Ogura Cytoplasmic Male Sterility Research Articles

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.

Subunit 6 of the Fo-ATP synthase complex from cytoplasmic male-sterile radish: RNA editing and NH2-terminal protein sequencing

RNA editing and NH2-terminal processing of subunit 6 (atp6) of the mitochondrial Fo-ATPase complex has been investigated for the normal (fertile) and Ogura (male-sterile) radish cytoplasms to determine if previously identified differences between the Ogura atp6 locus and its normal radish counterpart are associated with cytoplasmic male sterility. Analysis of cDNA clones from five different sterile and fertile radish lines identified one C-to-U transition, which results in the replacement of a proline with a serine, in several of the lines. No editing of atp6 transcripts was observed in two lines, Scarlet Knight (normal radish) and sterile CrGC15 (Ogura radish). This is the first example of a naturally occurring plant mitochondrial gene that is not edited. The Ogura atp6 polypeptide is synthesized with a predicted NH2-terminal extension of 174 amino acids in contrast to the nine amino acid extension found in normal radish. In spite of the lack of similarity between the two extensions, NH2-terminal sequence analysis indicates that both polypeptides are processed to yield identical core proteins with a serine as the NH2-terminal residue. These results indicate that ATPase subunit 6 is synthesized normally in Ogura radish, and that it is unlikely that the atp6 locus is associated with Ogura cytoplasmic male sterility.

Effect of Ogura male-sterile cytoplasm on the performance of cabbage hybrid variety. II. Commercial characteristics

The main constraint of using Ogura male-sterile cytoplasm in Brassica is the induction of leaf yellowing at low temperature and the low seed set. But whether or not the cytoplasm can disturb the general commerical performance of the plants is not well-known. Therefore, this work was carried out with the purpose of evaluating Ogura cytoplasmic male-sterile cabbage hybrids and compare them to genomic similar male-fertile ones and to the most popular cabbage hybrid cultivated in Brazil. Harvest data showed an effect of the cytoplasm on reducing plant and head weight, core length and head length and width, although not altering heading index, shape and core indexes, nor head compactness. On the other hand, it was observed also that a careful selection of the parental lineages can produce male-sterile hybrids as good as their male-fertile analogues. All the evaluated hybrids were similar or better than the check for the characteristics under study.

Effect of Ogura male-sterile cytoplasm on the performance of cabbage hybrid variety

Cabbage hybrid seeds are commercially produced by means of self-incompatibility. This system may show some instability mainly under tropical conditions, where cytoplasmic male sterility can be an alternative approach for hybrid seeds production. However, cabbage hybrids holding Ogura male-sterile cytoplasm show some irregularities during development. By assessing some characteristics during the growing cycle of male-sterile cabbage hybrids and comparing them to genomic similar male-fertile ones and to the most common cabbage hybrid cultivated in Brazil, it was observed that the male-sterile hybrids had the same vigour, uniformity, number of leaves, resistance to Xanthomonas campestris pv. campestris, and earliness as their male-fertile counterparts and performed better than the commercial check hybrid for some of these characteristics. Although male-sterile hybrids showed yellowing of leaves, some parental combinations succeeded in overcoming or strongly reducing this cytoplasmic effect.

Molecular and biological studies on male sterile cytoplasm in Cruciferae. II. The origin of Ogura male sterile cytoplasm inferred from the segregation pattern of male sterility in the F1 progeny of wild and cultivated radishes (Raphanus sativus L.)

To determine the origin of Ogura male sterile cytoplasm in radish (Raphanus sativus L.), wild and cultivated radishes were crossed. Three types of progeny resulted from the F1 hybrids between the wild radish from ‘Kushikino’ with Ogura-type mtDNA and the cultivars (‘Uchiki-Gensuke’ or ‘Comet’). The segregation patterns of the male sterility were compared with those of Ogura cytoplasm. The male sterility induced in the F1 hybrid was maintained by crossing with ‘Uchiki-Gensuke’, that maintains Ogura male sterility. In the two types of progeny, in which ‘Comet’ (a restorer of Ogura cytoplasm) was used as one of the parents, both fertile and sterile plants segregated at the predicted ratio on the assumption that a single dominant fertility restoring gene exists in the restorer. From these results, we concluded that the Ogura cytoplasm is identical to that of the wild radish, and the former originated in a population of Japanese wild radish.

The role of coxI-associated repeated sequences in plant mitochondrial DNA rearrangements and radish cytoplasmic male sterility.

