Nuclear Restorer Genes Research Articles

Development of PCR-based markers linked to a restorer gene for cytoplasmic male sterility in radish (Raphanus sativus L.)

A random amplified polymorphic DNA (RAPD) marker named OPC06-1900 was previously found linked to a fertility restorer gene (Rfw) for cytoplasmic male sterility (CMS) in radish (Raphanus sativus L.). The RAPD marker was converted to a dominant sequence characterized amplified region (SCAR) marker SCC06-1894 by molecular cloning and nucleotide sequencing. A BLAST search revealed that the SCAR marker SCC06-1894 showed significant homology to the corresponding regions of Arabidopsis and Brassica sulfate transporter genes. The presence of the intron and exon of the DNA fragment SCC06-1894 was demonstrated by comparing RT-PCR and PCR products. Thus, allele-specific oligonucleotide primers were designed to amplify the SCAR marker SCC06-415. PCR test with F2 plants and sequence analysis showed that SCC06-1894 and SCC06-415 were allelic, linked to Rfw/rfw gene at 8.0 cM. Nine oligonucleotide primers were designed based on a single radish nuclear restorer gene mRNA. A survey of these primer combinations by bulked segregant analysis (BSA) identified three polymorphisms. The three PCR-based markers were co-segregant in the coupling phase and distant from the Rfw gene by 1.4 cM. These specific markers distributed on both sides of the Rfw gene and will be helpful for breeding new rapseed (Brassica napus L.) restorer lines.

Spatial Distributions of Cytoplasmic Types and Sex Expression in Alaskan Populations ofSilene acaulis

Sex expression in gynodioecious plants is often governed by an interaction between maternally inherited cytoplasmic male sterility (CMS) genes and nuclear male fertility restorer genes. Previous studies have documented an association between females and maternally inherited molecular markers in the cytoplasmic genomes in natural populations, suggesting that these markers are associated with different CMS types. We analyzed the spatial distributions of mitochondrial and chloroplast alleles and the sexual phenotype of individuals within five Alaskan populations of Silene acaulis. Within two populations, mitochondrial alleles, but not sex, were clustered at a scale ≤2 m. We suggest that this pattern is generated by short‐distance dispersal of mitochondrial alleles (and CMS) through seed and longer‐distance dispersal of nuclear restorers through both pollen and seed. We also investigated whether the association between mitochondrial alleles and sex ratio changed through time (across size classes), as might be...

Consequences of prairie fragmentation on the progeny sex ratio of a gynodioecious species, Lobelia spicata (Campanulaceae)

Habitat fragmentation of prairie ecosystems has resulted in increased isolation and decreased size of plant populations. In large populations, frequency-dependent selection is expected to maintain genetic diversity of sex determining factors associated with gynodioecy, that is, nuclear restorer genes that reverse cytoplasmic male sterility (nucleocytoplasmic gynodioecy). However, genetic drift will have a greater influence on small isolated populations that result from habitat fragmentation. The genetic model for nucleocytoplasmic gynodioecy implies that the proportion of female progeny produced by hermaphroditic and female plants will show more extreme differences in populations with reduced allelic diversity, and that restoration of male function will increase with inbreeding. We investigated potential impacts of effects resulting from reduced population sizes by comparison of progeny sex ratios produced by female and hermaphroditic plants in small and large populations of the gynodioecious prairie species, Lobelia spicata. A four-way contingency analysis of the impact of population size, population sex ratio, and maternal gender on progeny sex ratios showed that progeny sex ratios of hermaphroditic plants were strongly influenced by population size, whereas progeny sex ratios of female plants were strongly influenced by population sex ratio. Further, analysis of variation in progeny-type distribution indicated decreased restoration and increased loss of male function in smaller and isolated populations. These results are consistent with reduced allelic diversity or low allelic frequency at restorer loci in small and isolated populations. The consequent decrease in male function has the potential to impede seed production in these fragmented prairies.

