Variation of female frequency and cytoplasmic male-sterility gene frequency among natural gynodioecious populations of wild radish (Raphanus sativus L.)

In gynodioecious plant populations, sex determination often involves both cytoplasmic male-sterility (CMS) genes and specific nuclear genes that restore male function. How gynodioecy is maintained under the joint dynamics of CMS and restorer genes remains controversial. Although many theoretical models deal with interactions between CMS genes and restorer genes with sexual phenotypes and predict changes in their frequencies, it is difficult to observe the frequencies because no molecular markers have been established for either CMS or restorer genes in well-studied gynodioecious plants. This is the first report of the frequency of a CMS gene determined using a molecular marker in natural populations of a gynodioecious plant. Using a set of CMS gene-specific polymerase chain reaction primers, we compared female and CMS gene frequencies in 18 natural populations of Raphanus sativus. Female frequency was relatively low, ranging from 0 to 0.21. In contrast, the CMS gene frequency was highly variable among populations, ranging from 0 to 1. Estimated restorer gene frequency seemed less variable than observed CMS gene frequency, probably due to higher gene flow than in the CMS gene. Genetic drift may play a role in maintaining high variability of the CMS gene, although other possibilities are not excluded.     

The Dynamics of Gynodioecy in Plantago Lanceolata L. II. Mode of Action and Frequencies of Restorer Alleles

Male fertility in Plantago lanceolata is controlled by the interaction of cytoplasmic and nuclear genes. Different cytoplasmic male sterility (CMS) types can be either male sterile or hermaphrodite, depending on the presence of nuclear restorer alleles. In three CMS types of P. lanceolata (CMSI, CMSIIa, and CMSIIb) the number of loci involved in male fertility restoration was determined. In each CMS type, male fertility was restored by multiple genes with either dominant or recessive action and capable either of restoring male fertility independently or in interaction with each other (epistasis). Restorer allele frequencies for CMSI, CMSIIa and CMSIIb were determined by crossing hermaphrodites with ``standard' male steriles. Segregation of male steriles vs. non-male steriles was used to estimate overall restorer allele frequency. The frequency of restorer alleles was different for the CMS types: restorer alleles for CMSI were less frequent than for CMSIIa and CMSIIb. On the basis of the frequencies of male steriles and the CMS types an ``expected' restorer allele frequency could be calculated. The correlation between estimated and expected restorer allele frequency was significant.     

Modeling gynodioecy: novel scenarios for maintaining polymorphism

Nuclear-cytoplasmic gynodioecy is a breeding system of plants in which females and hermaphrodites co-occur in populations, and gender is jointly determined by cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. Persistent polymorphism at both CMS and nuclear-restorer loci is necessary to maintain this breeding system. Theoretical models have explained how nuclear-cytoplasmic gynodioecy can be stable for certain assumptions. However, recent advances in our understanding of the genetics, population biology, and molecular mechanisms of sex determination in nuclear-cytoplasmic gynodioecious species suggest the utility of new models with different underlying assumptions. In this article, we examine different negative pleiotropic fitness effects of nuclear restorers (costs of restoration) using genetic and population assumptions based on recent literature. Specifically, we model populations with two CMS types and separate nuclear restorer loci for each CMS type. Under these assumptions, both overdominance for fitness and frequency-dependent selection at nuclear-restorer loci can support nuclear-cytoplasmic gynodioecy. Costs of restoration can be either dependent or independent of the cytoplasmic background. Seed fitness costs are more vulnerable to fixation of CMS types than pollen costs. Survivorship costs are effective at maintaining polymorphism even when total reproductive effects are low. Overall, our models display differences in the stability of nuclear-cytoplasmic gynodioecy and predicted population sex ratios that should be informative to researchers studying gynodioecy in the wild.     