The gene coxI, encoding subunit I of mitochondrial cytochrome c oxidase, has been characterized from the normal (fertile) and Ogura (male-sterile) cytoplasms of radish to determine if a previously identified mitochondrial DNA rearrangement, and its associated transcriptional differences, could play a role in Ogura cytoplasmic male sterility (CMS). The normal and Ogura loci are virtually identical for 2.8 kb, including a 527-codon open reading frame whose product is approximately 95% identical to other plant COXI polypeptides. A rearrangement 120 bp 5' to the coding region results in different 5' transcript termini for the two genes. A comparison of several crucifer mitochondrial DNAs indicates that this rearrangement also occurs in the normal radish cytoplasm and is, therefore, not involved in Ogura CMS. Sequences present at the coxI locus belong to at least two different dispersed repeat families, members of which are also associated with other rearranged genes in radish.

Ogura cytoplasmic male sterility and triazine tolerant Brassica napus cv. Westar produced by protoplast fusion

Protoplasts of Brassica napus L. cv. Westar with the Ogura male-sterile cytoplasm were fused with protoplasts from Westar plants carrying triazine tolerance (TT) conferring cytoplasm. Of twenty-four plants recovered, one exhibited the novel Ogura cytoplasmic-male sterility (CMS), TT cybrid combination. A plant line with the normal B. napus chromosome number ( 2n = 38) was derived from this plant by fertilization with cv. Westar pollen. Unlike previously reported Ogura CMS/TT cybrids, this line showed good seed set and the maternal inheritance of the cytoplasmic traits has been documented over several sexual generations. No mitochondrial or chloroplast DNA recombination was detected. All other regenerants displayed the Ogura CMS characteristic and were susceptible to triazine.

Characterization of radish mitochondrial atpA: influence of nuclear background on transcription of atpA-associated sequences and relationship with male sterility

We have previously shown that the mitochondrial gene atpA, encoding the alpha subunit of F1 ATP synthase, is associated with DNA rearrangements and nuclear-specific transcript patterns in the male-sterile cytoplasm of Ogura radish. Here we present a detailed characterization of this gene from both the normal (fertile) and Ogura (male-sterile) cytoplasms of radish to determine if it is involved in Ogura cytoplasmic male sterility. The normal and Ogura radish atpA loci are virtually identical for 3.8 kb, including a 507 codon open reading frame whose product is approximately 92% identical to other plant ATPA polypeptides. Rearrangement breakpoints have been identified 613 bp 5' and 1663 bp 3' to the atpA coding region. The 5' rearrangement breakpoint is located within a repeated sequence that has been associated with other rearrangement events in radish mitochondria. The previously identified transcript difference results from transcription originating upstream of this rearrangement site. Although the presence of this transcript is affected by nuclear background, analyses in several different sterile and fertile nuclear backgrounds indicate that the presence of this transcript is not strictly correlated with male sterility. In addition, normal levels of ATPA polypeptide are present in sterile plants containing the Ogura cytoplasm.

ORGANELLE SEGREGATION FOLLOWING CAULIFLOWER PROTOPLASM FUSION

Cauliflower protoplasts with male fertile and Ogura male sterile cytoplasm were fused. Organelle reassortment and recombinant mitochondria were found in calli and plants regenerated from the fused protoplasts. Pretreatment (gamma-irradiation or iodoacetate) and protoplasm source (leaf or hypocotyl tissue) were manipulated in a series of fusions to determine their effects on organelle segregation. Some regenerated plants appear to combine Ogura male sterile mitochondria with normal Brassica chloroplasts. Plants with this organelle combination should be free of the cold temperature chlorosis due to incompatibility between the Brassica nucleus and the radish chloroplasts of the Ogura cytoplasm. These plants may have potential for improved cauliflower hybrid production.

Synthesis of male sterile, triazine-resistant Brassica napus by somatic hybridization between cytoplasmic male sterile B. oleracea and atrazine-resistant B. campestris

Fusion of leaf protoplasts from an inbred line of Brassica oleracea ssp. botrytis (cauliflower, n=9) carrying the Ogura (R1) male sterile cytoplasm with hypocotyl protoplasts of B. campestris ssp. oleifera (cv "Candle", n=10) carrying an atrazine-resistant (ATR) cytoplasm resulted in the production of synthetic B. napus (n=19). Thirty-four somatic hybrids were produced; they were characterized for morphology, phosphoglucose isomerase isoenzymes, ribosomal DNA hybridization patterns, chromosome numbers, and organelle composition. All somatic hybrids carried atrazine-resistant chloroplasts derived from B. campestris. The mitochondrial genomes in 19 hybrids were examined by restriction endonuclease and Southern blot analyses. Twelve of the 19 hybrids contained mitochondria showing novel DNA restriction patterns; of these 12 hybrids, 5 were male sterile and 7 were male fertile. The remaining hybrids contained mitochondrial DNA that was identical to that of the ATR parent and all were male fertile.