Cell‐specific regulation of a Brassica napus CMS‐associated gene by a nuclear restorer with related effects on a floral homeotic gene promoter

Cytoplasmic male sterility (CMS) is a maternally inherited defect in pollen production specified by novel mitochondrial genes. It can be suppressed by nuclear restorer (Rf) genes which normally downregulate expression of a CMS-associated novel mitochondrial gene. Two forms of Brassica napus CMS, nap and pol, are associated with related chimeric genes, orf222 and orf224, respectively. We show that in pol and nap CMS, anther locule development is asynchronous and asymmetric, that one or more locules within each anther may fail to develop entirely and that CMS anthers display polarity in locule development. We show, by in situ hybridization, that orf222 transcripts accumulate in sterile anthers prior to development of morphological differences between CMS and restored stamens, and remain preferentially localized to microsporangia. In fertility-restored anthers, however, orf222 transcript levels remain low throughout development. Some sporogenous and meiotic cells differentiate within CMS anthers and form functional pollen despite retaining high orf222 transcript levels, suggesting that the effect of orf222 expression in blocking pollen development is limited to an early and specific stage. Transcripts of other mitochondrial genes, exemplified by atp6 and cob, and of the nuclear-encoded ATP synthase gamma subunit, accumulate preferentially in the microsporangia of both sterile and fertile anthers. Thus nuclear fertility restoration reduces orf222 transcript levels in a gene and tissue-specific manner. We observe differences between CMS and fertile plants in the timing and patterning of APETALA3 promoter activity that suggest a possible basis for the developmental abnormalities of CMS flowers.

A simple method to control the seed purity of japonica hybrid rice varieties using PCR‐based markers

AbstractCytoplasmic male sterility (CMS) by the cms‐bo cytoplasm and its restoration by the nuclear restorer gene, Rf‐1, are used for seed production of japonica hybrid rice varieties. To produce pure hybrid seeds, a prerequisite is to properly manage the seed purity of parental lines, especially CMS lines. In this study, three dominant polymerase chain reaction (PCR)‐based markers (M1, M2 and M3) were developed to detect mutual contamination in seed batches of CMS lines, maintainer lines, restorer lines and hybrids. M1 detected the mitochondrial sequence that was present in the cytoplasm of common japonica varieties and absent in the cms‐bo cytoplasm. M2 and M3 detected the chromosomal sequence related to the Rf‐1 allele in restorer lines and the rf‐1 allele in common japonica varieties, respectively. By the strategic use of these markers, japonica hybrids and their parental lines could be efficiently distinguished from each other. Furthermore, sensitivity tests for the three markers with a series of crude DNA samples prepared from polished grains demonstrated that these markers could detect one contaminating grain among 500 or 1000 grains. Therefore, the bulk PCR analyses with the markers developed here probably make it possible to control the seed purity of japonica hybrids properly by selecting appropriate seed batches of their parental lines quickly and efficiently.

Preferential expression of a HLP homolog encoding a mitochondrial L14 ribosomal protein in stamens of common wheat

Interaction between nucleus and cytoplasm has essential roles in plant development, including that of floral organs. We isolated a wheat homolog Whlp of Arabidopsis HUELLENLOS PARALOG ( HLP) gene encoding a mitochondrial (mt) ribosomal protein L14. Transient expression analysis using the green fluorescent protein (GFP) fusion protein showed that 50 amino residues located on the N-terminal of the wheat HLP homolog (WHLP) protein acted as a mt-targeting signal (MTS). Expression patterns of the Whlp gene were compared among floral organs of alloplasmic lines of wheat, in which intrinsic cytoplasms were replaced by the cytoplasm of a wild relative Aegilops crassa. In these alloplasmic lines, pistillody (homeotic transformation of stamens into pistil-like organs) is induced by the alien cytoplasm in the absence of nuclear restorer genes. The Whlp transcripts preferentially accumulated in stamens compared with pistils, leaves, and roots. The expression level of Whlp in the pistillate stamens of the alloplasmic lines was similar to that in genuine pistils of both euplasmic lines and fertile alloplasmic lines. The result suggested that the elevated expression of the Whlp gene plays a role in aiding the development of male reproductive organ but not in the determination of its whorl identity. A comparable expression pattern was observed in another nuclear-encoded mt ribosomal protein gene but not in a mt-encoded gene. The different expression patterns of different mt ribosomal protein genes suggest that the abundance of mt ribosomal proteins is differentially regulated in the organ/tissue development in wheat.

