Intra- and inter-specific variations in the mitochondrial gene orf138 of Ogura-type male-sterile cytoplasm from Raphanus sativus and Raphanus raphanistrum

In order to gain a better understanding of the evolution of Ogura male-sterile cytoplasm in radish, a large-scale sequence analysis of mitochondrial orf138 was conducted using 107 Japanese wild radishes, 29 cultivated radishes and seven Raphanus raphanisturum. A single approximately 0.8-kb fragment containing the orf138 locus was amplified from each plant by PCR, and the nucleotide sequence of an entire coding region of orf138 was determined by direct-sequencing procedures. An identical sequence to the published orf138 (Type A) was identified in Japanese wild radish, including a single plant in a population near Kagoshima prefecture where Ogura (1968) first found ’Ogura male-sterile radish’. Thus, it was confirmed that the ’Ogura male-sterile cytoplasm’ was derived from Japanese wild radish, with a Type A orf138 sequence, growing in this area. A total of six nucleotide changes and a single insertion/deletion (indel) were found in orf138 from both wild and cultivated radishes. By a combination of mutations, the orf138 sequences of the 143 radish plants were classified into nine types. Based on the pattern of mutations and the distribution of orf138 variants, it was concluded that the orf138 variants are derived from Type B or C, after Ogura-type cytoplasm was introduced from R. raphanistrum into Japanese wild radish. Received: 19 December 2000 / Accepted. 26 January 2001     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. III. Distribution of Ogura-type cytoplasm among Japanese wild radishes and Asian radish cultivars

The distribution of Ogura male-sterile cytoplasm among Japanese wild radish populations and Asian cultivated radishes was studied by means of polymerase chain reaction (PCR)-aided assays using mitochondrial atp6 and orf138 loci as molecular markers. Three separate PCR experiments were performed to amplify the target sequences in normal-type atp6, Ogura-type atp6, and Ogura-specific orf138, and the cytoplasm of each plant was classified as either normal or Ogura. Among 217 wild radish plants, 93 had both Ogura-type atp6 and orf138 (or its modified form), whereas 124 had normal-type atp6. Of the 93 plants with Ogura-type cytoplasm, only a single plant showed male sterility. A complete linkage between Ogura-type atp6 and orf138 loci was found in Japanese wild radishes, confirming our findings that Ogura-type cytoplasm is distributed widely among Japanese wild radish populations. A modified form of orf138 (orf138-S) was identified in a few wild radish populations in a limited area of Japan, and the nucleotide sequence of the orf138-S revealed a 39-bp deletion shared in common with Kosena male-sterile cytoplasm. Among the 44 Asian cultivars analyzed, 40 were determined to have normal cytoplasm since all 4 plants tested in each cultivar showed the same PCR amplification profiles as that of Uchiki-Gensuke, a reference cultivar with normal cytoplasm. The plants with Ogura-type cytoplasm (or its modified form) were found in 1, 1, and 2 cultivars from Tibet, Japan, and Taiwan, respectively. Except for 1 cultivar from Taiwan, those with Ogura-type cytoplasm included a few plants having male sterility. The multiple and independent introduction of Ogura-type cytoplasm from the wild radish in Asia into these cultivars is suggested.     

Multiple origins of cultivated radishes as evidenced by a comparison of the structural variations in mitochondrial DNA of Raphanus.

Configurations of mitochondrial coxI and orfB gene regions were analysed by polymerase chain reaction (PCR) in three wild and one cultivated species of Raphanus. A total of 207 individual plants from 60 accessions were used. PCR with five combinations of primers identified five different amplification patterns both in wild and cultivated radishes. While the mitochondrial DNA (mtDNA) type of Ogura male-sterile cytoplasm was distinguishable from the normal type, the mtDNAs of normal radishes were further classified into four types. The variations were common to wild and cultivated radishes, although contrasting features were found depending on the region of cultivation. These results provide evidence that cultivated radishes have multiple origins from various wild plants of Raphanus.     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. I. The origin and distribution of Ogura male-sterile cytoplasm in Japanese wild radishes (Raphanus sativus L.) revealed by PCR-aided assay of their mitochondrial DNAs

