Genetic analyses supported by molecular methods provide evidence of a new genic (st1) and a new cytoplasmic (st2) male sterility in Allium schoenoprasum L.

Spontaneous mutations leading to male sterility have been described for many different crops and are of great importance to hybrid breeding, provided that their inheritance is resolved. This paper describes an efficient method to characterise male sterilities with respect to cytoplasmic factors that might be causally related to them. The differentiation of cytoplasmic (CMS) and genic (GMS) male sterility is achieved by a specific transfer of nuclear male sterility factors to different cytoplasm types which have previously been distinguished by means of RFLP analyses using mitochondrial gene probes. The nuclear sterility factors of Allium schoenoprasum used, st1 and st2, showed a monogenic recessive inheritance in their original cytoplasms. While st1 was expressed in four different cytoplasm types, st2 did not show itself in a cytoplasm type differing from the original. Therefore, the st1-sterility is a GMS, while a cytoplasmic factor is necessary for the occurrence of st2-sterility. This cytoplasmic factor was verified by a reciprocal cross, and the CMS system was completed by the selection of maintainer genotypes. Neither of these new sterilities were influenced by high temperatures or tetracycline. The benefits of a new CMS system to practical breeding and the advantages and disadvantages of the environmental influences on the expression of male sterility are discussed. Received: 24 November 1999 / Accepted: 3 December 1999     

Cytoplasmic male sterility in plants: molecular evidence and the nucleocytoplasmic conflict

A much-debated issue in plant evolutionary biology concerns the maintenance of a high frequency of male sterility in natural populations. For the past decade, a theoretical framework has been provided by the concept of nucleocytoplasmic conflict. Recent molecular studies on cytoplasmic male sterility indicate that novel chimeric genes, resulting from duplications and rearrangements of mitochondrial DNA sequences, are involved In its control. Thus, male sterility, which is phenotypically the loss of the male function, is encoded by a new mitochondrial function at the molecular level. Molecular data are in agreement with theoretical models that consider cytoplasmic male sterility as a stage in the coevolution between nucleus and mitochondria, and not simply as a deleterious mitochondrial mutation.     

Mitochondrial genome diversity in connection with male sterility in Allium schoenoprasum L.

Mitochondrial genome diversity in chives (Allium schoenoprasum L.) was investigated with respect to different forms of male sterility. Cytoplasmic male-sterile (CMS) and restored genotypes of the known CMS system, compared to plants of the wi-, the st1- and the st2-sterility types and additional fertile plants of different origin were examined by means of RFLP analyses using mitochondrial gene probes. Besides the (S)-cytoplasm of the CMS system four additional cytoplasms were distinguished that differed in the organisation of their mitochondrial genomes. There is consequently a high degree of variability of the mitochondrial genome in chives, especially when compared with the closely related onion. A possible function of the atp9 gene in generating the different cytoplasm types of chives is discussed in relation to the origin of known CMS sequences in other plant species. The existence of different cytoplasm types offers the opportunity for further characterisation of the wi-, st1- and st2-sterility systems with respect to cytoplasmic factors which might be causally related to them. Whether these new sterilities are CMS or GMS (genic male sterilities) is of interest to plant breeders in order that restrictions on the genetic basis used in hybrid seed production be avoided. Received: 6 July 1999 / Accepted: 6 September 1999     

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.     

The nucleo-mitochondrial conflict in cytoplasmic male sterilities revisited

Cytoplasmic male sterility (CMS) in plants is a classical example of genomic conflict, opposing maternally-inherited cytoplasmic genes (mitochondrial genes in most cases), which induce male sterility, and nuclear genes, which restore male fertility. In natural populations, this type of sex control leads to gynodioecy, that is, the co-occurrence of female and hermaphroditic individuals within a population. According to theoretical models, two conditions may maintain male sterility in a natural population: (1) female advantage (female plants are reproductively more successful than hermaphrodites on account of their global seed production); (2) the counter-selection of nuclear fertility restorers when the corresponding male-sterility-inducing cytoplasm is lacking. In this review, we re-examine the model of nuclear-mitochondrial conflict in the light of recent experimental results from naturally occurring CMS, alloplasmic CMS (appearing after interspecific crosses resulting from the association of nuclear and cytoplasmic genomes from different species), and CMS plants obtained in the laboratory and carrying mitochondrial mutations. We raise new hypotheses and discuss experimental models that would take physiological interactions between cytoplasmic and nuclear genomes into account.     