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.

Multiple genetic contributions to plant performance in Thymus vulgaris

Summary 1 Diverse genetic factors can affect plant performance. Here we evaluate how maternal and paternal origin, maternal and offspring sexual phenotype and degree of outcrossing jointly influence performance of gynodioecious Thymus vulgaris . 2 In uniform conditions we quantified size and survival of second-generation (G 2 ) offspring produced by selfing and outcrossing on the open-pollinated offspring (G 1 ) of G 0 females from four populations that differ in sex ratio, selfing rate and chemical type. We controlled for environmental variation, maternal effects and plant age. 3 Offspring with maternal parents from populations dominated by plants that synthesize phenolic monoterpenes had greater survival and size than those from populations with non-phenolic chemotypes. In addition, outcrossing among populations with pollen donors from non-phenolic populations significantly reduced progeny performance. These results suggest chemotype-mediated local adaptation. 4 The progeny of hermaphrodites showed significant inbreeding depression, which despite the pollination treatment × family and pollination treatment × population interactions, showed no consistent variation in relation to population inbreeding levels. 5 For a given level of outcrossing, the progeny of hermaphrodites showed greater among-family and among-population variation in plant size than those of females. Variation in nuclear restorer gene composition across cytoplasmic backgrounds may cause this variation. 6 In T. vulgaris , diverse genetic factors jointly affect performance, a result with general implications for understanding fitness variation in natural populations.

Development of PCR-based markers linked to dominant genes for male-fertility restoration in Pampa CMS of rye (Secale cereale 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.

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

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.

Characterization of the aldehyde dehydrogenase gene families of Zea mays and Arabidopsis.

Cytoplasmic male sterility is a maternally transmitted inability to produce viable pollen. Male sterility occurs in Texas (T) cytoplasm maize as a consequence of the premature degeneration of the tapetal cell layer during microspore development. This sterility can be overcome by the combined action of two nuclear restorer genes, rf1 and rf2a. The rf2a gene encodes a mitochondrial aldehyde dehydrogenase (ALDH) that is capable of oxidizing a variety of aldehydes. Six additional ALDH genes were cloned from maize and Arabidopsis. In vivo complementation assays and in vitro enzyme analyses demonstrated that all six genes encode functional ALDHs. Some of these ALDHs are predicted to accumulate in the mitochondria, others in the cytosol. The intron/exon boundaries of these genes are highly conserved across maize and Arabidopsis and between mitochondrial and cytosolic ALDHs. Although animal, fungal, and plant genomes each encode both mitochondrial and cytosolic ALDHs, it appears that either the gene duplications that generated the mitochondrial and the cytosolic ALDHs occurred independently within each lineage or that homogenizing gene conversion-like events have occurred independently within each lineage. All studied plant genomes contain two confirmed or predicted mitochondrial ALDHs. It appears that these mitochondrial ALDH genes arose via independent duplications after the divergence of monocots and dicots or that independent gene conversion-like events have homogenized the mitochondrial ALDH genes in the monocot and dicot lineages. A computation approach was used to identify amino acid residues likely to be responsible for functional differences between mitochondrial and cytosolic ALDHs.

Genetics of sex determination in the gynodioecious species Lobelia siphilitica: evidence from two populations.

In order to determine whether interactions between multiple sex-determining genes might be partly responsible for the wide variation in female frequency among populations of Lobelia siphilitica, we used progeny sex ratios from field-collected plants and from controlled crosses within and between two populations. We demonstrate that multiple cytoplasmic male-sterility types are present in a gynodioecious population where female frequency exceeds 50%. These male-sterility types each have corresponding nuclear alleles that can restore pollen fertility. Restoration of one male-sterility type appears to be controlled by a single, dominant allele, but restoration of a second cytoplasmic type is not easily explained with simple genetic models -- perhaps multiple nuclear loci and/or epistatic interactions are involved. In addition, the crosses show that pollen from some hermaphrodite plants in a population containing no females restores male fertility to plants from a geographically distant gynodioecious population that have male-sterile cytoplasm. Furthermore, some plants in that hermaphrodite population carry a male-sterile cytoplasm. Taken together, these results fit theoretical predictions that female frequency might be highly variable among populations when sex is determined by interactions between several nuclear and cytoplasmic genetic factors, some of which may not be present in all populations. The data also illustrate the need for more theoretical and empirical work investigating the evolutionary impact of nuclear restorer genes with complex action, and explaining the existence of nuclear restorers and cytoplasmic male-sterility genes in a population where females are very rare.

