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.     

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.     

The male-sterility polymorphism of Silene vulgaris: analysis of genetic dat from two populations and comparison with Thymus vulgaris.

Results are given of genetic studies of male sterility using plants from two natural populations from Sussex, England. Both populations have substantial frequencies of females, approximately 0.25 in population 1 and 0.60 in population 3. As in the few other gynodioecious populations studied in detail, many genetic factors are present. In population 1, there are at least two, and more likely three, different cytoplasmic types, one of which appears to produce male sterility in progeny from any hermaphrodite pollen donor; in other words restorer alleles for this cytoplasm are rare or absent from the population. The other two populations can be carried in hermaphrodites that have the dominant restorers. In population 1, there are also probably three restorer loci with complementary recessive male-sterility alleles, as well as a locus with duplicate action, which cannot produce male sterility unless the plant is also homozygous for the recessive allele at another locus. The results from population 3 are quite similar, though there was no evidence in this population for an unrestored sterility cytoplasm. A similar joint nucleocytoplasmic model with multiple restorers fits data from Thymus vulgaris.     

Nuclear-cytoplasmic male-sterility in diploid dandelions

Male-sterility was found in diploid dandelions from two widely separated populations from France, and its inheritance was analysed by crossing a diploid male-sterile dandelion to diploid sexuals and triploid apomicts. Nuclear genetic variation, found in full-sib families, segregated for male-fertility, partial male-sterility, and full male-sterility, and also segregated for small-sized versus normally sized pollen. The crossing results are best explained by a cytoplasmic male-sterility factor in combination with two dominant restorer genes. Involvement of the cytoplasmic male-sterility factor was further investigated by chloroplast haplotyping. Male-sterility was exclusively associated with a rare chloroplast haplotype (designated 16b). This haplotype was found in seven male-sterile plants and one (apparently restored) male-fertile individual but does not occur in 110 co-existing male-fertile plants and not in several hundreds of individuals previously haplotyped. Apomicts with cytoplasmic male sterility were generated in some test crosses. This raises the question as to whether the male sterility found in natural dandelion apomicts, is of cytoplasmic or of nuclear genetic nature. As many breeding systems in Taraxacum are involved in shaping population structure, it will be difficult to predict the evolutionary consequences of nuclear-cytoplasmic male-sterility for this species complex.     

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.     

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.     

Cytoplasmic male sterility in Mimulus hybrids has pleiotropic effects on corolla and pistil traits

The mechanisms underlying genetic associations have important consequences for evolutionary outcomes, but distinguishing linkage from pleiotropy is often difficult. Here, we use a fine mapping approach to determine the genetic basis of association between cytonuclear male sterility and other floral traits in Mimulus hybrids. Previous work has shown that male sterility in hybrids between Mimulus guttatus and Mimulus nasutus is due to interactions between a mitochondrial gene from M. guttatus and two tightly linked nuclear restorer alleles on Linkage Group 7, and that male sterility is associated with reduced corolla size. In the present study, we generated a set of nearly isogenic lines segregating for the restorer region and male sterility, but with unique flanking introgressions. Male-sterile flowers had significantly smaller corollas, longer styles and greater stigmatic exsertion than fertile flowers. Because these effects were significant regardless of the genotypic composition of introgressions flanking the restorer region, they suggest that these floral differences are a direct byproduct of the genetic incompatibility causing anther abortion. In addition, we found a non-significant but intriguing trend for male-sterile plants to produce more seeds per flower than fertile siblings after supplemental pollination. Such pleiotropic effects may underlie the corolla dimorphism frequently observed in gynodioecious taxa and may affect selection on cytoplasmic male sterility genes when they initially arise.     

Allelic relationship of four male sterility genes and nucleo-cytoplasmic interactions in the expression of male sterility in pearl millet,Pennisetum americanum (L.) leeke

