Nuclear Restorer Genes Research Articles

A P-type pentatricopeptide repeat protein ZmRF5 promotes 5' region partial cleavages of atp6c transcripts to restore the fertility of CMS-C maize by recruiting a splicing factor.

A fast evolution within mitochondria genome(s) often generates discords between nuclear and mitochondria, which is manifested as cytoplasmic male sterility (CMS) and fertility restoration (Rf) system. The maize CMS-C trait is regulated by the chimeric mitochondrial gene, atp6c, and can be recovered by the restorer gene ZmRf5. Through positional cloning in this study, we identified the nuclear restorer gene, ZmRf5, which encodes a P-type pentatricopeptide repeat (PPR) family protein. The over-expression of ZmRf5 brought back the fertility to CMS-C plants, whereas its genomic editing by CRISPR/Cas9 induced abortive pollens in the restorer line. ZmRF5 is sorted to mitochondria, and recruited RS31A, a splicing factor, through MORF8 to form a cleaving/restoring complex, which promoted the cleaving of the CMS-associated transcripts atp6c by shifting the major cleavage site from 480th nt to 344 th nt for fast degradation, and preserved just right amount of atp6c RNA for protein translation, providing adequate ATP6C to assembly complex V, thus restoring male fertility. Interestingly, ATP6C in the sterile line CMo17A, with similar cytology and physiology changes to YU87-1A, was accumulated much less than it in NMo17B, exhibiting a contrary trend in the YU87-1 nuclear genome previously reported, and was restored to normal level in the presence of ZmRF5. Collectively these findings unveil a new molecular mechanism underlying fertility restoration by which ZmRF5 cooperates with MORF8 and RS31A to restore CMS-C fertility in maize, complemented and perfected the sterility mechanism, and enrich the perspectives on communications between nucleus and mitochondria.

Fujian cytoplasmic male sterility and the fertility restorer gene OsRf19 provide a promising breeding system for hybrid rice

Cytoplasmic male sterility (CMS) determined by mitochondrial genes and restorer of fertility (Rf) controlled by nuclear-encoded genes provide the breeding systems of many hybrid crops for the utilization of heterosis. Although several CMS/Rf systems have been widely exploited in rice, hybrid breeding using these systems has encountered difficulties due to either fertility instability or complications of two-locus inheritance or both. In this work, we characterized a type of CMS, Fujian Abortive cytoplasmic male sterility (CMS-FA), with stable sporophytic male sterility and a nuclear restorer gene that completely restores hybrid fertility. CMS is caused by the chimeric open reading frame FA182 that specifically occurs in the mitochondrial genome of CMS-FA rice. The restorer gene OsRf19 encodes a pentatricopeptide repeat (PPR) protein targeted to mitochondria, where it mediates the cleavage of FA182 transcripts, thus restoring male fertility. Comparative sequence analysis revealed that OsRf19 originated through a recent duplication in wild rice relatives, sharing a common ancestor with OsRf1a/OsRf5, a fertility restorer gene for Boro II and Hong-Lian CMS. We developed six restorer lines by introgressing OsRf19 into parental lines of elite CMS-WA hybrids; hybrids produced from these lines showed equivalent or better agronomic performance relative to their counterparts based on the CMS-WA system. These results demonstrate that CMS-FA/OsRf19 provides a highly promising system for future hybrid rice breeding.

Did doubly uniparental inheritance (DUI) of mtDNA originate as a cytoplasmic male sterility (CMS) system?

Animal and plant species exhibit an astonishing diversity of sexual systems, including environmental and genetic determinants of sex, with the latter including genetic material in the mitochondrial genome. In several hermaphroditic plants for example, sex is determined by an interaction between mitochondrial cytoplasmic male sterility (CMS) genes and nuclear restorer genes. Specifically, CMS involves aberrant mitochondrial genes that prevent pollen development and specific nuclear genes that restore it, leading to a mixture of female (male-sterile) and hermaphroditic individuals in the population (gynodioecy). Such a mitochondrial-nuclear sex determination system is thought to be rare outside plants. Here, we present one possible case of CMS in animals. We hypothesize that the only exception to the strict maternal mtDNA inheritance in animals, the doubly uniparental inheritance (DUI) system in bivalves, might have originated as a mitochondrial-nuclear sex-determination system. We document and explore similarities that exist between DUI and CMS, and we propose various ways to test our hypothesis.

