水稻两种细胞质雄性不育“三系”及其F1杂交种线粒体DNA的RFLP分析

对水稻BT型和WA型细胞质的雄性不育系,相应保持系和恢复系以及杂种的mtDNA用12个线粒体探针进行了RFLP分析,结果如下(1)BT型和WA型不育系的mtDNA在组织结构上存在差异;(2)不育系的mtDNA与其保持系间存在显著差异,推测mtDNA与水稻的cms有关;(3)atp9探针检测到WA型不育系与F1之间的多态性,Frag36探针检测到BT型不育系与F1之间的多态性,Frag9探针检测到WA型和BT型不育系与其F1之间的多态性,证明核恢复基因影响mtDNA的结构;(4)对mtDNA的结构变异与细胞质雄性不育的关系进行了分析与探讨.     

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).     

高等植物细胞质雄性不育分子机理的研究进展

从线粒体DNA、叶绿体DNA和线粒体质粒DNA方面较详细地阐述了高等植物细胞质雄性不育的分子机理及最新进展;探讨了细胞核DNA和细胞质DNA之间的相互关系。     

Molecular basis of cytoplasmic male sterility and fertility restoration in rice

Cytoplasmic male sterility (CMS) is a maternally inherited trait that causes dysfunctions in pollen and anther development. CMS is caused by the interaction between nuclear and mitochondrial genomes. A product of a CMS-causing gene encoded by the mitochondrial genome affects mitochondrial function and the regulation of nuclear genes, leading to male sterility. In contrast, the RESTORER OF FERTILITY gene (Rf gene) in the nuclear genome suppresses the expression of the CMS-causing gene and restores male fertility. An alloplasmic CMS line is often bred as a result of nuclear substitution, which causes the removal of functional Rf genes and allows the expression of a CMS-causing gene in mitochondria. The CMS/Rf system is an excellent model for understanding the genetic interactions and cooperative functions of mitochondrial and nuclear genomes in plants, and is also an agronomically important trait for hybrid seed production. In this review article, pollen and anther phenotypes of CMS, CMS-associated mitochondrial genes, Rf genes, and the mechanism that causes pollen abortion and its agronomical application for rice are described.     

Asymmetric protoplast fusion aimed at intraspecific transfer of cytoplasmic male sterility (CMS) in Lolium perenne L.

Summary Techniques have been developed for the production of cybrids in Lolium perenne (perennial ryegrass). Gamma-irradiated protoplasts of a cytoplasmically male-sterile breeding line of perennial ryegrass (B200) were fused with iodoacetamide-treated protoplasts of a fertile breeding line (Jon 401). After fusion 25 putative cybrid calli were characterized to determine mitochondrion type and composition of the nuclear genome. Analysis of phosphoglucoisomerase isozyme profiles and determination of the ploidy level by flow cytometry indicated that all of the calli tested essentially contained the nuclear DNA of the fertile line. However, the presence of parts of the nuclear DNA from the sterile line could not be excluded. Southern blotting of total DNA isolated from the parental lines and putative cybrids combined with hybridizations using the mitochondrial probes cox1 and atp6 revealed that the mitochondria of the calli originated from the fertile line (5 calli), the sterile line (5 calli) or from both parental lines (15 calli). The hybridization patterns of the mtDNA from the cybrid calli showed extensive quantitative and qualitative variation, suggesting that fusion-induced inter- or intramolecular mitochondrial recombination had taken place.     