Summary Male sterility genes isolated in four inbred lines of pearl millet were found allelic. The differences between male fertile and male sterile phenotypes is mainly due to a single gene. Presence of a dominant gene (Ms) resulted in male fertility and double recessiveness (ms ms) in male sterility. However, genic male sterility (GMS) in Pennisetum is not a simply inherited case of monogenic recessive condition but is influenced by cytoplasmic and several nuclear factors. In a male sterile, the stage at which the male sterility gene is expressed during the development of the male gametophyte resulting in breakdown of the cells is influenced by cytoplasmic and other nuclear factors. Two types of cytoplasm, C-1 and C-2, are recognized. Presence of any two recessive male sterility alleles in C-1 led to breakdown of male development before differentiation of an archesporium in the anther (Arc-type); in C-2 cytoplasm, degeneration started during meiosis with fusion of meiocytes and syncyte formation (Syn-type), or at post-meiotic stages terminating in abortion of microspores before first pollen mitosis (PGM type). The triggering of activity of recessive male sterility genes in C-2 cytoplasm appeared to be regulated by two nuclear factors, R ₁ and R ₂ with duplicate gene action. Recessiveness for both the R factors in C-2 cytoplasm resulted in PGM-type expression. The action of R ₁ and R ₂ is specific to C-2 cytoplasm. Mutation of cytoplasm from C-1 to C-2 and C-2 to C-1 was observed.     

Genetics of Male Sterility in Gynodioecious Plantago Coronopus. I. Cytoplasmic 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) in each of six locations. The crosses were planned to test for cytoplasmic inheritance of male sterility. In four locations significant differences between reciprocal crosses were observed. The progenies of these reciprocal crosses were used in a crossing scheme designed to test whether these reciprocal differences were caused by different cytoplasmic types between the plants. In all four locations, the existence of at least two cytoplasmic types could be shown. Moreover, the results of the crosses between locations showed that the same two cytoplasmic types were present in all four locations. We therefore argue that there is only limited cytoplasmic variation in P. coronopus. In each cytoplasmic type a series of intermediate sex forms occurred. A marked difference in restoration level existed between the two cytoplasmic types. Plants with cytoplasmic type 2 hardly segregated male steriles, in contrast to plants with cytoplasmic type 1.     

THE EFFECT OF POLLEN VERSUS SEED FLOW ON THE MAINTENANCE OF NUCLEAR‐CYTOPLASMIC GYNODIOECY

Gynodioecy, where females co‐occur with hermaphrodites, is a relatively common sexual system in plants that is often the result of a genetic conflict between maternally inherited male sterility genes in the mitochondrial genome and the biparentally inherited male fertility restorer genes in the nucleus. Previous models have shown that nuclear‐cytoplasmic gynodioecy can be maintained under certain conditions by negative frequency‐dependent selection, but the effect of other evolutionary processes such as genetic drift and population subdivision is only partially understood. Here, we investigate the joint effects of frequency‐dependent selection, drift, and migration through either pollen or seeds on the maintenance of nuclear‐cytoplasmic gynodioecy in a subdivided population. We find that the combination of drift and selection causes the loss of gynodioecy under scenarios that would maintain it under the influence of selection alone, and that both seed and, more surprisingly, pollen flow can maintain the polymorphism. In particular, although pollen flow could not avoid the loss of cytoplasmic polymorphism within demes, it allowed the maintenance of nuclear‐cytoplasmic polymorphism at the metapopulation level.     

Mapping of genes for male-fertility restoration in ’Pampa’ CMS winter rye (Secale cereale L.)

Hybrid rye breeding and seed production is based on the cytoplasmic male sterility (CMS)-inducing Pampa (P)-cytoplasm. For restoring male fertility in the hybrids, dominant, nuclear restorer genes are necessary. However, current pollinator lines are only partial restorers. Effective restorers were recently detected in the German inbred line L18 and in materials originating from the Argentinian rye cultivar Pico Gentario and an Iranian primitive rye accession called IRAN IX. F₂ populations were developed for each of these three restorer sources to map the responsible genes by means of RFLP (restriction fragment length polymorphism) markers. For this purpose, homo- and heterologous DNA probes were used leading to 101 polymorphic marker loci in total. For phenotypic evaluation, 100 to 134 randomly chosen plants from each of the populations were cloned and grown at two or three locations with two plants each. Segregation ratios of pollen fertility in the F₂ populations with L18 and IRAN IX were in accordance with a monogenic dominant inheritance. The segregation pattern for Pico Gentario indicated complementary gene action. Major dominant restorer genes were detected on chromosomes 1RS (L18) and 4RL (Pico Gentario, IRAN IX). The gene on 1RS explained 54% of the phenotypic variation and that on 4 RL 59% and 68% in the Pico Gentario and IRAN IX populations, respectively. Additionally, three minor genes from L18 were identified on chromosomes 3RL, 4RL and 5R. In the Pico Gentario population, a dominant modifier gene contributed by the female parent was found on chromosome 6R. This gene significantly enhanced the expression of the major restorer gene but on its own was not able to restore any degree of fertility. The map-distances between the major restorer loci and at least one flanking marker were small in all three F₂ populations (5–6 cM). In Pico Gentario an unfavorable linkage exists between the major restorer gene and a QTL for plant height. Since highly effective restorers are scarce in actual breeding populations, the major restorer genes detected on chromosomes 1 RS and 4RL should be introgressed into actual restorer lines. This is facilitated by using the closely linked molecular markers described. Received: 10 February 2000 / Accepted: 31 March 2000<@head-com-p1a.lf>Communicated by G. Wenzel     

