Multiple CMS-restorer gene polymorphism in gynodioecious Plantago coronopus

The mode of inheritance of the male sterility trait is crucial for understanding the evolutionary dynamics of the sexual system gynodioecy, which is the co-occurrence of female and hermaphrodite plants in natural populations. Both cytoplasmic (CMS) and nuclear (restorer) genes are known to be involved. Theoretical models usually assume a limited number of CMS genes with each a single restorer gene, while reality is more complex. In this study, it is shown that in the gynodioecious species Plantago coronopus two new CMS-restorer polymorphisms exist in addition to the two that were already known, which means four CMS-restorer systems at the species level. Furthermore, three CMS types were shown to co-occur within a single population. All new CMS types showed a multilocus system for male fertility restoration, in which both recessive and dominant restorer alleles occur. Our finding of more than two co-occurring CMS-restorer systems each with multiple restorer genes raises the question how this complex of male sterility systems is maintained in natural populations.     

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.     

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.     

Relative fitness of females and hermaphrodites in a natural gynodioecious population of wild radish,Raphanus sativus L. (Brassicaceae): comparison based on molecular genotyping

In many gynodioecious species, sex determination involves both cytoplasmic male‐sterility (CMS) genes and nuclear genes that restore male function. Differences in fitness among genotypes affect the dynamics of those genes, and thus that of gynodioecy. We used a molecular marker to discriminate between hermaphrodites with and without a CMS gene in gynodioecious Raphanus sativus. We compared fitness through female function among the three genotypes: females, hermaphrodites with the CMS gene and those without it. Although there was no significant difference among the genotypes in seed size, hermaphrodites without the CMS gene produced significantly more seeds, and seeds with a higher germination rate than the other genotypes, suggesting no fitness advantage for females and no benefit to bearing the CMS gene. Despite the lack of fitness advantage for females in the parameter values we estimated, a theoretical model of gynodioecy shows it can be maintained if restorer genes impose a cost paid in pollen production. In addition, we found that females invest more resources into female reproduction than hermaphrodites when they become larger. If environmental conditions enable females to grow larger this would facilitate the dynamics of CMS genes.     

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.     

植物CMS/Rf系统的基因克隆及其结构特征

细胞质雄性不育和恢复系统（CMS/Rf）在植物杂种优势利用中已被广泛应用。为阐明恢复基因在这一系统中的作用机理,众多研究者开展了恢复基因的定位和克隆研究。近年来,4个植物恢复基因的成功克隆有力地推动了这一研究领域的发展。本文综述了植物恢复基因的定位、克隆以及育性恢复分子机理的研究进展,并讨论了恢复基因在植物分子育种上的应用。     

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.     

Offspring sex ratio under inbreeding and outbreeding in a gynodioecious plant

Silene vulgaris is a gynodioecious plant native to Eurasia and now found throughout much of North America. Using hermaphrodite plants from three geographic regions (Stamford, NY; Broadway,VA; and Giles Co., VA) and four local populations within each region, we employed a hierarchical crossing design to explore the geographic structure of sex determining genes. Sex determination in this species is cytonuclear involving multiple cytoplasmic male sterility and nuclear restorer loci. Due to dominance effects within nuclear restorer loci, self-fertilization of hermaphrodites heterozygous at restorer loci should produce some homozygous recessive female offspring. Female offspring may also result from outcrossing among related individuals. At greater geographic and genetic distances, mismatches between cytoplasmic and nuclear sex determining genes should also produce high frequencies of female offspring if coevolution between cytoplasmic and nuclear sex determining alleles occurs independently among widely separated populations. We found evidence of dominance effects among nuclear restorer loci but no evidence of nuclear-cytoplasmic mismatches at the regional level. Of 63 maternal lines, 55 produced at least one female offspring when self-fertilized. Outcrossing within populations produced significantly fewer female offspring than self-fertilization. Outcrossing among regions produced the lowest proportion of female offspring, significantly fewer than outcrossing among populations within regions. Regions responded differently to among-region outcrossing with pollen donors from the two Virginia regions producing far fewer female offspring with New York dams than crosses among New York populations. These results indicate that nuclear restoration is complex, involving multiple loci with epistatic interactions and that most hermaphrodites in nature are heterozygous at one or more restorer locus. Further, regional differences in restorer frequencies indicate significant genetic structure for sex determining genes at large geographic scales, perhaps reflecting invasion history.     

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。     

植物细胞质雄性不育及其育性恢复的分子生物学研究进展

植物细胞质雄性不育（CMS）和恢复系统在作物杂交种子生产中具有重要的意义。综述了目前已发现的与植物CMS相关的线粒体DNA位点,育性恢复基因对CMS相关DNA位点表达的影响,育性恢复基因的分子标记定位、克隆,及育性恢复分子机理等方面的研究进展,并讨论了恢复基因在植物分子育种上的应用。     

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.     