Distribution of fertility-restorer genes for wild-abortive and Honglian CMS lines of rice in the AA genome species of genus Oryza

BACKGROUND AND AIMS: Rice (Oryza sativa) is one of the most important cereal plants in the world. Wild-abortive (WA) and Honglian (HL) cytoplasmic male sterility (CMS) have been used extensively in the production of hybrid seeds. Although a variable number of fertility-restorer genes (Rf) for WA and HL-CMS have been identified in various cultivars, information on Rf in Oryza species with the AA-genome is sparse. Therefore the distribution and heredity of Rf for WA and HL-CMS in wild rice species of Oryza with the AA-genome were investigated. METHODS: Fertility-restorer genes for WA and HL-CMS in wild rice species with the AA-genome were investigated by following the fertility of microspores identified by I2-KI staining and by following the seed-setting rate of spikelets. A genetic model of Rf in some selected restorer accessions was analysed based on the fertility segregation of BC1F1 populations. KEY RESULTS: Fertility analysis showed that 21 out of 35 HL-type F1s, and 13 out of 31 WA-type F1s were scored as fertile. The frequency of Rf in wild rice was 60% for HL-CMS and 41.9% for WA-CMS, respectively. The fertility-restorer accessions, especially those with complete restoring ability, aggregated mainly in two species of O. rufipogon and O. nivara. The wild rice accessions with Rf for HL-CMS were distributed in Asia, Oceania, Latin American and Africa, but were centered mainly in Asia, whilst the wild restorer accessions for WA-CMS were limited only to Asia and Africa. Apart from one restorer accession that possessed two pairs of Rf for WA-CMS, all of the other nine tested wild restorer accessions each contained only a single Rf for WA-CMS or HL-CMS. Allele analysis indicated that there existed at least three Rf loci for the WA and HL-CMS systems. CONCLUSIONS: These data support the hypothesis that fertility-restorer genes exist widely in Oryza species with the AA-genome, and that Rf in Oryza sativa originated from the Oryza rufipogon/Oryza nivara complex, the ancestor of cultivated rice in Asia. The origin and evolution of Rf is tightly linked to that of CMS in wild rice, and fertility of a given CMS type is controlled by several Rf alleles in various wild restorer accessions.     

植物细胞质雄性不育及其育性恢复的分子基础

植物细胞质雄性不育是广泛存在于高等植物中的现象,其表现为母性遗传、花粉败育,但雌蕊正常。细胞质雄性不育在杂交种子生产中起着重要作用,研究其分子作用机制有利于更有效地利用细胞质雄性不育。随着一些不育基因和恢复基因相继被克隆,人们对一些细胞质雄性不育和恢复系统的分子作用机理已经有一定了解。本文综述了近年来对植物细胞质雄性不育基因和恢复基因作用机理研究的进展。     

植物细胞质雄性不育及其育性恢复的分子基础

植物细胞质雄性不育是广泛存在于高等植物中的现象, 其表现为母性遗传、花粉败育, 但雌蕊正常。细胞质雄性不育在杂交种子生产中起着重要作用, 研究其分子作用机制有利于更有效地利用细胞质雄性不育。随着一些不育基因和恢复基因相继被克隆, 人们对一些细胞质雄性不育和恢复系统的分子作用机理已经有一定了解。本文综述了近年来对植物细胞质雄性不育基因和恢复基因作用机理研究的进展。     

Development of PCR-based markers linked to dominant genes for male-fertility restoration in Pampa CMS of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Cytoplasmatic male sterility (CMS) is the basis for commercial hybrid seed production of rye. Nuclear restorer genes are indispensable for a complete restoration of fertility of the CMS lines. The drawbacks of current European restorer lines require the utilisation of new genetic resources that have been recently detected in an Iranian primitive rye population (IRAN IX) and an Argentinean landrace (Pico Gentario). The introgression of these effective restorer genes (Rfp1 and Rfp2, respectively) into breeding material can be facilitated by marker-assisted selection. Using two F(2) populations based on crosses between the non-restorer inbred line Lo6 and the restorer IRAN IX, as well as Pico Gentario, RAPDs and AFLPs were screened and led to a closely linked marker set for each of these genes. The conversion of the closest markers into fragment-specific sequence-characterised amplified region (SCAR) markers resulted in flanking ranges of 2.9 cM (Rfp1) and 5.2 cM (Rfp2). The application of these markers in backcross programmes is discussed.     

SEX RATIO VARIATION AMONG GYNODIOECIOUS POPULATIONS OF SEA BEET: CAN IT BE EXPLAINED BY NEGATIVE FREQUENCY‐DEPENDENT SELECTION?