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。     

Unveiling the molecular arms race between two conflicting genomes in cytoplasmic male sterility?

Cytoplasmic male sterility can be thought of as the product of a genetic conflict between two genomes that have different modes of inheritance. Male sterilizing factors, generally encoded by chimeric mitochondrial genes, can be down-regulated by specific nuclear restorer genes. The recent cloning of a restorer gene in rice and its comparison with restorer genes cloned in petunia and radish could be regarded as the beginning of a general molecular scenario in this peculiar arms race.     

Identification of a mitochondrial protein associated with cytoplasmic male sterility in petunia.

The petunia fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxII and urfS portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXII but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines. The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility.     

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.     

Mitochondrial genome organization and cytoplasmic male sterility in plants

Plant mitochondrial genomes are much larger and more complex than those of other eukaryotic organisms. They contain a very active recombination system and have a multipartite genome organization with a master circle resolving into two or more subgenomic circles by recombination through repeated sequences. Their protein coding capacity is very low and is comparable to that of animal and fungal systems. Several subunits of mitochondrial functional complexes, a complete set of tRNAs and 26S, 18S and 5S rRNAs are coded by the plant mitochondrial genome. The protein coding genes contain group II introns. The organelle genome contains stretches of DNA sequences homologous to chloroplast DNA. It also contains actively transcribed DNA sequences having open reading frames. Plasmid like DNA molecules are found in mitochondria of some plants Cytoplasmic male sterility in plants, characterized by failure to produce functional pollen grains, is a maternally inherited trait. This phenomenon has been found in many species of plants and is conveniently used for hybrid plant production. The genetic determinants for cytoplasmic male sterility reside in the mitochondrial genome. Some species of plants exhibit more than one type of cytoplasmic male sterility. Several nuclear genes are known to control expression of cytoplasmic male sterility. Different cytoplasmic male sterility types are distinguished by their specific nuclear genes(rfs) which restore pollen fertility. Cytoplasmic male sterility types are also characterized by mitochondrial DNA restriction fragment length polymorphism patterns, variations in mitochondrial RNAs, differences in protein synthetic profiles, differences in sensitivity to fungal toxins and insecticides, presence of plasmid DNAs or RNAs and also presence of certain unique sequences in the genome. Recently nuclear male sterility systems based on (i) over expression of agrobacterialrol C gene and (ii) anther specific expression of an RNase gene have been developed in tobacco andBrassica by genetic engineering methods.     

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.     

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.     

Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryza sativa L.)

A rice nuclear gene, Rf-1, restores the pollen fertility disturbed by the BT-type male sterile cytoplasm, and is widely used for commercial seed production of japonica hybrid varieties. Genomic fragments carrying Rf-1 were identified by conducting chromosome walking and a series of complementation tests. Isolation and analysis of cDNA clones corresponding to the fragments demonstrated that Rf-1 encodes a mitochondrially targeted protein containing 16 repeats of the 35-aa pentatricopeptide repeat (PPR) motif. Sequence analysis revealed that the recessive allele, rf-1, lacks one nucleotide in the putative coding region, presumably resulting in encoding a truncated protein because of a frame shift. Rice Rf-1 is the first restorer gene isolated from cereal crops that has the property of reducing the expression of the cytoplasmic male sterility (CMS)-associated mitochondrial gene like many other restorer genes. The present findings may facilitate not only elucidating the mechanisms of male sterility by the BT cytoplasm and its restoration by Rf-1 but also isolating other restorer genes from cereal crops, especially rice.