A new type of cytoplasmic male sterility in rye (Secale cereale L.): analysis of mitochondrial DNA

Summary Mitochondrial (mt) DNA of a new type of rye cytoplasm (Gülzow, G) that induces cytoplasmic male sterility (CMS) was analyzed and compared with rye mtDNAs of different origins MtDNA of the G type was easily distinguishable from mtDNA of another CMS source, Pampa (P) type, and from mtDNA of fertile lines with respect to restriction fragment patterns and hybridization with mitochondrial genes. The results of the molecular analyses indicate a close, but not identical relationship between the mtDNA of the G type cytoplasm and that of cv Pluto.     

Cytoplasmic male sterility in rapeseed (Brassica napus L.)

Summary Restriction patterns of chloroplast (cp) and mitochondrial (mt) DNA in Brassica napus rapeseed reveal the alloplasmic nature of cytoplasmic male sterility in this crop. Both the Shiga and Bronowski systems probably exploit cytoplasmic diversity in B. napus cultivars arising from introgression of cytoplasm from the other rapeseed species, B. campestris. Nuclear genes specific to these systems do not cause sterility in maintainers (Bronowski and Isuzu-natane) because they have a campestris cytoplasm, but give rise to sterility in napus cytoplasms. In the course of hybridization to napus cultivars a line with the triazine resistant cytoplasm (a campestris cytoplasm) has undergone an alteration in the mt genome rendering its restriction pattern more similar than previously to that of napus. The alteration may be an inversion between 7.2 and 3.4 kb in length.     

Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny

Summary A circular supercoiled mitochondrial DNA plasmid P₁ (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P₂) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P₁ was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P₁ and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P₁ and P₂. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.     

Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.)

Summary The development of microspore mother cells (MMC) and tapetum in male-fertile and male-sterile anthers of Beta vulgaris L. was compared at the electron microscope level. These studies were complemented by morphometric analyses of mitochondria in both tissues through successive stages of microsporogenesis. The earliest irregularities in the ultrastructure of male-sterile anthers were noted within the tapetum at the tetrad stage. These disturbances were initially expressed by a slight reduction in mitochondrial size and the appearance of concentric configurations of endoplasmic reticulum. As development proceeded, a further decrease in mitochondrial size become more conspicuous and was accompanied by a reduction in ribosome population and a failure of the tapetum to produce Ubisch bodies. This failure to produce Ubisch bodies is reflected in the underdevelopment of sterile microspore exine.     

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

High-rate spontaneous reversion to cytoplasmic male sterility in sugar beet: a characterization of the mitochondrial genomes

Summary Among the fertile sugar beet lines with nuclear sterility maintenance genes, rf, in a homozygous recessive state, sublines capable of reverting spontaneously at a high rate to sterility were identified. Of 24 related fertile sublines studied, 6 were found to spontaneously revert to sterility with a frequency of about 19%. Genetic analysis confirmed the cytoplasmic nature of spontaneously arising sterility. Reversion to sterility in these sublines was accompanied by alterations in the mitochondrial genome structure: loss of the autonomously replicating minicircle c (1.3 kb) and changes in the restriction patterns of high-molecular-weight mitochondrial DNA (mtDNA). Southern hybridixation analysis with cloned minicircle c as a probe revealed no integration of this DNA molecule into the main mitochondrial and nuclear genomes of the revertants. Comparative BamHI and EcoRI restriction analysis of the mtDNA from the sterile revertants and fertile parental subline showed that the spontaneous reversion is accompanied by extensive genomic rearrangement. Southern blot analysis with cloned -subunit of F₁-ATPase (atpA) and cytochrome c oxidase subunit II (COX II) genes as probes indicated that the changes in mtDNA accompanying spontaneous reversion to sterility involved these regions. The mitochondrial genomes of the spontaneous revertants and the sterile analogue were shown to be identical.     

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.     

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.     

Genetic analyses supported by molecular methods provide evidence of a new genic (st1) and a new cytoplasmic (st2) male sterility in Allium schoenoprasum L.

Spontaneous mutations leading to male sterility have been described for many different crops and are of great importance to hybrid breeding, provided that their inheritance is resolved. This paper describes an efficient method to characterise male sterilities with respect to cytoplasmic factors that might be causally related to them. The differentiation of cytoplasmic (CMS) and genic (GMS) male sterility is achieved by a specific transfer of nuclear male sterility factors to different cytoplasm types which have previously been distinguished by means of RFLP analyses using mitochondrial gene probes. The nuclear sterility factors of Allium schoenoprasum used, st1 and st2, showed a monogenic recessive inheritance in their original cytoplasms. While st1 was expressed in four different cytoplasm types, st2 did not show itself in a cytoplasm type differing from the original. Therefore, the st1-sterility is a GMS, while a cytoplasmic factor is necessary for the occurrence of st2-sterility. This cytoplasmic factor was verified by a reciprocal cross, and the CMS system was completed by the selection of maintainer genotypes. Neither of these new sterilities were influenced by high temperatures or tetracycline. The benefits of a new CMS system to practical breeding and the advantages and disadvantages of the environmental influences on the expression of male sterility are discussed. Received: 24 November 1999 / Accepted: 3 December 1999     

Differences between,and possible origins of,the cytoplasms found in fertile and male-sterile onions (Allium cepa L.)