Ogura male-sterile cytoplasm was surveyed in common Japanese radish cultivars and in wild radishes growing in various localities in Japan. Mitochondrial (mt) DNA rearrangement involving the atp6 gene was used as a molecular marker. To detect the mtDNA rearrangement, polymerase chain reactions (PCR) were designed to amplify the upstream region of the atp6 gene. The oligonucleotides homologous to the following three regions were synthesized: (1) trnfM, (2) ORF105 and (3) atp6. PCRs were conducted with a pair of the first and the third primers to detect normal mtDNA, and with the second and the third primers for Ogura-type mtDNA. All 15 Japanese cultivars yielded an amplification product which was the same as that of normal mtDNA, whereas some wild radishes gave the product specific to Ogura mtDNA. Twenty-four populations of wild radish were classified into three groups according to the frequency of Ogura-type mtDNA: (1) in ten populations, all four plants analyzed per population had normal type mtDNA, (2) in five populations, only plants with Ogura-type mtDNA were found, and (3) nine populations included both normal and Oguratype mtDNAs. There were no geographical restrictions and no cline in the distribution of the plants with Ogura-type mtDNA. These results suggested that the Ogura-type male-sterile cytoplasm originated in wild radishes.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Discovery of a novel cytoplasmic male-sterility and its restorer lines in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

A male-sterile (MS) radish (Raphanus sativus L.) was found in an accession collected from Uzbekistan. Unlike Ogura MS radishes in which no pollen grain is typically visible during anthesis, a small number of pollen grains stuck together in the dehiscing anthers was observed in the newly identified MS radish. Fluorescein diacetate tests and scanning electron micrographs showed that pollen grains in the new MS radish were severely deformed and non-viable. Cytological examination of pollen development stages showed a clear difference in the defective stage from that seen in Ogura male-sterility. Reciprocal cross-pollination with diverse male-fertile lines indicated that pollen grains of the new MS radish were completely sterile, and the female organs were fully fertile. When the new MS radish and Ogura MS lines were cross-pollinated with a set of eight breeding lines, all F₁ progeny originating from crosses with the new MS radish were male-sterile. In contrast, most of the F₁ progeny resulting from crosses with Ogura MS lines were male-fertile. These results demonstrated that factors associated with induction of the newly identified male-sterility are different from those of Ogura male-sterility. The lack of restorer lines for the newly identified male-sterility led us to predict that it might be a complete cytoplasmic male-sterility without restorer-of-fertility genes in nuclear genomes. However, cross-pollination with more diverse radish germplasm identified one accession introduced from Russia that could completely restore fertility, proving the existence of restorer-of-fertility gene(s) for the new male-sterility. Meanwhile, the PCR amplification profile of molecular markers for the classification of radish mitochondrial genome types revealed that the new MS radish contained a novel mitotype.     

Identification of a novel mitochondrial genome type and development of molecular markers for cytoplasm classification in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

Plant mitochondrial genomes have complex configurations resulting from the multipartite structures and highly rearranged substoichiometric molecules created by repetitive sequences. To expedite the reliable classification of the diverse radish (Raphanus sativus L.) cytoplasmic types, we have developed consistent molecular markers within their complex mitochondrial genomes. orf138, a gene responsible for Ogura male-sterility, was detected in normal cultivars in the form of low-copy-number substoichiometric molecules. In addition to the dominant orf138-atp8 Ogura mitochondrial DNA (mtDNA) organization, three novel substoichiometric organizations linked to the atp8 gene were identified in this study. PCR amplification profiles of seven atp8- and atp6-linked sequences were divided into three groups. Interestingly, the normal cytoplasm type, which had previously been considered a single group, showed two patterns by PCR amplification. The most prominent difference between the two normal mtDNAs was size variation within four short-repeat sequences linked to the atp6 gene. This variation appeared to be the result of a double crossover, mediated by these homologous, short-repeat sequences. Specific PCR amplification profiles reflecting the stoichiometry of different mtDNA fragments were conserved within cultivars and across generations. Therefore, the specific sequences detected in these profiles were used as molecular markers for the classification of diverse radish germplasm. Using this classification system, a total of 90 radish cultivars, or accessions, were successfully assigned to three different mitotypes.     

Complete mitochondrial genome sequence and identification of a candidate gene responsible for cytoplasmic male sterility in radish (Raphanus sativus L.) containing DCGMS cytoplasm

A novel cytoplasmic male sterility (CMS) conferred by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its restorer-of-fertility gene (Rfd1) was previously reported in radish (Raphanus sativus L.). Its inheritance of fertility restoration and profiles of mitochondrial DNA (mtDNA)-based molecular markers were reported to be different from those of Ogura CMS, the first reported CMS in radish. The complete mitochondrial genome sequence (239,186 bp; GenBank accession No. KC193578) of DCGMS mitotype is reported in this study. Thirty-four protein-coding genes and three ribosomal RNA genes were identified. Comparative analysis of a mitochondrial genome sequence of DCGMS and previously reported complete sequences of normal and Ogura CMS mitotypes revealed various recombined structures of seventeen syntenic sequence blocks. Short-repeat sequences were identified in almost all junctions between syntenic sequence blocks. Phylogenetic analysis of three radish mitotypes showed that DCGMS was more closely related to the normal mitotype than to the Ogura mitotype. A single 1,551-bp unique region was identified in DCGMS mtDNA sequences and a novel chimeric gene, designated orf463, consisting of 128-bp partial sequences of cox1 gene and 1,261-bp unidentified sequences were found in the unique region. No other genes with a chimeric structure, a major feature of most characterized CMS-associated genes in other plant species, were found in rearranged junctions of syntenic sequence blocks. Like other known CMS-associated mitochondrial genes, the predicted gene product of orf463 contained 12 transmembrane domains. Thus, this gene product might be integrated into the mitochondrial membrane. In total, the results indicate that orf463 is likely to be a casual factor for CMS induction in radish containing the DCGMS cytoplasm.     