This study is devoted to assess sex ratio variation among 33 populations of the gynodioecious Beta vulgaris ssp. maritima in Brittany (France) and to explore the causes of this variation. We showed that three different CMS (cytoplasmic male sterility) cytotypes occurred in populations, but strongly differed for their frequencies and the frequency of their associated nuclear restorer alleles (which counteract the effect of CMS and restore male fertility). No correlation was found between CMS and restorer frequencies within populations, which has been previously interpreted as a result of stochasticity. However, neutral genetic variation did not indicate recent population bottlenecks in studied populations. Moreover, no significant correlation was found between female frequency or variance and current population size. Consequently, stochastic processes could not be the major cause of sex ratio variation. Alternatively, empirical estimations of the variation of females, CMS genes and nuclear restorer allele's frequencies were compared to theoretical predictions based on a frequency‐dependent selection model of gynodioecy. In particular, we showed that an absence of correlation between CMS and restorer frequencies could also occur without stochasticity. The large variation of sex ratio in Beta vulgaris could thus be explained by frequency‐dependent selection acting on CMS genes and restorer alleles.     

Effects of CMS types and restorer alleles on plant performance in Plantago lanceolata L.: an indication for cost of restoration

In gynodioecious species, male steriles co-occur with hermaphrodites. Usually, the male sterile trait is maternally inherited, hence it is called Cytoplasmic Male Sterility (CMS). Nuclear loci restore male fertility in combination with their ‘own’ specific cytoplasmic types. In theory, two fitness components are important for the maintenance of this breeding system: a fitness advantage of the male steriles, and costs of restoration. The costs of restoration are alleged negative pleiotropic effects of restorer alleles. In this study the effects of different CMS types on plant performance and the cost of restoration were assessed in two experiments with Plantago lanceolata L. Biomass production differed significantly between the CMS types studied. In order to assess the costs of restoration, hermaphrodites with or without restorer alleles for a CMS type other than its own were compared. The studied restorer alleles caused a reduction in weight per seed, but the number of seeds produced was unaffected. The estimated cost of restoration measured as reduction of seed biomass was 13% for restorer alleles for CMSI. However, in the second experiment no pleiotropic effects of restorer alleles were detected, either because the assumptions for the experimental set-up were not valid or the costs of restoration may not always be expressed.     

Paternal leakage sustains the cytoplasmic polymorphism underlying gynodioecy but remains invasible by nuclear restorers

Cytoplasmic male sterility (CMS) in plants often results in gynodioecious populations, composed of hermaphrodites and male-sterile females. All models of gynodioecy assume maternal inheritance of the cytoplasmic alleles and postulate a variety of negatively frequency-dependent mechanisms to maintain the cytoplasmic polymorphisms observed in many natural populations. However, in some plant species, mitochondria are transmitted at least occasionally by pollen, a process called paternal leakage. We show that even a small amount of paternal leakage is sufficient to sustain a permanent, stable cytoplasmic polymorphism. Because only hermaphrodites provide pollen in gynodioecious species, the effects of paternal leakage are biased and occur more often from the non-CMS male-fertile haplotype to the CMS male-sterile haplotype. We also show that a nuclear restorer disrupts the polymorphic cytoplasmic equilibrium, leading to fixation of both the CMS allele and the restorer. Although a dominant nuclear restorer fixes, it fixes much more slowly than in the standard CMS models. Although a stable cytonuclear polymorphism is possible with "matching alleles" nuclear restoration, oscillations to low frequencies present a risk of loss by drift. Paternal leakage enhances the stability of joint cytonuclear polymorphism by reducing the chance that a CMS allele is lost by drift.     

A cost of restoration of male fertility in a gynodioecious species,Lobelia siphilitica

Abstract.— Models allowing the coexistence of females and hermaphrodites in gynodioecious populations assume a simple genetic system of sex determination, a seed fitness advantage of females (compensation), and a negative pleiotropic effect of nuclear sex-determining genes on fitness (cost of restoration). In Lobelia siphilitica , sex is determined by both mitochondrial genes causing cytoplasmic male sterility (CMS) and nuclear genes that restore fertility when present with specific CMS haplotypes (nuclear restorers). I tested for a cost of restoration in L. siphilitica by measuring restored hermaphrodites for five fitness components and estimating the number of nuclear restorers by crosses with females carrying CMS1 and CMS2. A cost of restoration appears as a significant negative coefficient (B) in the regression model explaining fitness. I found that hermaphrodites carrying more nuclear restorer genes for CMS2 (or restorer genes of greater effect) have lower pollen viability (B =– 1.08, P = 0.001). This pollen viability cost of restoration in L. siphilitica supports the theoretical prediction that negative pleiotropic effects of restorers will exist in populations of gynodioecious species containing females. The existence of such a cost supports the view that gynodioecy can be a stable breeding system in nature.     