Single-cell RNA sequencing of meiocytes and microspores reveals the involvement of the Rf4 gene in redox homeostasis of CMS-C maize

Normal microsporogenesis is determined by both nuclear and mitochondrial genes. In maize C-type cytoplasmic male sterility, it is unclear how the development of meiocytes and microspores is affected by the mitochondrial sterility gene and the nuclear restorer gene. In this study, we sequenced the transcriptomes of single meiocytes (tetrad stage) and early mononucleate microspores from sterile and restorer lines. The numbers of expressed genes varied in individual cells and fewer than half of the expressed genes were common to the same cell types. Four comparisons revealed 3379 differentially expressed genes (DEGs), with 277 putatively associated with mitochondria, 226 encoding transcription factors, and 467 possibly targeted by RF4. KEGG analysis indicated that the DEGs in the two lines at the tetrad stage were involved predominantly in carbon metabolism and in amino acid biosynthesis and metabolism, whereas the DEGs during the transition from the tetrad stage to the early mononucleate stage were associated mostly with regulation of protein metabolism, fatty acid metabolism, and anatomical structure morphogenesis. Thus, meiocyte and microspore development was affected by the surrounding cells and the restorer gene, and the restorer gene helped restore the redox homeostasis of microspores and the normal cellular reconstruction during the transition.

Genetic resources in Finnish landrace rye (Secale cereale) and experimental evolving of its spring-habit from winter rye

Rye was the most important grain crop in Finland towards the end of the 19th century. Rye was largely grown in burned lands, kaski (slash-and-burn) and kytö, in the past. In the primary form of kytö, shoveled topsoil was heated on fires slowly combusting extraneous wood, brushwood or reed, or in the secondary form, a dry upper layer of organic topsoil of field was burned in a prescribed frontal mode in situ. The kytö selected against the brittle spike type, largely eliminating the weedy seed banks in the soil. Likewise, seed handling, especially the common cleaning with a pohdin -device further eliminated partially brittle spike types and selected against weedy rye. Rye was a cash-crop for the peasants in the past and was mainly attempted to be exported as seed. The commonly used smoky riihi-drying sanitized and conserved grains, which retained germinabilty, and in part increased demand for seed abroad. The grains produced on burned lands were fortified with minerals, including the minor elements, and good winter-hardiness occurred in the Finnish rye. The immigrant Finns were probably the first since 1638 to grow rye from seeds brought along with them to New Sweden in North America, where de-domesticated or feral rye became a weed problem in the 1950s. Some genetically variable landraces could be sown during different times of the year, thanks to segregate plants adapted to different sowing-times. Sowing of a winter rye landrace in May, the season of spring grain sowing, enabled selection of spring-habit mutants or segregants, which could be used to establish a true-breeding spring stock of rye shown experimentally. In the past, mid-summer sowing could occur with co-cultivation, e.g. with the traditional slash-and-burn turnip as the first season crop, or the autumn seedling of rye could be used as pasture. The Finnish rye populations frequently had cytoplasmic male sterility (CMS) and nuclear restorer genes of anther fertility effective in the CMS. A non-leaky CMS and a leaky CMS (with male fertility in the late stems) are shown. Homozygosity obtained through forced self-pollination in a Finnish rye revealed unnoticed genes, such as dwarfs. A local rye population originating from Putkosjärvi area (64° 27’ N), in Ristijärvi Municipality, evidently devoid of the frequently contaminating weed, rye brome (Bromus secalinus), is thought to present an uncontaminated, ancient Finnish rye. The rye brome has contaminated growth in Finland at least since the Iron Age. Morphological variants, like brown spike or glume color and awnlessness were detected in the landrace. Two of 18 Finnish landraces were found to carry accessory or B chromosomes in a study in 1964. B chromosomes are known to interfere with the expression of some genes, perhaps also ensuring variation in the vernalisation needs of the plants.

