2 populations containing CMS-T-like cytoplasm co-segregated perfectly with jnurf13 genotypes. Allelic segregation distortion was detected in two F2 populations using the jnurf13 maker. The results of this study were in conflict with a previous model for inheritance of fertility restoration in the CMS-T system. Therefore, we proposed a new model based on the data analyzed with the jnurf13 marker, which was in linkage disequilibrium with restorer-of-fertility genes for both CMS systems.

HOW SELFING, INBREEDING DEPRESSION, AND POLLEN LIMITATION IMPACT NUCLEAR-CYTOPLASMIC GYNODIOECY: A MODEL

Gynodioecy, the co-occurrence of females and hermaphrodites, is often due to conflicting interactions between cytoplasmic male sterility genes and nuclear restorers. Although gynodioecy often occurs in self-compatible species, the effect of self-pollination, inbreeding depression, and pollen limitation acting differently on females and hermaphrodites remains poorly known in the case of nuclear-cytoplasmic gynodioecy (NCG). In this study, we model NCG in an infinite population and we study the effect of selfing rate, inbreeding depression, and pollen limitation on the maintenance of gynodioecy and on sex ratios at equilibrium. We found that selfing and inbreeding depression have a strong impact, which depends on whether restorer cost acts on male or female fitness. When cost affects male fitness, the strength of cost has no effect, whereas selfing and inbreeding depression only impact gynodioecy by modifying the value of the female advantage. When cost affects female fitness, selfing facilitates NCG and reduces the role of strength of the cost, even when no inbreeding depression occurs, whereas inbreeding depression globally restricts the maintenance of the polymorphism. Finally, we found that pollen limitation could additionally strongly modify the dynamic of gynodioecy. We discuss our findings in the light of empirical data available in gynodioecious species.

Inheritance of fertility restorer gene for cytoplasmic male-sterility in B. napus and identification of closely linked molecular markers to it

Here we report identification and inheritance of a restorer gene for the restoration of cytoplasmic male sterility in Brassica napus. Analysis of 373 plants in F2 segregating population resulted from the crosses of cms PI-ARIT121 with four different restorer inbred lines found 288 male fertile and 85 male sterile progeny in the expected 3–1 ratio indicating a single dominant gene controlling fertility restoration. This observation has also been confirmed in a back cross population of 136 individuals with 66 male fertile and 70 male sterile with the expected 1–1 ratio. Analysis of two F2 populations with SRAP molecular marker revealed that two primer combinations, Me2Em5 and Me1Mo1 were linked to the restorer gene with 1 % recombination frequency. Sequencing of the linked SRAP markers from the polyacrylamide gel and blast search revealed no significant match with the known restorer gene found in B. napus. By designing new primers in the direction of the restorer gene doing chromosome walk, we found 100 % linked marker to the restorer gene and named as PPR121. Sequencing of this gene indicated to be in the same locus with other restorer genes found in B. napus, but a little different sequence may indicates evolution of new restorer gene for the cms-ARIT121. NCBI searches indicate close similarity to genes encoding the pentatricopeptide proteins that have functions in almost all stages of gene expression, including messenger RNA transcription, splicing, processing, editing, translation and stability.

ORFH79 impairs mitochondrial function via interaction with a subunit of electron transport chain complex III in Honglian cytoplasmic male sterile rice

Cytoplasmic male sterility (CMS) has attracted great interest because of its application in crop breeding. Despite increasing knowledge of CMS, not much is understood about its molecular mechanisms. Previously, orfH79 was cloned and identified as the CMS gene in Honglian rice, but how the ORFH79 protein causes pollen abortion is still unknown. Through bacterial two-hybrid library screening, P61, a subunit of the mitochondrial electron transport chain (ETC) complex III, was selected as a candidate that interacts with ORFH79. Bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation (coIP) assays verified their interaction inside mitochondria. Blue native polyacrylamide gel electrophoresis (BN-PAGE) and western blotting showed ORF79 and P61 colocalized in mitochondrial ETC complex III of CMS lines. Compared with the maintainer line, Yuetai B (YB), a significant decrease of enzyme activity was detected in mitochondrial complex III of the CMS line, Yuetai A (YA), which resulted in decreased ATP concentrations and an increase in the reactive oxygen species (ROS) content. We propose that the CMS protein, ORFH79, can bind to complex III and decrease its enzyme activity through interaction with P61. This defect results in energy production dysfunction and oxidative stress in mitochondria, which may work as retrograde signals that lead to abnormal pollen development.

TESTING MODELS OF SEX RATIO EVOLUTION IN A GYNODIOECIOUS PLANT: FEMALE FREQUENCY COVARIES WITH THE COST OF MALE FERTILITY RESTORATION

In many gynodioecious species, cytoplasmic male sterility genes (CMS) and nuclear male fertility restorers (Rf) jointly determine whether a plant is female or hermaphrodite. Equilibrium models of cytonuclear gynodioecy, which describe the effect of natural selection within populations on the sex ratio, predict that the frequency of females in a population will primarily depend on the cost of male fertility restoration, a negative pleiotropic effect of Rf alleles on hermaphrodite fitness. Specifically, when the cost of restoration is higher, the frequency of females at equilibrium is predicted to be higher. To test this prediction, we estimated variation in the cost of restoration across 26 populations of Lobelia siphilitica, a species in which Rf alleles can have negative pleiotropic effects on pollen viability. We found that L. siphilitica populations with many females were more likely to contain hermaphrodites with low pollen viability. This is consistent with the prediction that the cost of restoration is a key determinant of variation in female frequency. Our results suggest that equilibrium models can explain variation in sex ratio among natural populations of gynodioecious species.

Sex‐specific fitness variation in gynodioecious Beta vulgaris ssp. maritima: do empirical observations fit theoretical predictions?

In gynodioecious species, in which hermaphroditic and female plants co-occur, the maintenance of sexual polymorphism relies on the genetic determination of sex and on the relative fitness of the different phenotypes. Flower production, components of male fitness (pollen quantity and pollen quality) and female fitness (fruit and seed set) were measured in gynodioecious Beta vulgaris spp. maritima, in which sex is determined by interactions between cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. The results suggested that (i) female had a marginal advantage over hermaphrodites in terms of flower production only, (ii) restored CMS hermaphrodites (carrying both CMS genes and nuclear restorers) suffered a slight decrease in fruit production compared to non-CMS hermaphrodites and (iii) restored CMS hermaphrodites were poor pollen producers compared to non-CMS hermaphrodites, probably as a consequence of complex determination of restoration. These observations potentially have important consequences for the conditions of maintenance of sexual polymorphism in B. vulgaris and are discussed in the light of existing theory on evolutionary dynamics of gynodioecy.