Plant Mitochondrial Genome Evolution and Cytoplasmic Male Sterility

Mitochondria are responsible for providing energy currency to life processes in the molecular form of ATP and are therefore typically referred to as the power factories of cells. Plant mitochondria are also relevant to the common phenomenon of cytoplasmic male sterility, which is agronomically important in various crop species. Cytoplasmic male sterility (CMS) is a complex trait that may be influenced by patterns of mitochondrial genome evolution, and by intergenomic gene transfer among the organellar and nuclear compartments of plant cells. Here, we review patterns and processes that shape plant mitochondrial genomes, some relevant interactions between organelles, and the general features shared by the majority of cytoplasmic male-sterile genes in plants to further the goal of understanding CMS.     

Metabolically engineered male sterility in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Male sterility is of special interest as a mechanism allowing hybrid breeding, especially in important crops such as rapeseed (Brassica napus). Male sterile plants are also suggested to be used as a biological safety method to prevent the spread of transgenes, a risk that is high in the case of rapeseed due to the mode of pollination, out-crossing by wind or insects, and the presence of related, cross-pollinating species in the surrounding ecosystem in Europe. Different natural occurring male sterilities and alloplasmic forms have been tried to be used in rapeseed with more or less success. Due to the difficulties and limitations with these systems, we present a biotechnological alternative: a metabolically engineered male sterility caused by interference with anther-specific cell wall-bound invertase. This is an essential enzyme for carbohydrate supply of the symplastically isolated pollen. The activity of this enzyme is reduced either by antisense interference or by expressing an invertase inhibitor under control of the anther-specific promoter of the invertase with the consequence of a strong decrease of pollen germination ability.     

Mitochondrial DNA polymorphism induced by protoplast fusion in Cruciferae

Summary The mitochondrial genomes of five rapeseed somatic hybrid plants, which combine in a first experimentBrassica napus chloroplasts and a cytoplasmic male sterility trait coming fromRaphanus sativus, and in a second experiment chloroplasts of a triazine resistantB. compestris and a cytoplasmic male sterility trait fromR. sativus, were analyzed by restriction endonucleases. Restriction fragment patterns indicate that these genomes differ from each other and from both parents. The presence of new bands in the somatic hybrid mitochondrial DNA restriction patterns is evidence of mitochondrial recombination in somatic hybrid cells. In both parental and somatic hybrid plants large quantitative variations in a mitochondrial plasmid-like DNA have been observed. Our results suggest that the cytoplasmic support for male sterility is located in the chromosomal mitochondrial DNA instead of the plasmid-like DNA.     

A Case of Gene Fragmentation in Plant Mitochondria Fixed by the Selection of a Compensatory Restorer of Fertility-Like PPR Gene

The high mutational load of mitochondrial genomes combined with their uniparental inheritance and high polyploidy favors the maintenance of deleterious mutations within populations. How cells compose and adapt to the accumulation of disadvantageous mitochondrial alleles remains unclear. Most harmful changes are likely corrected by purifying selection, however, the intimate collaboration between mitochondria- and nuclear-encoded gene products offers theoretical potential for compensatory adaptive changes. In plants, cytoplasmic male sterilities are known examples of nucleo-mitochondrial coadaptation situations in which nuclear-encoded restorer of fertility (Rf) genes evolve to counteract the effect of mitochondria-encoded cytoplasmic male sterility (CMS) genes and restore fertility. Most cloned Rfs belong to a small monophyletic group, comprising 26 pentatricopeptide repeat genes in Arabidopsis, called Rf-like (RFL). In this analysis, we explored the functional diversity of RFL genes in Arabidopsis and found that the RFL8 gene is not related to CMS suppression but essential for plant embryo development. In vitro-rescued rfl8 plantlets are deficient in the production of the mitochondrial heme–lyase complex. A complete ensemble of molecular and genetic analyses allowed us to demonstrate that the RFL8 gene has been selected to permit the translation of the mitochondrial ccmF_N2 gene encoding a heme–lyase complex subunit which derives from the split of the ccmF_N gene, specifically in Brassicaceae plants. This study represents thus a clear case of nuclear compensation to a lineage-specific mitochondrial genomic rearrangement in plants and demonstrates that RFL genes can be selected in response to other mitochondrial deviancies than CMS suppression.     

Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice

Wild abortive cytoplasmic male sterility has been extensively used in hybrid seed production in the tropics. Using protoplast fusion between cytoplasmic male sterile and fertile maintainer lines; we report here, transfer of wild abortive cytoplasmic male sterility to the nuclear background of RCPL1-2C, an advance breeding line which also served as maintainer of this cytoplasm. In total, 27 putative cybrids between V20A and RCPL1-2C and 23 lines between V20A and V20B were recovered and all of them were sterile. DNA blots prepared from the mitochondrial DNA of the cybrid lines from both the sets were probed with orf155 that is known to exhibit polymorphism between the mitochondrial DNA of the male-sterile and fertile maintainer lines. Hybridization of orf155 to 1.3 kb HindIII-digested mitochondrial DNA fragment of the cybrids showed transfer of mitochondrial DNA from wild abortive cytoplasmic male-sterile line to the maintainers, viz. RCPL 1-2C and V20B. Expression of male sterility was confirmed by the presence of sterile pollen grains and the lack of seed setting due to selfing in all the cybrid lines. These cybrids, on crossing with respective fertile maintainers set seeds that in turn, produced sterile BC1 plants. DNA blots from HindIII-digested mitochondrial DNA of these BC1 plants when probed with orf155 again exhibited localization of orf155 in wild abortive cytoplasm-specific 1.3 kb HindIII-digested mitochondrial DNA fragments. This demonstrated that the cytoplasmic male sterility transferred through protoplast fusion retained intact female fertility and was inherited and expressed in BC1 plants. Fusion-derived CMS lines, on pollination with pollen grains from restorer, showed restoration of fertility in all the lines. The results demonstrate that protoplasts fusion can be used for transferring maternally inherited traits like cytoplasmic male sterility to the desired nuclear background which can, in turn, be used in hybrid seed production programme of rice in the tropical world.     

Complex determination of male sterility in Thymus vulgaris L.: genetic and molecular analysis

Summary Nucleocytoplasmic determination of male sterility in Thymus vulgaris L. has been assumed in all papers attempting to explain the remarkably high frequencies of male steriles found in natural populations of this species. This paper provides strong evidence that both nuclear and cytoplasmic genes are involved in the determination of male sterility of this species, giving a complex inheritance. Interpopulation and intrapopulation crosses have shown that the ratio of females versus hermaphrodites among offsprings varied widely from 10 to 11. Furthermore, interpopulation crosses consistently yielded a higher frequency of females than intrapopulation crosses. Nucleocytoplasmic inheritance was demonstrated by an absence of male fertiles in backcrosses and asymmetrical segregation in reciprocal crosses. Molecular analysis of the mitochondrial DNA of some of the parents used in crosses suggested the involvement of different cytoplasms in the inheritance of male steriliy.     

Mitochondrial DNA modifications associated with cytoplasmic male sterility in rice

Summary Mitochondrial DNA was isolated from fertile and cytoplasmic male sterile lines of rice. Restriction analysis showed specific modifications in the male sterile cytoplasm. In addition to the major mitochondrial DNA, three small plasmid-like DNA molecules were detected by agarose gel electrophoresis in both cytoplasms. An additional molecule was specifically found in the sterile cytoplasm. These mitochondrial DNA modifications support the hypothesis of the mitochondrial inheritance of the cytoplasmic male sterility in rice.     

Founder effects and sex ratio in the gynodioecious Thymus vulgaris L.

Thymus vulgaris is a gynodioecious species (in which females and hermaphrodites coexist) with a highly variable frequency of females among natural populations (5–95%) and a high average female frequency (60%). Sex determination involves both cytoplasmic genes responsible for male sterility, i.e. the female phenotype, and specific nuclear factors responsible for the restoration of male fertility, and thus a hermaphrodite phenotype. In this study, molecular markers of the mitochondrial genome have been used to quantify the cytoplasmic diversity in 11 clumps of individuals observed in four recently founded populations. The very low diversity within patches in conjunction with the strong diversity among patches strongly suggests that clumps of individuals are the result of single matrilinear families. In clumps that contain mainly females, all the analysed females showed the same cytoplasmic pattern. This pattern differed from that shown by neighbouring hermaphrodites, indicating that the determination of sex is locally cytoplasmic. A comparison of genetic diversity before and after fire in one population showed that disturbances may cause a reduction in genetic diversity and a concurrent induction of local cytoplasmic determination of sex. Such cytoplasmic determination of sex in colonizing populations, together with the greater seed set of females, may largely improve the colonizing ability of the species.     

水稻广亲和性遗传的再研究

水稻广亲和基因的利用是克服亚种间杂种不育性的重要途径。但在广亲和性的遗传上不同研究者的结论不尽一致。以3种有广亲和品种参加的三交组合为研究材料,研究了品种Ketan Nangka的广亲和性遗传。结果表明水稻亚种杂交F1同时存在着雄性不育和雌性不育,但雄性不育对小穗育性的作用大小因组合而异;无论是在雄性不育位点还是雄性不育位点上,Ketan Nangka均具有相对应的中性基因（广亲和基因）;广亲和性的遗传特点与所用的籼粳测验品种间的杂种不育性密切相关;S－5位点的广亲和基因遗传符合单位点孢子体－配子体互作模型。     

Mitochondrially-targeted expression of a cytoplasmic male sterility-associated <Emphasis Type="Italic">orf220</Emphasis> gene causes male sterility in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Background  

The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility.     

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.