Characterization of cytoplasmic female sterility in an alloplasmic and monosomic addition line of Brassica rapa carrying the cytoplasm and one chromosome of Diplotaxis tenuifolia.

Alloplasmic plants exhibit various phenotypic changes such as cytoplasmic male sterility (CMS). We have been attempting to produce an alloplasmic Brassica rapa CMS line (2n = 20) carrying Diplotaxis tenuifolia cytoplasm (cyt-Dt) for several years, but a single extra chromosome always remained in all lines produced. We confirmed a D. tenuifolia-specific band in the alloplasmic line carrying D. tenuifolia cytoplasm by RAPD analysis, indicating that the additional chromosome was derived from D. tenuifolia. Here, we observed the phenotypic characteristics of the alloplasmic B. rapa monosomic addition line, named (cyt-Dt) B. rapa MAL, and investigated why a single extra chromosome is required in its genetic background for viability. When the (cyt-Dt) B. rapa MALs were crossed with pollen of several B. rapa lines, approximately 50% of the ovules attracted pollen tubes, and all the progeny had the additional chromosome. These results suggested that only the female gametes with n = 11 rather than n = 10 were fertilized and developed into mature seeds, and that cytoplasmic female sterility was overcome by nuclear restorer gene(s) derived from the cytoplasmic donor species.

Patterns of Genome-Wide Nucleotide Diversity in the Gynodioecious Plant Thymus vulgaris Are Compatible with Recent Sweeps of Cytoplasmic Genes.

Gynodioecy is a sexual dimorphism where females coexist with hermaphrodite individuals. In most cases, this dimorphism involves the interaction of cytoplasmic male sterility (CMS) genes and nuclear restorer genes. Two scenarios can account for how these interactions maintain gynodioecy. Either CMS genes recurrently enter populations at low frequency via mutation or migration and go to fixation unimpeded (successive sweeps), or CMS genes maintain polymorphism over evolutionary time through interactions with a nuclear restorer allele (balanced polymorphism). To distinguish between these scenarios, we used transcriptome sequencing in gynodioecious Thymus vulgaris and surveyed genome-wide diversity in 18 naturally occurring individuals sampled from populations at a local geographic scale. We contrast the amount and patterns of nucleotide diversity in the nuclear and cytoplasmic genome, and find ample diversity at the nuclear level (π = 0.019 at synonymous sites) but reduced genetic diversity and an excess of rare polymorphisms in the cytoplasmic genome relative to the nuclear genome. Our finding is incompatible with the maintenance of gynodioecy via scenarios invoking long-term balancing selection, and instead suggests the recent fixation of CMS lineages in the populations studied.

A novel source of cytoplasmic male sterility in Calibrachoa pubescens

Calibrachoa pubescens is a species native from the south of Brazil, Uruguay and northeast Argentina. An accession identified as 7.3.1.1 was collected at San Martín Department in Corrientes Province, Argentina and is included in the calibrachoa breeding program at the Institute of Floriculture, INTA. This accession is male sterile and produces male-sterile progeny, characterized by the lack of pollen production. Male sterility may be controlled by nuclear or cytoplasmic genes with each type presenting a different mode of inheritance. The objective of this research was to present a novel source of cytoplasmic male sterility in Calibrachoa pubescens. Crosses were made in the greenhouse between the male-sterile line 7.3.1.1 as the female parent and seven male-fertile lines of diverse origin. F1s were backcrossed and self-pollinated. Individual plants of the progenies were classified as male-fertile or male-sterile according to pollen viability. Analyses of observed segregations showed that male sterility observed in 7.3.1.1 results from the interaction of a male sterile cytoplasm and nuclear restorer genes. A novel male sterile cytoplasm is now available for breeders. Main advantages of cytoplasmic male sterility in ornamentals breeding are: efficient hybrid production, increased flower longevity, avoidance of pollen allergens and control of plant invasiveness.