Consequences of inbreeding for offspring fitness and gender in Silene vulgaris,a gynodioecious plant

Abstract In gynodioecious plants, hermaphrodite and female plants co‐occur in the same population. In these systems gender typically depends on whether a maternally inherited cytoplasmic male sterility factor (CMS) is counteracted by nuclear restorer alleles. These restorer alleles are often genetically dominant. Although plants of the female morph are obligatorily outcrossing, hermaphrodites may self. This selfing increases homozygosity and may thus have two effects: (1) it may decrease fitness (i.e. result in inbreeding depression) and (ii) it may increase homozygosity of the nuclear restorer alleles and therefore increase the production of females. This, in turn, enhances outcrossing in the following generation. In order to test the latter hypothesis, experimental crosses were conducted using individuals derived from four natural populations of Silene vulgaris, a gynodioecious plant. Treatments included self‐fertilization of hermaphrodites, outcrossing of hermaphrodites and females using pollen derived from the same source population as the pollen recipients, and outcrossing hermaphrodites and females using pollen derived from different source populations. Offspring were scored for seed germination, survivorship to flowering and gender. The products of self‐fertilization had reduced survivorship at both life stages when compared with the offspring of outcrossed hermaphrodites or females. In one population the fitness of offspring produced by within‐population outcrossing of females was significantly less than the fitness of offspring produced by crossing females with hermaphrodites from other populations. Self‐fertilization of hermaphrodites produced a smaller proportion of hermaphroditic offspring than did outcrossing hermaphrodites. Outcrossing females within populations produced a smaller proportion of hermaphrodite offspring than did crossing females with hermaphrodites from other populations. These results are consistent with a cytonuclear system of sex determination with dominant nuclear restorers, and are discussed with regard to how the mating system and the genetics of sex determination interact to influence the evolution of inbreeding depression.     

Genetic determination of male sterility in gynodioecious Silene nutans

Gynodioecy, the coexistence of female and hermaphrodite plants within a species, is often under nuclear–cytoplasmic sex determination, involving cytoplasmic male sterility (CMS) genes and nuclear restorers. A good knowledge of CMS and restorer polymorphism is essential for understanding the evolution and maintenance of gynodioecy, but reciprocal crossing studies remain scarce. Although mitochondrial diversity has been studied in a few gynodioecious species, the relationship between mitotype diversity and CMS status is poorly known. From a French sample of Silene nutans, a gynodioecious species whose sex determination remains unknown, we chose the four most divergent mitotypes that we had sampled at the cytochrome b gene and tested by reciprocal crosses whether they carry distinct CMS genes. We show that gynodioecy in S. nutans is under nuclear–cytoplasmic control, with at least two different CMSs and up to four restorers with epistatic interactions. Female occurrence and frequency were highly dependent on the mitotype, suggesting that the level of restoration varies greatly among CMSs. Two of the mitotypes, which have broad geographic distributions, represent different CMSs and are very unequally restored. We discuss the dynamics of gynodioecy at the large-scale meta-population level.     

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.     

Sex-biased seed predation and the maintenance of females in a gynodioecious plant

We investigated genetic and ecological factors contributing to the maintenance of females in populations of the gynodioecious plant, Sidalcea hendersonii (Malvaceae). Our crossing experiments indicated that male sterility is controlled by a dominant nuclear allele. Nuclear determination of sex theoretically requires much higher female fitness to account for the high frequency of female plants often observed during surveys of S. hendersonii populations. Females were, in fact, shown to have higher fitness in an experimental population, producing more viable seed and surviving offspring than hermaphrodite plants. In natural populations, however, differences in viable seed production were not evident unless seed predation was considered. Sex-biased seed predation appears to play an important role in the maintenance of gynodioecy in S. hendersonii. In populations with high female frequencies, weevil larvae destroyed significantly more seeds from hermaphrodite plants than females, substantially reducing their seed production. To our knowledge, this study provides the first evidence that sex-biased predation may be responsible for high female frequencies in natural populations of a gynodioecious species.     

几种农作物细胞质雄性不育恢复基因的定位和分子标记研究进展

利用杂种优势提高作物产量时, 生产杂交种的主要授粉控制系统是细胞质雄性不育及其恢复系统。在杂交品种的选育过程中, 优良恢复系选育至关重要。为了高效并准确地鉴定选择恢复材料, 同时更深入地研究恢复基因的作用机理, 近年来植物细胞质雄性不育恢复基因分子标记研究受到了广泛重视。本文综述了主要农作物水稻、油菜、小麦、棉花和玉米等细胞质雄性不育类型恢复基因的定位和分子标记研究进展, 并讨论了恢复基因的精确定位和分子标记鉴定在基因克隆和分子标记辅助选择育种中的意义和应用前景。     

Mitochondrial DNA diversity, population structure, and gender association in the gynodioecious plant Silene vulgaris

A highly variable mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) locus is used to assess the population structure of mitochondrial genomes in the gynodioecious plant Silene vulgaris at two spatial scales. Thirteen mtDNA haplotypes were identified within 250 individuals from 18 populations in a 20-km diameter region of western Virginia. The population structure of these mtDNA haplotypes was estimated as thetaST = 0.574 (+/- 0.066 SE) and, surprisingly, genetic differentiation among populations was negatively correlated with geographic distance (Mantel r = -0.246, P < 0.002). Additionally, mtDNA haplotypes were spatially clumped at the scale of meters within one population. Gender in S. vulgaris is determined by an interaction between autosomal male fertility restorers and cytoplasmic male sterility (CMS) factors, and seed fitness is affected by an interaction between gender and population sex ratio; thus, selection acting on gender could influence the distribution of mtDNA RFLP haplotypes. The sex ratio (females:hermaphrodites) varied among mtDNA haplotypes across the entire metapopulation, possibly because the haplotypes were in linkage disequilibrium with different CMS factors. The gender associated with some of the most common haplotypes varied among populations, suggesting that there is also population structure in male fertility restorer genes. In comparison with reports of mtDNA variation from other published studies, we found that S. vulgaris exhibits a large number of mtDNA haplotypes relative to that observed in other species.