Atrazine-resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B. napus

Somatic hybridization between Brassica oleracea ssp. botrytis (cauliflower, 2n=18), carrying the Ogura (R1) male-sterile cytoplasm and B. napus (2n= 38), carrying a male-fertile, atrazine-resistant (ATR) cytoplasm, yielded three hybrids (2n=56) and six cauliflower cybrids (2n=18), which were selected for resistance to the herbicide in vitro. The hybrids and cybrids were male fertile and self-compatible. They contained both chloroplasts and mitochondria from the ATR cytoplasm. We found no evidence for mtDNA recombination in any of the regenerated plants. Selfed progeny of the B. oleracea atrazine-resistant cybrids were evaluated for tolerance to the herbicide in the field. Resistant plants exposed to 0.56-4.48 kg/ha (0.5-4.0 pounds/acre) atrazine in the soil showed no damage at any herbicide level, whereas plants of a susceptible alloplasmic line were severely damaged at the lowest level of herbicide application and killed at all higher levels. These atrazine-resistant cauliflower may have potential horticultural use, especially in fields where atrazine carry over is a serious problem.

Genotypic Variability in the Frequency of Plant Regeneration from Leaf Protoplasts of Four Brassica spp. and of Raphanus sativus

Abstract More than 65 different genotypes, including cultivars and inbred lines, from five cruciferous species (Brassica oleracea L., B. campestris L., B. napus L., B. juncea L., and Raphanus sativus L.) were tested for their in vitro response of leaf protoplasts. Protoplasts were cultured in three liquid media and the resulting colonies were placed on seven test regeneration media. Significant differences among the species were found in plating efficiency in the frequency of shoot regeneration. Two broad response groups were identified: 1) Cultivars from B. oleracea and B. napus—these generally yielded protoplasts that were able to divide, form colonies at high frequencies, and regenerate shoots at variable frequencies; and 2) cultivars of the other species evaluated, which typically exhibited low plating efficiencies and little, if any, shoot regeneration. Evaluation for the effect of the cytoplasmic constitution of a few B. oleracea breeding lines on in vitro performance indicated that protoplasts carrying the Ogura (R1) male-sterile cytoplasm regenerated shoots at slightly lower frequencies than the corresponding alloplasmic-fertile lines. Genotypes exhibiting high frequency of shoot formation in one medium also had efficient shoot regeneration in other media as well, while genotypes with low shoot regeneration responded consistently in the different media used. This consistency in response indicates that genotype plays a critical role in determining the success of leaf protoplast culture in the crucifers.

Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid

A linear mitochondrial plasmid reported to be associated with cytoplasmic male sterility in the genus Brassica was analyzed. A protein was found to be associated with the 5' ends of the plasmid. The entire plasmid was cloned by the homopolymer tailing technique via free hydroxyl groups present at its 3' ends. DNA sequence analysis of the cloned plasmid revealed a perfect terminal inverted repeat of 325 base pairs. Southern hybridization and restriction enzyme mapping analysis confirmed colinearity of the native plasmid and the clone, which showed significant homology with organelle DNA but not with nuclear DNA. Under high-stringency hybridization conditions, an internal 4.6 kb fragment of the 11.5 kb plasmid hybridized to the main mitochondrial genome in several species. Although the hybridization signal was weaker, the chloroplast genome also showed homology to the mitochondrial plasmid. The plasmid was undetectable at a molar ratio of less than 1/10 000 of the main mitochondrial genome in some lines of Brassica and Raphanus that contain the Ogura male sterile cytoplasm (cms). The absence of the plasmid in these sterile lines demonstrates that the plasmid is not required for the expression and maternal inheritance of male sterility.

Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B. campestris

An atrazine-resistant, male-fertile Brassica napus plant was synthesized by fusion of protoplasts from the diploid species B. oleracea and B. campestris. Leaf protoplasts from B. oleracea var. italica carrying the Ogura male-sterile cytoplasm derived from Raphanus sativus were fused with etiolated hypocotyl protoplasts of atrazine-resistant B. campestris. The selection procedure was based on the inability of B. campestris protoplasts to regenerate in the media used, and the reduction of light-induced growth of B. oleracea tissue by atrazine. A somatic hybrid plant that differed in morphology from both B. oleracea and B. campestris was regenerated on medium containing 50 μM atrazine. Its chromosome number was 36-38, approximately that of B. napus. Furthermore, nuclear ribosomal DNA from this hybrid was a mixture of both parental rDNAs. Southern blot analyses of chloroplast DNA and an assay involving tetrazolium blue indicated that the hybrid contained atrazine-resistant B. campestris chloroplasts. The hybrid's mitochondrial genome was recombinant, containing fragments unique to each parent, as well as novel fragments carrying putative crossover points. Although the plant was female-sterile, it was successfully used to pollinate B. napus.