Comparative genomic analysis of the compound Brassica napus Rf locus.

BackgroundThe plant trait of cytoplasmically-inherited male sterility (CMS) and its suppression by nuclear restorer-of-fertility (Rf) genes can be viewed as a genetic arms race between the mitochondrial and nuclear genomes. Most nuclear Rf genes have been shown to encode P-type pentatricopeptide repeat proteins (PPRs). Phylogenetic analysis of P-class PPRs from sequenced plants genomes has shown that Rf-proteins cluster in a distinct clade of P-class PPRs, RFL-PPRs, that display hallmarks of positive evolutionary selection. Genes encoding RFL-PPRs (RFLs) within a given plant genome tend to be closely related both in sequence and position, but a detailed understanding of how such species-specific expansion occurs is lacking. In the canola, (oilseed rape) species Brassica napus, previous work has indicated the nuclear restorer genes for the two native forms of CMS, Rfn (for nap CMS) and Rfp (pol CMS), represent alternate haplotypes, or alleles, of a single nuclear locus.ResultsFine genetic mapping indicates that Rfn does indeed localize to the same genomic region as Rfp. We find this region is enriched in RFL genes, three of which, based on their position and expression, represent potential candidates for Rfn; one of these genes, designated PPR4, is a preferred candidate in that it is not expressed in the nap CMS line. Comparison of the corresponding regions of the genomes of B. rapa, B. oleracea, Arabidopsis thaliana and A. lyrata provides insight into the expansion of this group of RFL genes in different lines of evolutionary descent.ConclusionsUnlike other nuclear restorer loci containing multiple RFL genes, the RFL genes in the Rf region of B. napus are not present in tandem arrays but rather are dispersed in genomic location. The genes do not share similar flanking non-coding regions and do not contain introns, indicating that they have duplicated primarily through a retrotransposition-mediated process. In contrast, segmental duplication has been responsible for the distribution of the 10 sequences we annotated as RFL genes in the corresponding region of the A. lyrata genome. Our observations define the Brassica Rf locus and indicate that different mechanisms may be responsible for the proliferation of RFL genes even among closely related genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3117-0) contains supplementary material, which is available to authorized users.

The effect of disease on the evolution of females and the genetic basis of sex in populations with cytoplasmic male sterility.

The evolution of separate males and females is an important evolutionary transition that has occurred multiple times in flowering plants. While empirical studies have stressed the potential importance of natural enemies and organismal interactions in the evolution of separate sexes, there has been no treatment of natural enemies in the theoretical literature. We investigated the effects of disease on the evolution of females in gynodioecious populations composed of females and hermaphrodites, where sex is determined by the interaction of cytoplasmic male sterility (CMS) and nuclear restorer genes. When females are significantly more resistant than hermaphrodites, disease drives an increase in the frequency of females and sex determination becomes nuclear, creating the pre-conditions for the evolution of separate males and females. However, when females are only moderately more resistant, disease drives changes in the frequency of CMS and restorer alleles, but has little effect on the frequency of females. We discuss our results in the context of the evolution of mating systems and cyto-nuclear epistasis.

Influence of Soil Moisture and Air Temperature on the Stability of Cytoplasmic Male Sterility (CMS) in Maize (<i>Zea mays</i> L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait that suppresses the production of viable pollen. CMS is a useful biological tool for confinement strategies to facilitate coexistence of genetically modified (GM) and non-GM crops in case where it is required. The trait is reversible and can be restored to fertility in the presence of nuclear restorer genes (Rf genes) and by environmental impacts. The aim of this study was to investigate the influence of the level of irrigation on the stability of CMS maize hybrids under defined greenhouse conditions. Additionally the combination of irrigation and air temperature was studied. Three CMS maize hybrids were grown with different levels of irrigation and in different temperature regimes. Tassel characteristics, pollen production and fertility were assessed. The CMS stability was high in hot air temperatures and decreased in lower temperatures. The level of irrigation had no major effect on the level of sterility. The extent of these phenomena was depending on the genotype of CMS maize and should be known before using CMS for coexistence purposes.