A cytonuclear incompatibility causes anther sterility in Mimulus hybrids

Multilocus interactions (also known as Dobzhansky-Muller incompatibilities) are thought to be the major source of hybrid inviability and sterility. Because cytoplasmic and nuclear genomes have conflicting evolutionary interests and are often highly coevolved, cytonuclear incompatibilities may be among the first to develop in incipient species. Here, we report the discovery of cytoplasm-dependent anther sterility in hybrids between closely related Mimulus species, outcrossing M. guttatus and selfing M. nasutus. A novel pollenless anther phenotype was observed in F2 hybrids with the M. guttatus cytoplasm (F2G) but not in the reciprocal F2N hybrids, F1 hybrids or parental genotypes. The pattern of phenotypic segregation in the F2G hybrids and two backcross populations fit a Mendelian single-locus recessive model, allowing us to map the underlying nuclear locus to a small region on LG7 of the Mimulus linkage map. Anther sterility was associated with a 20% reduction in flower size in backcross hybrids and we mapped a major cytoplasm-dependent corolla width QTL with its peak at the anther sterility locus. We argue that the cytonuclear anther sterility seen in hybrids reflects the presence of a cryptic cytoplasmic male sterility (CMS) and restorer system within the hermaphroditic M. guttatus population and therefore name the anther sterility locus restorer-of-male-fertility (RMF). The genetic mapping of RMF is a first step toward testing hypotheses about the molecular basis, individual fitness consequences, and ecological context of CMS and restoration in a system without stable CMS-restorer polymorphism (i.e., gynodioecy). The discovery of cryptic CMS in a hermaphroditic wildflower further suggests that selfish cytoplasmic evolution may play an important, but often undetected, role in shaping patterns of hybrid incompatibility and interspecific introgression in plants.     

Genetic analysis of fertility-restorer genes in rice

Wild-abortive (WA), Honglian (HL) and Baro-II (BT) are three important cytoplasmic male sterility (CMS) types in rice. It is essential to investigate genetic mode and allelism of fertility restorer (Rf) genes and the relationship between Rf and CMS. Fertility of the all test-cross F₁ plants shows that the restorer-maintainer relationship is similar for HL-CMS and BT-CMS, while that is variance for WA-CMS and HL-CMS (or BT-CMS), respectively. Genetic analysis of Rf genes indicates that HL-or BT-CMS are controlled by single dominant Rf gene and WA-CMS is controlled by one or two pairs of dominant Rf genes, which reflects the characters of the gametophytic and sporophytic restoration CMS type. It is concluded that there are at least three Rf loci in different accessions with Rf genes for each CMS type.     

Genetics of Male Sterility in Gynodioecious Plantago Coronopus. II. Nuclear Genetic Variation

Inheritance of male sterility was studied in the gynodioecious species Plantago coronopus using five plants and their descendants from an area of ~50 m(2) from each of four locations. In each location, crosses between these five plants yielded the entire array of possible sex phenotypes. Both nuclear and cytoplasmic genes were involved. Emphasis is placed on the nuclear (restorer) genetics of two cytoplasmic types. For both types, multiple interacting nuclear genes were demonstrated. These genes carried either dominant or recessive restorer alleles. The exact number of genes involved could not be determined, because different genetic models could be proposed for each location and no common genetic solution could be given. At least five genes, three with dominant and two with recessive restorer allele action, were involved with both cytoplasmic types. Segregation patterns of partially male sterile plants suggested that they are due to incomplete dominance at restorer loci. Restorer genes interact in different ways. In most instances models with independent restorer gene action were sufficient to explain the crossing results. However, for one case we propose a model with epistatic restorer gene action. There was a consistent difference in the segregation of male sterility between plants from the two cytoplasmic types. Hermaphrodites of cytoplasmic type 2 hardly segregated male steriles, in contrast to plants with cytoplasmic type 1.     