Mitochondrial genome organization and cytoplasmic male sterility in plants

Plant mitochondrial genomes are much larger and more complex than those of other eukaryotic organisms. They contain a very active recombination system and have a multipartite genome organization with a master circle resolving into two or more subgenomic circles by recombination through repeated sequences. Their protein coding capacity is very low and is comparable to that of animal and fungal systems. Several subunits of mitochondrial functional complexes, a complete set of tRNAs and 26S, 18S and 5S rRNAs are coded by the plant mitochondrial genome. The protein coding genes contain group II introns. The organelle genome contains stretches of DNA sequences homologous to chloroplast DNA. It also contains actively transcribed DNA sequences having open reading frames. Plasmid like DNA molecules are found in mitochondria of some plants Cytoplasmic male sterility in plants, characterized by failure to produce functional pollen grains, is a maternally inherited trait. This phenomenon has been found in many species of plants and is conveniently used for hybrid plant production. The genetic determinants for cytoplasmic male sterility reside in the mitochondrial genome. Some species of plants exhibit more than one type of cytoplasmic male sterility. Several nuclear genes are known to control expression of cytoplasmic male sterility. Different cytoplasmic male sterility types are distinguished by their specific nuclear genes(rfs) which restore pollen fertility. Cytoplasmic male sterility types are also characterized by mitochondrial DNA restriction fragment length polymorphism patterns, variations in mitochondrial RNAs, differences in protein synthetic profiles, differences in sensitivity to fungal toxins and insecticides, presence of plasmid DNAs or RNAs and also presence of certain unique sequences in the genome. Recently nuclear male sterility systems based on (i) over expression of agrobacterialrol C gene and (ii) anther specific expression of an RNase gene have been developed in tobacco andBrassica by genetic engineering methods.     

Variable presence of the 1.94kb mitochondrial plasmid in maize S cytoplasm and its relationship to cytoplasmic male sterility

Mitochondrial DNA (mtDNA) was isolated from over 100 different maize nucleo-cytoplasmic combinations. DNA preparations were assayed for the presence of the 1.94kb mitochondrial plasmid by agarose gel electrophoresis and hybridization to a recombinant clone of the plasmid. The plasmid was present in all tested inbreds which carried N, male fertile, cytoplasm or the cytoplasmically male sterile (cms) groups,cms-T andcms-C. However, members of thecms-S group differed with respect to the presence of the plasmid. Cytoplasms I, J and S possessed the plasmid, whereas cytoplasms B, CA, D, G, H, IA, ME, ML, PS, RD and VG did not.Cms-S group lines which had spontaneously reverted to fertility (nuclear and cytoplasmic revertants) did not exhibit a concomitant change in 1.94kb plasmid levels, although all such lines showed the previously reported alteration in levels of the linear mtDNAs, S1 and S2. The presence or absence of the plasmid was not correlated with (i) frequency of reversion to fertility, (ii) the degree of male sterility expressed, (iii) the presence or absence of standard nuclear restorer to fertility genes and (iv) nuclear genotype. Latin American races carrying RU cytoplasm possessed the plasmid, as did sweet corn varieties. The relevance of the data tocms and evolution of thecms-S group is discussed.     

BT型细胞质雄性不育水稻及其三系的线粒体DNA研究

用RAPD技术对BT型水稻胞质雄性不育系秀A及其保持系秀B、恢复系湘晴以及杂种F1代的线粒体DNA进行了比较分析。结果表明不育系与其保持系间存在显著差异;不育系与其F1之间mtDNA也存在差异。在引物OPJ－08的扩增产物中,秀A扩增出一条分子量为800bp的多态性片段,在引物OPK－10的扩增产物中,杂种F1扩增出一条分子量为900bp的片段。把这两片段回收、克隆并制备探针,OPJ－08800的Southern杂交结果显示不育系与其F1杂交图谱存在多态性;OPK－10900的Suthern杂交结果显示不育系与其保持系同存在差异。推测这两片段与育性可能有一定的联系。     

Mitochondrial DNA modifications associated with cytoplasmic male sterility in rice

Summary Mitochondrial DNA was isolated from fertile and cytoplasmic male sterile lines of rice. Restriction analysis showed specific modifications in the male sterile cytoplasm. In addition to the major mitochondrial DNA, three small plasmid-like DNA molecules were detected by agarose gel electrophoresis in both cytoplasms. An additional molecule was specifically found in the sterile cytoplasm. These mitochondrial DNA modifications support the hypothesis of the mitochondrial inheritance of the cytoplasmic male sterility in rice.     

Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion

Male sterility conferred by ogu cytoplasm of Raphanus sativus has been transferred to Brassica juncea cv RLM 198 from male-sterile B. napus through repeated backcrossing and selection. The male-sterile B. juncea is, however, highly chlorotic and late. It has low female (seed) fertility and small contorted pods. To rectify these defects, protoplasts of the male sterile were fused with normal RLM 198 (green, self fertile). Four dark green, completely male-sterile plants were obtained and identified as putative cybrids. All the plants were backcrossed three times with RLM 198. Mitochondrial and chloroplast DNA analysis of backcross progeny confirmed hybridity of the cytoplasm. The restriction pattern of the chloroplast DNA of progeny plants of three cybrids (Og 1, Og 2, Og 3) was similar to that of the green self-fertile RLM 198 and indicated that the correction of chlorosis resulted from chloroplast substitution. The chloroplast DNA of the lone progeny plant of the fourth cybrid (Og 10) could not be analyzed because the plant was stunted and had only a few leaves. When total cellular DNA was probed with mitochondrial probes coxI and atpA it was found that the cybrids had recombinant mitochondria. The chlorosis-corrected plants were early flowering and had vastly improved seed fertility.     

Two new types of cytoplasmic male sterility found in wild Beta beets

Summary Mitochondrial (mt) and chloroplast (ct) DNAs from sugar beet carrying normal fertile and different cytoplasmic male sterile (cms) cytoplasms were compared by restriction analysis and for the occurrence of minicircles. One of the cms materials had the Owen cms cytoplasm currently used for hybrid production in sugar beet; the other three cms materials were derived from wild Beta beets. The mtDNAs from two of the latter cms types (C 7051, C 8640) differed from both the Owen and the fertile cytoplasms in fragment patterns seen after restriction enzyme analysis and in minicircle composition. The third cms type (C 8684) differed from the Owen cytoplasm in mini-circle composition, but restriction enzyme analysis revealed no differences. The presence of the different minicircles was confirmed by Southern hybridization using minicircle-specific clones. All bands hybridized as predicted by gel electrophoresis except a band in the cms type C 8640, which migrated in a similar manner as the c.c.c. form of the a minicircle. This band hybridized only faintly to a minicircle a-specific probe and could be removed by treatment with nuclease S1. In contrast to the large mtDNA variation, restriction analysis of ctDNA detected little variation between cytoplasms. The molecular characterization of the new sources of cms supports the results of previous crossings. Two of the cytoplasms are not only of independent origin, but are also most likely functionally different and thus may be of value in future production of hybrid sugar beet varieties.     

3种小麦细胞质雄性不育系及其杂种线粒体DNA的RFLP分析

对细胞质分别来源于提莫菲维（Ｔ．ｔｉｍｏｔｈｅｅｖｉｉ）,粘果山羊草（Ａｅ．ｋｏｔｓｃｈｙｉ）,偏凸山羊草（Ａｅ．ｖｅｎｙｒｉｃｏｓａ）的３种普通小麦的雄性不育系,相应保持系和恢复系及其上的ｍｔＤＮＡ用１２个线粒体基因探针进行了ＲＦＬＰ分析,结果为：⑴Ｔ、Ｋ、Ｖ型不育系的ｍｔＤＮＡ在组织结构上存在显著差异;⑵Ｔ、Ｋ、Ｖ不育系的ｍｔＤＮＡ与共同的保持系间显著不同,失测ｍｔＤＮＡ与小麦ｃｍｓ有关;⑶在     

On the mechanism of cytoplasmic male sterility in the 447 line of Vicia faba

The trait of cytoplasmic male sterility, expressed in plants bearing the 447 cytoplasm of Vicia faba, is uniquely and positively correlated with the presence of a linear double-stranded RNA molecule (dsRNA) 16.7 kb in size. Restriction enzyme digestion profiles of mitochondrial DNA isolated from fertile and cytoplasmic malesterile (CMS) lines do show a limited number of specific differences in fragment intensities and mobilities. However, mitochondria isolated from the progeny of the cross CMS × Restorer line contain DNA with an identical restriction profile as the male-sterile parent: moreover, subsequent generations are completely and permanently fertile, even upon segregation of the nuclear restoration gene. Southern hybridizations, using cDNA clones as probes, reveal homology between the CMS-associated dsRNA and the nuclear genome of both sterile and fertile lines. The regions cloned, representing approximately 22% of the total dsRNA sequence, show no homology to organelle DNA. We have not been able to stably transmit the dsRNA to fertile lines of V. faba or any other plant species, using a variety of standard virological techniques.     