Fine mapping and candidate gene analysis of the nuclear restorer gene Rfp for pol CMS in rapeseed (Brassica napus L.)

The Polima (pol) system of cytoplasmic male sterility (CMS) in rapeseed is widely used in China for commercial hybrid seed production. Genetic studies have shown that its fertility restorer gene (Rfp) is monogenic dominant. For fine mapping of the Rfp gene, a near isogenic line comprising 3,662 individuals of BC(14)F(1) generation segregating for the Rfp gene was created. Based on the sequences of two SCAR markers, SCAP0612ST and SCAP0612EM2, developed by Zhao et al. (Genes Genom 30(3):191-196, 2008) and the synteny region of Brassica napus and other Brassica species, 13 markers strongly linked with the Rfp gene were identified. By integrating three of these markers to the published linkage map, the Rfp gene was mapped on linkage group N9 of B. napus. Using these markers, the Rfp locus was narrowed down to a 29.2-kb genomic region of Brassica rapa. Seven open reading frames (ORFs) were predicted in the target region, of these, ORF2, encoding a PPR protein, was the most likely candidate gene of Rfp. These results lay a solid foundation for map-based cloning of the Rfp gene and will be helpful for marker-assisted selection of elite CMS restorer lines.

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.

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

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

Modified complementation test of male sterility mutants in pepper (Capsicum annuum L.): preliminary study to convert male sterility system from GMS to CMS

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS systems.

RAPD Analysis on Variation of Mitochondrial DNA for Cytoplasmic-Nuclear Male Sterile Lines in Wheat

In order to produce a good F1 hybrid variety in wheat, it is necessary to explore a new male-sterile cytoplasm and its nuclear restore gene(s). Four alloplasmic male sterile lines of wheat with Aegilops cytoplasm were developed to identify mitochondrial DNA (mtDNA) variation that could potentially be associated with cytoplasmic male sterility (CMS). mtDNA isolated from the Aegilops species, the respective male sterile lines and F1 hybrids were analyzed by RAPD markers. Reproducible polymorphisms were detected between the Aegilops species and male sterile lines. Above results indicated that mtDNA variation existed in the cytoplasm donors and male sterile lines resulted from genetic interactions between common wheat nucleus and Aegilops cytoplasm, and have affected the structure of the mitochondrial genome. Similar results were also obtained in male sterile lines and fertility-restored F1 hybrids. These demonstrated the variation of mtDNA in fertility restoration by the combination of the fertility restorer gene(s), and fertility restoration involved a strong influence of nuclear restorer genes on mtDNA organization. The variation of mtDNA in Aegilops species, their respective CMS lines and fertility-restored F1 hybrids may reflect the fertility divergence.

Identification of a novel chimeric gene, orf725, and its use in development of a molecular marker for distinguishing among three cytoplasm types in onion (Allium cepa L.)

A novel chimeric gene with a 5' end containing the nearly complete sequence of the coxI gene and a 3' end showing homology with chive orfA501 was isolated by genome walking from two cytoplasm types: CMS-S and CMS-T, both of which induce male-sterility in onion (Allium cepa L.). In addition, the normal active and variant inactive coxI genes were also isolated from onions containing the normal and CMS-S cytoplasms, respectively. The chimeric gene, designated as orf725, was nearly undetectable in normal cytoplasm, and the copy number of the normal coxI gene was significantly reduced in CMS-S cytoplasm. RT-PCR results showed that orf725 was not transcribed in normal cytoplasm. Meanwhile, the normal coxI gene, which is essential for normal mitochondrial function, was not expressed in CMS-S cytoplasm. However, both orf725 and coxI were transcribed in CMS-T cytoplasm. The expression of orf725, a putative male-sterility-inducing gene, was not affected by the presence of nuclear restorer-of-fertility gene(s) in male-fertility segregating populations originating from the cross between a male-sterile plant containing either CMS-T or CMS-S and a male-fertile plant whose genotypes of nuclear restorer gene(s) might be heterozygous. The specific stoichiometry of orf725 and coxI in the mtDNA of the three cytoplasm types was consistent among diverse germplasm. Therefore, a molecular marker based on the relative copy numbers of orf725 and coxI was designed for distinguishing among the three cytoplasm types by one simple PCR. The reliability and applicability of the molecular marker was shown by testing diverse onion germplasm.