High-resolution mapping of the Brassica napus Rfp restorer locus using Arabidopsis-derived molecular markers

The two forms of cytoplasmic male sterility (CMS) native to the oilseed rape or canola species Brassica napus, nap and pol, have novel features that may provide insight into the molecular mechanisms through which CMS/nuclear restorer systems evolve. One such feature is the finding that the distinct nuclear restorer genes for the two systems represent different alleles or haplotypes of the same nuclear locus. Improved understanding of how these systems have evolved will require molecular cloning and characterization of this novel locus. We have employed an approach that exploits the regional co-linearity between the Arabidopsis and Brassica genomes to construct a high-resolution genetic map of the nuclear restorer for the pol system, Rfp. Specifically, Arabidopsis-derived sequences have been used as a set of ordered RFLP probes to localize Rfp to a region of the B. napus genome equivalent to a 115 kb interval on Arabidopsis chromosome 1. Based on the known relationship of physical distances between orthologous segments of Arabidopsis and Brassica chromosomes, it is anticipated that the B. napus restorer locus is now mapped to sufficient resolution to permit its isolation and characterization.     

Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.     

普通小麦D2型CMS系的育性基因及其遗传特性

通过研究普通小麦D^2型CMS－育性恢复体系中育性基因的种类及其遗传特性。结果表明：（1）D^2型不育系具有较好的不育性保持与恢复特征,在一般的普通小麦品种（系）中具有广泛的恢复（基因）源、可恢复度高（恢复度超过50％的品种或品质占到33．61％）,也能较容易地转育出新的不育系（完全保持不育性的品种或品系占到25．21％）,这一特征明显优于现有T、K、V型等不育系。（2）D^2型不育系的不育性受核内不育基因和抑制基因控制,相应的核基因型分为Al（不育基因）、A2（不育基因＋抑制基因）两类;恢复纱的恢复性受核内主效恢复基因、微效恢复基因和抑制基因控制,相应的核基因型分为C1（主效恢复基因）、C2(驻效恢复基因＋微效恢复基因）、C3（微效恢复基因）、C4（主效恢复基因＋抑制基因）、C5（主效恢复基因＋微效恢复基因＋抑制基因）、C6（微效恢复基因＋抑制基因）6种。环境条件的变化对育性基因、尤其是微效恢复基因和抑制基因的遗传效应有不同程度的影响。D^2型不育有效杂交组合的模型为：A1＋C1`A1 C2、A2＋C2。（3）D^2型不育系等位恢复基因的遗传表现为不完全显性,非等位恢复基因的遗传表现出积效应,这正是强恢复系德育的理论依据之一。     

A field guide to models of sex-ratio evolution in gynodioecious species

Gynodioecious plant species, species in which individuals are females or hermaphrodites, are ideal systems for studying connections between genetics, ecology, and long‐term evolutionary changes because sex determination can be complex, involving cytoplasmic and/or nuclear genes, and sex ratio is often variable across landscapes. Field data are needed to evaluate the many theories concerning this breeding system. In order to facilitate the gathering of relevant data, this paper introduces the four types of gynodiocy (nuclear, nuclear‐cytoplasmic and stochastic gynodioecy plus subdioecy), describes example species and expected patterns, discusses the various forces that drive the evolution of female frequencies, and gives concrete advice on where to start collecting data for different systems. For species in which females are relatively rare, we recommend reciprocal crosses to determine if sex‐determination is nuclear or nuclear‐cytoplasmic along with a search for correlations between female frequencies and ecological factors. For species in which females are common and sex ratios are highly variable, we recommend looking at female offspring sex ratios to determine if females are primarily produced in ephemeral epidemics. In the course of this discussion, we argue that the majority of natural gynodioecious species will have complex sex determination in which multiple cytoplasmic male sterility (CMS) genes interact with multiple nuclear restorers of fertility. Sex‐ratio evolution in such species will be primarily influenced by fitness differences among hermaphrodites (costs of restoration) and less influenced by fitness differences between the sexes (compensation). Metapopulation dynamics alone may explain population sex ratios of species in which females are associated with marginal environments or hybrid zones; however, we feel that in most cases equilibrium forces within populations and metapopulation dynamics among populations each explain portions of the sex‐ratio pattern.