Transfer of the Brassica tournefortii cytoplasm to B. napus for the production of cytoplasmic male sterile B. napus

The donor-recipient fusion method was used to combine the cytoplasm of Brassica lournefortii with the nucleus of B. napus for the production of cytoplasmic male sterile (CMS) plants. X-ray-irradiated mesophyll protoplasts of B. tournefortii were fused with iodoacetamide (lOA)-inactivated hypocotye protoplasts of B. napus . Selective conditions of IOA concentrations and X-ray doses were determined, which resulted in recovery of fusion products and inhibition of further growth of unfused parental cells. In total, 54 plants were obtained from different fusion experiments, of which 25 were verified as cybrids or partial hybrids. Mitochondrial DNA (mtDNA) analyses using 5 mitochondrial gene probes revealed that 20 of the 25 fusion-derived plants had mtDNA either identical, or with varying degrees of similarity, to B. lournefortii . These plants were classified into four groups on the basis of pollen viability and number. Seven plants were categorised as male sterile since they did not produce pollen or had non-viable pollen. Of the male sterile plants, five had a mtDNA pattern identical to B. tournefortii and a nuclear DNA content corresponding to B. napus . The nuclear-mito-chondrial constitution of these plants thus indicates that the combination of B. tournefortii cytoplasm with the B. napus nucleus results in CMS. Furthermore, mtDNA analysis of the two additional male sterile plants which displayed a rearranged mtDNA, revealed that the only mtDNA similarity shared among all male sterile plants was specific for B. tournefortii atp6 pattern. This indicates that the atp6 region of B. tournefortii may be involved in the expression of CMS.     

Expression of ogu cytoplasmic male sterility in cybrids of Brassica napus

Summary A light and electron microscopic investigation revealed that ogu cytoplasmic male sterility (CMS) in cybrids of Brassica napus is primarily a deficiency of the tapetum and clearly time and site specific. Three patterns of ogu CMS were found, and specific conclusions drawn. First, the partially male fertile cybrid 23 was highly variable. It sometimes produced heterogeneous stamens with an endothecium formed exclusively around the fertile locules, thus delineating each microsporangium as a functional unit. The second type, including cybrids 27, 58 and 85, on the contrary, was stable and completely male sterile. In the four locules of normal length, microspores were observed to die at the vacuolate polarized stage while the tapetum disappeared prematurely through excessive vacuolization by the end of meiosis followed by a rapid autolysis during the tetrad or early free microspore stage. The subepidermal layer of the locule wall failed to form characteristic thickenings. The male-sterile stamens were completely indehiscent. At the time of anthesis they contained only collapsed empty exines adhering to each other. These cybrids, 27, 58 and 85, were closest to the ogu CMS trait of radish and seemed to be the best suited for further use in plant breeding. The third pattern was found in cybrids 77 and 118, which besides showing abortion of the microsporangia also showed a feminization of the stamens. We suggest that this feminization might be due to an alloplasmic situation associating Brassica napus nuclear genes with the mitochondrial DNA of radish.     

The effects of cytoplasmic male sterility on cytonuclear disequilibria in hybrid zones

Male sterility is studied in hybrid zones by different measures of cytonuclear disequilibria, D, D₁, D₂, and D₃. Of particular interest are the dynamics of disequilibria as the system evolves to equilibrium. Our first model, the hybrid swarm model, yields equilibrium results identical to those observed in a model with random mating. In our second model of a hybrid zone, predictions of the sign pattern of disequilibrium values can be made based on migration values. A characteristic sign pattern may help to distinguish cytoplasmic male sterility (CMS) from other mechanisms of selection. Our simple CMS model with migration is successfully fit to cytonuclear data on a hybrid population of cottonwoods.     

Mitochondrial gene expression and cytoplasmic male sterility in sorghum

Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.Analysis of native mitochondrial DNA by agarose gel electrophoresis revealed the presence of two plasmid-like DNA species of molecular weight 5.3 and 5.7 kb in the cytoplasmic male sterile lines Kafir nucleus in cytoplasm IS1112 and Yellow Feterita nucleus in M35-1 cytoplasm. Thus there is a positive correlation between the synthesis of the 82 000 mol. wt. polypeptide and the presence of the additional DNA species.     

高等植物胞质雄性不育及育性恢复的分子生物学研究进展

综述了几种植物胞质雄性不育的分子机理研究进展,着重介绍了与细胞质雄性不育相关的线粒体连锁位点的分析及育性恢复的几种假说,并对今后的研究进行了讨论。     

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.