Restricted distribution of orf687 as the pollen fertility restorer gene for Ogura male sterility in Japanese wild radish

In radish, the mitochondrial gene orf138 found in Ogura cytoplasm is responsible for male sterility, whereas the nuclear restorer gene (Rf gene) inhibits the expression of orf138. To investigate the distribution of the Rf gene for Ogura male sterile cytoplasm in Japanese wild radish, 226 plants collected from 15 regions in Japan and two additional sites of Korea were used as materials. PCR analysis indicated that 42% of the wild radishes possessed orf138. On the other hand, 207 plants (91.6%) were judged to have an Rf gene by the observation of pollen fertility in wild radishes themselves and in hybrids obtained by crosses with a male sterile variety having Ogura cytoplasm. For plants having restorer function, we analyzed the nucleotide sequence of the Rf gene, orf687, identified to date. Among the 207 plants having the Rf gene, 199 amplified the orf687 fragment of the expected size by PCR. PCR products were further analyzed by mismatch-specific endonuclease digestion and PCR-RFLP analysis. As a result, plants that showed an identical RFLP pattern to the known Rf type of orf687 were restricted to 30 plants (14.5% of the 207 plants) collected mainly in populations of southern region of Japan. It was also found that most of the plants (148 plants; 71.5% of 207 plants) possessed the sequence of the orf687 corresponding to the rf genotype lacking the restorer function. We conclude that the Rf gene widely distributed in Japanese wild radish is not orf687.

Molecular markers linked to theRf2fertility restorer gene in cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants do not produce viable pollen. Fertility in plants with CMS can be recovered by nuclear restorer genes. Most restorer genes cloned so far are members of the pentatricopeptide repeat (PPR) protein family. The objective of our study was to use the CMS-D8 and restoration (Rf2) system of cotton (Gossypium hirsutum L.) to develop more DNA markers for the Rf2 gene. In a backcross population with 112 plants, segregation of male fertility was 1 fertile : 1 sterile. Three new RAPD markers were identified for Rf2, one of which was converted to a CAPS marker. In addition, 2 AFLP markers and 1 SSR marker were identified to be linked to the fertility restorer gene (Rf2). PPR motif primers were designed based on the conserved PPR motifs and used in combination with AFLP primers to test the mapping population, and 1 PPR-AFLP marker was identified. A linkage map with 9 flanking markers including 1 from a previous study was constructed.

Towards Positional Cloning in Brassica napus: Generation and Analysis of Doubled Haploid B. rapa Possessing the B. napus pol CMS and Rfp Nuclear Restorer Gene

The Polima (pol) system of cytoplasmic male sterility (CMS) and its fertility restorer gene Rfp are used in hybrid rapeseed production in Brassica napus. To facilitate map-based cloning of the Rfp gene, we have successfully transferred the pol cytoplasm and Rfp from the amphidiploid B. napus to the diploid species B. rapa and generated a doubled haploid pol cytoplasm B. rapa population that segregates for the Rfp gene. This was achieved through interspecific crosses, in vitro rescue of hybrid embryos, backcrosses, and microspore culture. Male fertility conditioned by Rfp was shown to co-segregate in this population with Rfp-specific mitochondrial transcript modifications and with DNA markers previously shown to be linked to Rfp in B. napus. The selfed-progeny of one doubled haploid plant were confirmed to be characteristic B. rapa diploids by cytogenetic analysis. Clones recovered from a genomic library derived from this plant line using the RFLP probe cRF1 fell into several distinct physical contigs, one of which contained Rfp-linked polymorphic restriction fragments detected by this probe. This indicates that chromosomal DNA segments anchored in the Rfp region can be recovered from this library and that the library may therefore prove to be a useful resource for the eventual isolation of the Rfp gene.