A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.)

ABSTRACT: BACKGROUND: Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138. RESULTS: Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences. CONCLUSIONS: Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.     

Low-copy-number molecules are produced by recombination, actively maintained and can be amplified in the mitochondrial genome of Brassicaceae: relationship to reversion of the male sterile phenotype in some cybrids

A PCR analysis of mitochondrial (mt) genomes of cybrid rapeseed plants revealed substoichiometric concentrations of molecules bearing different configurations of the gene (orf138) responsible for Ogura cytoplasmic male sterility (CMS). These sub-stoichiometric molecules are also present in plants bearing the unmodified Ogura cytoplasm. In one cybrid family, which shows reversion of the male sterile phenotype, we observed changes in the respective proportions of these molecules. The phenotypic (sterility-fertility) reversion occurs as a result of a modification of the equilibrium state between the different forms of the orf138 gene and is very probably determined by the level of expression of this gene. Stable situations are always characterized by one predominant form; the others, when present, exist in substoichiometric amounts. We report results indicating that the different forms of the orf138 gene are continuously interconverted by recombination and that an active mechanism is involved in the maintenance of some substoichiometric molecules.     

Direct transfer of a cold-tolerant Ogura male-sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion

Cold tolerant cytoplasmic male-sterile (CMS) cabbage (Brassica oleracea var. capitata) was produced by the fusion of leaf protoplasts from fertile cabbage and cold-tolerant Ogura CMS broccoli lines. The cabbage lines tested showed great variation in plant regeneration from unfused protoplasts; three with high regenerability were selected as the fusion partners. Several procedures for eliminating the nuclear DNA of the broccoli fusion partner were tested. Diploid cabbage plants were identified by flow cytometry and morphological characters. Gamma-irradiation (30 krad) was the most successful procedure; isolation of cytoplasts from broccoli leaf protoplasts, followed by gamma-irradiation of the cytoplast fraction, also produced diploids. UV-irradiation of the broccoli protoplasts was less effective. PCR using primers for an Ogura CMS-specific mitochondrial DNA sequence permitted the identification of cybrids likely to be CMS. Over 200 diploid plants with the CMS-specific sequence were obtained from 66 independent fusion products and three cabbage lines. Plants were ready for transfer into soil within 8 months after fusion. The plants identified as CMS by PCR produced male-sterile flowers. Our procedures permit the transfer of a desirable male-sterile cytoplasm into cabbage much more rapidly than conventional backcrossing procedures. Received: 4 June 1996 / Accepted: 2 August 1996     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

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.     

Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility

Cytoplasmic male sterility is a maternally inherited trait in higher plants that prevents the production of functional pollen. Ogura cytoplasmic male sterility in radish (Raphanus sativus) is regulated by the orf138 mitochondrial locus. Male fertility can be restored when orf138 accumulation is suppressed by the nuclear Rfo locus, which consists of three genes putatively encoding highly similar pentatricopeptide repeat proteins (PPR-A, -B, and -C). We produced transgenic rapeseed (Brassica napus) plants separately expressing PPR-A and PPR-B and demonstrated that both encoded proteins accumulated preferentially in the anthers of young flower buds. Immunodetection of ORF138 showed that, unlike PPR-B, PPR-A had no effect on the synthesis of the sterility protein. Moreover, immunolocalization experiments indicated that complete elimination of ORF138 from the tapetum of anthers correlated with the restoration of fertility. Thus, the primary role of PPR-B in restoring fertility is to inhibit ORF138 synthesis in the tapetum of young anthers. In situ hybridization experiments confirmed, at the cellular level, that PPR-B has no effect on the accumulation of orf138 mRNA. Lastly, immunoprecipitation experiments demonstrated that PPR-B, but not PPR-A, is associated with the orf138 RNA in vivo, linking restoration activity with the ability to directly or indirectly interact with the orf138 RNA. Together, our data support a role for PPR-B in the translational regulation of orf138 mRNA.