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.

Post-transcriptional and developmental regulation of a CMS-associated mitochondrial gene region by a nuclear restorer gene.

Transcripts of the mitochondrial gene region orf224/atp6, which is associated with the Polima or pol cytoplasmic male sterility (CMS) of Brassica napus, differ among fertile, sterile and nuclear-restored plants. We show here that the effects of the restorer gene Rfp on orf224/atp6 transcripts varies among different floral organs. Relative to monocistronic atp6 transcripts, levels of the dicistronic transcripts spanning orf224 and atp6 are dramatically reduced in petals, stamens and carpels, but not sepals, of restored flowers. In pol CMS plants, the relative levels of different orf224/atp6 transcripts are similar among the floral organs. Analysis of guanylyltransferase-labeled mtRNA indicates that only the dicistronic 2.2 and 1.9 kb orf224/atp6 transcripts carry an initiator 5' terminus; hence the 1.4 and 1.3 kb transcripts of restored plants, as well as the 1.1 kb atp6 transcript common to all genotypes, are generated by RNA processing and not de novo initiation. Although steady-state levels of dicistronic transcripts in flower buds are lower in restored than in sterile plants, run-on transcription experiments show that these transcripts are synthesized at the same rate in both types of flowers. These findings imply that the restorer gene acts by conditioning the removal of sequences from the 5' end of dicistronic transcripts in a developmentally regulated manner. Run-on transcription experiments indicate that the single 1.1 kb atp6 transcript of nap cytoplasm is also generated by removal of sequences from the 5' end of a precursor. We suggest that specific endonucleolytic cleavage of a precursor RNA, followed by non-specific 3' to 5' exonuclease action, may represent a common mechanism for tailoring transcripts in plant mitochondria.

Nuclear and Cytoplasmic Interactions for Petaloid Male-sterile Accessions of Wild Carrot (Daucus carota L.)

Male-sterile carrot plants with petaloid-stamens were discovered in wild populations in the vicinity of Guelph, Ontario, Canada and inheritance of phenotype was assessed. Genetic analyses were also conducted to identify nuclear restorer genes of petaloid cytoplasmic male sterility (CMS) from wild carrot, and determine if Guelph, Cornell and Wisconsin cytoplasmic male-sterile accessions interacted similarly with alleles of restorer genes from different sources. Petaloid male-sterility was inherited cytoplasmically for the new Guelph accessions, and nuclear genotypes which normally produce `green' or `white' petaloid-stamens in combination with the Cornell cytoplasm also produced corresponding phenotypes when backcrossed to Guelph cytoplasmic accessions. Two duplicate restorer genes were identified from wild carrot. Dominant alleles of single restorer genes from three sources, one from wild carrot and two from cultivated germplasm, restored stamen production to Guelph, Cornell and Wisconsin petaloid cytoplasmic accessions, suggesting that the cytoplasms were genetically identical.

Restorer genes for different forms of Brassica cytoplasmic male sterility map to a single nuclear locus that modifies transcripts of several mitochondrial genes.

The oilseed rape plant, Brassica napus, possesses two endogenous male sterile cytoplasms, nap and pol. Previous studies have shown that nuclear restoration of pol cytoplasmic male sterility (CMS) is conditioned by a gene, Rfp, that is also involved in modifying transcripts of the pol CMS-associated orf224/atp6 mtDNA region. We now find that the nap nuclear restorer gene Rfn apparently is identical to Mmt, a gene that conditions the modification of transcripts from several different mtDNA regions, including one that is associated with nap CMS and contains orf222, a chimeric gene related to orf224. Mmt, in turn, is found to be allelic to Rfp, suggesting that restorer genes for the two cytoplasms represent different alleles or haplotypes of a single nuclear locus. This view is supported by restriction fragment length polymorphism mapping studies that indicate that Rfn and Rfp map to the same chromosomal position. Thus, in contrast to CMS in other species, different forms of Brassica CMS are restored by alleles of a single nuclear locus, and the restoration properties of these alleles reflect their involvement in the modification of transcripts of corresponding CMS-associated mtDNA regions. A survey of 51 varieties from 8 Brassica and Sinapis species failed to find evidence of Rfn(Mmt) in other than fertility-restored, nap cytoplasm B. napus. This suggests that Rfn(Mmt) arose in Brassica with nap cytoplasm and that the necessity for fertility restoration may have provided the selective pressure for its origin and maintenance.

