A rapid,single leaf,nucleic acid assay for determining the cytoplasmic organelle complement of rapeseed and related Brassica species

Summary An assay is described whereby Eco RI restriction fragment length polymorphisms of mitochondrial and chloroplast DNAs can definitively identify cytoplasms of interest in Brassica crop development. Restrictable mitochondrial and chloroplast DNA is extracted from as little as 2–3 g and 0.5 g leaf tissue, respectively, and the donor plants are able to continue to develop in a normal manner. An unknown cytoplasm can be identified in three days, which is a considerable saving in time and labor compared to the several years required by traditional methods. The assay is very inexpensive and should be established as a routine procedure in laboratories involved in sexual or somatic Brassica hybrid production.     

Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid

Summary 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.     

Comparison of restriction endonucleases and sources of probes for their efficiency in detecting restriction fragment length polymorphisms in lettuce (Lactuca sativa L.)

Summary Five accessions of members of the C group of male sterile maize cytoplasms (BB, C, ES, PR, and RB) in two nuclear backgrounds (A619 and A632) were examined to elucidate the nature of mitochondrial genome diversity within a related group of cytoplasms. Cosmid and plasmid clones carrying single copy and recombinationally active sequences from N and S cytoplasms of maize were used as probes. Although restriction patterns are quite similar, each of the five could be discriminated by evidence of sequence duplication and recombination, deletion of recombinationally active sequences of N, normal cytoplasm, population of mini-circular DNAs, and by restriction patterns. Each member of the group carried a 1,913 bp minicircular mtDNA, while all entries but RB carried a 1,445 bp minicircular mtDNA. Members of the C group clearly are not molecularly identical; evolution of the group included principal genome reorganization involving sequence duplication/deletion events, apparently independent of the cms trait.Cooperative Investigations of Agricultural Research Service, U.S. Department of Agriculture, and Institute of Food and Agricultural Sciences, University of FloridaMention of trademark, proprietary product, or vendor does not constitute a guarantee of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

No evidence for a pleiotropic relationship between male sterility and cyanide-resistant respiration in Plantago lanceolata

Gynodioecy, the coexistence of hermaphrodites and male steriles, is frequent in populations of Plantago lanceolata L. A condition for the maintenance of gynodioecy in an obligatory outbreeding species like this is an increase in female fitness in male steriles compared with hermaphrodites. One of the possible underlying mechanisms, a lower cyanide-resistant respiration in male steriles, which could lead to a higher metabolic efficiency, was investigated. For the experiments adult plants were used, because the effects which compensate for male sterility have been found in characters like seed production and longevity. No general correlation between sex phenotype and cyanide-resistant respiration capacity, or with any other respiration component, was found. Only in a single cross a strong correlation between cyanide-resistant respiration activity and sex phenotype was established, male steriles possessing the higher activity. The conclusion from these experiments is that there is no pleiotropic relationship between respiration levels and sex phenotype. The strongly significant correlation mentioned is ascribed to chromosomal linkage.     

DNA polymorphism in Allium cepa cytoplasms and its implications concerning the origin of onions

Summary Mitochondrial and chloroplast DNA was isolated from fertile and cytoplasmic male sterile cultivars of cultivated onions. Restriction fragment length polymorphism led to the distinction between cytoplasms S and M. Mitochondrial DNA patterns from S cytoplasms appeared dentical and characterized mostly male sterile lines. An open-pollinated variety was found to bear this cytoplasm and thought to be the origin of S types. Mitochondrial DNA patterns from M cytoplasms were subdivided into four types, M₁ and M₂ corresponding to normal N cytoplasm, M₃ and M₄ probably corresponding to T cytoplasms. S and M cytoplasms were also distinguished by chloroplast DNA restriction patterns. Our results confirm previous genetic distinction between S, N and T cytoplasms.     

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

Summary 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.     

Small mitochondrial DNA molecules of wild abortive cytoplasm in rice are not necessarily associated with CMS

Summary Mitochondrial DNA was isolated from leaf tissue of both the cytoplasmic male sterile line of Indica rice variety V41, which carries wild abortive (WA) cytoplasm, and from the corresponding maintainer line. In addition to the main mitochondrial DNA, four small plasmid-like DNA molecules were detected in both the male sterile and fertile lines. Restriction analysis of total mitochondrial DNA from the male sterile and fertile lines showed DNA fragments unique to each. Our findings suggest that the four small mitochondrial DNA (mtDNA) molecules are conserved when WA cytoplasm is transferred into different nuclear backgrounds. However, there is no simple correlation between the presence/ absence of small mitochondrial DNA molecules and the expression of WA cytoplasmic male sterility (CMS).     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary In the ogu cytoplasmic male-sterile (CMS) line of Brassica napus, stamen morphology was influenced by temperature conditions. Under a high temperature regime (27° C/23° C; day/ night) CMS stamens had a near-normal morphology, but microsporogenesis proceeded to a maximum of the microspore stage. However, compared to the normal stamens, the occurrence of sporopollenin-like deposits in the tapetum and deposition of exine on the microspores was sparse. Also, the tapetal cells of the CMS line were often highly vacuolate and failed to degenerate at the same stage as the normal. Ultrastructural changes in the mitochondrial matrix and cristae plus dilation of the endoplasmic reticulum, which occurred during development in sporogenous tissues of the normal line, were often lacking or mistimed in the mutant. Due to extensive variation, even between adjacent locules, the cytological differences between the normal and CMS anthers cannot be ascribed as the cause of male sterility in the ogu CMS line of B. napus, rather they may be the consequence of it.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

Correction of chlorophyll-defective male-sterile winter oilseed rape (Brassica napus) through organelle exchange: Characterization of the chlorophyll deficiency

Jarl, C. I., Ljungberg, U. K. and Bornman, C. H. 1988. Correction of chlorophyll-defective male-sterile winter oilseed rape ( Brassica napus ) through organelle exchange: Characterization of the chlorophyll deficiency. - Physiol. Plant. 72: 505–510.
As is known, the introduction of male-sterile Raphanus sativus L. cytoplasm into Brassica napus L. results in male-sterile oilseed rape plants, which display a temperature-related chlorophyll defect. The influences of temperature and irradiance on this defect were investigated. Compared to a line of normal (green phenotype) male-fertile oilseed rape, the male-sterile line had reduced chlorophyll content, fewer chloroplasts per cell, an altered ultrastructure of the chloroplasts and reduced activities of both photosystems, although the relative amounts of the photosystems and the chlorophyll a/b ratio were similar. The lower activity of the photosystems is explained by a decreased functional antennae size and a reduced efficiency in the interactions between the nuclear-encoded light-harvesting proteins and the reaction centres coded for by the plastome. Some thylakoid polypeptides differed in proportion between the male-fertile line with green phenotype and the male-sterile line with chlorotic phenotype. Characters, in which the two lines exhibited differences, are ascribed to difficulties in molecular communication between the oilseed rape nucleus and the radish cytoplasm, which are combined in the deficient male-sterile line.