No evidence for local adaptation between cytoplasmic male sterility and nuclear restorer genes in the gynodioecious species Thymus vulgaris L.

In Thymus vulgaris L., sex determination involves both the nuclear and the cytoplasmic genomes: the cytoplasm is responsible for male sterility (the female phenotype) whereas specific nuclear genes may restore male fertility (the hermaphrodite phenotype). The evolutionary dynamics of cytoplasmic male-sterility genes and nuclear restorer genes represents a coevolutionary conflict. Here we draw a parallel between this conflict and the coevolutionary interaction between hosts and parasites. Local adaptation of parasites to their hosts may be predicted under some situations of relative evolutionary rates of the two partners in this conflict and is often observed in natural populations. We tested for local adaptation between nuclear restorer genes and cytoplasmic male-sterility factors using 12 female plants that were hand-pollinated with pollen from hermaphrodites from more or less distant populations, and measuring progeny sex ratios. No significant effects of the geographical distance between maternal and paternal parents could be detected, although highly significant differences among individual females for the restorability of their cytoplasms were found. We suggest that the same evolutionary processes giving rise to individual differences among cytoplasmic male-sterility types might account for the absence of local adaptation between nuclear and cytoplasmic factors in this system, and also discuss the absence of local adaptation in the light of a recent model of local adaptation in host-parasite metapopulations.

No evidence for local adaptation between cytoplasmic male sterility and nuclear restorer genes in the gynodioecious species Thymus vulgaris L.

No evidence for local adaptation between cytoplasmic male sterility and nuclear restorer genes in the gynodioecious species Thymus vulgaris L.

Spatial Distribution of Male Sterility in Plantago maritima

Sexual polymorphism in angiosperms can be explained both by the functional responses of male and female function to autogamy and geitonogamy, and by the conflict between the nuclear and cytoplasmic genomes. In predominantly hermaphrodite species, cytoplasmically determined male sterility may persist in a population because of maternal inheritance, i.e. the loss of male function does not change the fitness of the cytoplasmic genome. However, in populations with cytoplasmic male sterility. male fertility is often restored by nuclear genes. Therefore, in populations with genetical substructure, the frequencies of the different sex-morphs will fluctuate depending on the presence of both the male sterile cytoplasms, and of their specific nuclear restorer genes. In Plantago maritima, we showed that the frequencies of male sterility were highest in regions with the highest population turnover rates and that male sterile individuals were more frequently found in the lower, less dense parts of the meadows. This indicates that male sterile cytoplasms have their highest probabilities to escape their nuclear restorer genes during recolonisation in disturbed regions within populations. We also found that male sterile individuals dispersed their seeds a little bit further than did the hermaphrodites. This can be interpreted as an adaptive response to the local occurrence of nuclear restorer genes.

Nuclear fertility restorer genes map to the same linkage group in cytoplasmic male-sterile bean

Fertility restoration in cytoplasmic malesterile plants (CMS) by nuclear restorer genes is one of the few useful systems for studying nuclear-mitochondrial interactions in higher eukaryotes. In CMS bean there exist multiple independently identified nuclear genes that restore fertility. Two restorer genes (Fr and Fr 2) have been characterized previously. We have genetically characterized two additional restorer genes; both restorers are single genes that behave similarly to Fr 2. We compared the linkage relationship of all four independently identified restorer genes to understand the relationship among these loci further. All four genes map to the same linkage group. It was not possible to distinguish between the two newly identified restorer genes and Fr 2. We suggest that they may be allelic.