Summary The DNA of the organellar genomes of Allium cepa has been examined to detect restriction fragment length polymorphisms. Differences can be shown between both the chloroplastal and mitochondrial genomes of the N and cms-S cytoplasms in their restriction fragment profiles. Southern blot analysis of the mtDNA profiles using probes containing defined mitochondrial genes also detected polymorphisms. No differences can be shown between the organellar genomes of the N and cms-T onions by either of these techniques. These data indicate different origins for the two sterility-conferring cytoplasms, suggesting autoplasmic and alloplasmic origins for the cms-T and cms-S cytoplasms, respectively. No evidence of the presence of virus-like particles was found in any of the cytoplasms.     

TheBrassica mitochondrial DNA plasmid and large RNAs are not exclusively associated with cytoplasmic male sterility

Summary More than 100 differentBrassica nucleo-cytoplasmic combinations were analysed for the presence or absence of the 11.3 kb mitochondrial plasmid. Contrary to some previous reports, no close association exists between the presence of the plasmid and cytoplasmic male sterility. Some novel abundant RNAs which copurified withBrassica mitochondria are described.     

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.     

Inheritance and molecular mapping of two fertility-restoring loci for Honglian gametophytic cytoplasmic male sterility in rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

The Honglian cytoplasmic male sterility (cms-HL) system, a novel type of gametophytic CMS in indica rice, is being used for the large-scale commercial production of hybrid rice in China. However, the genetic basis of fertility restoration (Rf) in cms-HL remains unknown. Previous studies have shown that fertility restoration is controlled by a single locus located on chromosome 10, close to the loci Rf1 and Rf4, which respond to cms-BT and cms-WA, respectively. To determine if the Rf locus for cms-HL is different from these Rf loci and to establish fine-scale genetic and physical maps for map-based cloning of the Rf gene, high-resolution mapping of the Rf gene was carried out using RAPD and microsatellite markers in three BCF₁ populations. The results of the genetic linkage analysis indicated that two Rf loci respond to cms-HL, and that these are located in different regions of chromosome 10. One of these loci, Rf5 , co-segregates with the SSR marker RM3150, and is flanked by RM1108 and RM5373, which are 0.9 cM and 1.3 cM away, respectively. Another Rf locus, designated as Rf6(t), co-segregates with RM5373, and is flanked by RM6737 and SBD07 at genetic distances of 0.4 cM. The results also demonstrated these loci are distinct from Rf1 and Rf4. A 105-kb BAC clone covering the Rf6(t) locus was obtained from a rice BAC library. The sequence of a 66-kb segment spanning the Rf6(t) locus was determined by a BLASTX search in the genomic sequence database established for the cultivar 93-11.Communicated by R. Hagemann     

A new cytoplasmic male sterile genotype in the sugar beet Beta vulgaris L.: a molecular analysis

Summary Mitochondrial DNA (mtDNA) from fertile (N) and possibly new cytoplasmic male sterile (CMS) genotypes was studied in the sugar beet Beta vulgaris L. It was found by restriction endonuclease analysis that BMC-CMS, a cytoplasm that was derived from the wild beet Beta maritima, contained a unique type of mtDNA which is distinguishable from both the N and S-CMS, the only other CMS genotype that is currently availabe in B. vulgaris L. The organization of three genes: coxI, coxII and cob, was analyzed by hybridization with heterologous probes from maize. These genes have a similar structure in N and BMC-CMS that is different from S-CMS. It is concluded that BMC-CMS is a novel CMS genotype in the sugar beet.     

Cytoplasmic male sterility in Vicia faba L.

Summary In many higher plants, nucleo-cytoplasmic interactions lead to pollen abortion. In Vicia faba, cytoplasmic male sterility is unstable as the cytoplasm appears to shift from a sterile to a fertile state. In this report, five flower phenotypes are defined but the study is focussed on the progenies obtained from intermediate, semi-sterile plants with the same homozygous nuclear constitution during five successive generations. The results could be interpreted by quantitative modifications of at least four different kinds of cytoplasmic determinants.     

Linkage between an isozyme marker and a restorer gene in radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Summary Co-segregation studies of isozyme markers and male fertility restoration showed that a restorer gene from radish was introduced into rapeseed along with an isozyme marker (Pgi-2). The radish chromosome segment carrying these genes was introgressed into rapeseed through homoeologous recombination, substituting for some of the rapeseed alleles. By crossing heterozygous restored plants to male-sterile lines and to maintainers, tight linkage was found between the restorer gene and the marker. The recombination fraction was estimated at 0.25 ± 0.02%. Although few restored plants lacked the radish isozyme marker, it was still possible to distinguish male-fertile from male-sterile plants by their PGI-2 patterns. Furthermore, homozygous and heterozygous restored plants could be separated by specific PGI-2 phenotypes. Thus, the Pgi-2 marker is now currently used in restorer breeding programs.     

Structural studies of microsporogenesis in fertile and male-sterile onions (Allium cepa L.) containing the cms-S cytoplasm

Summary The structure of anther tissues has been studied during microsporogenesis in male-sterile and -fertile onions. Three types of abnormal tapetal behaviour have been observed within the single line II/3ms containing the cms-S cytoplasm: type 1, the premature breakdown of the tapetum at the tetrad stage, type 2, the hypertrophy of the tapetum after the diad stage followed by its premature autolysis and, type 3, in which the tapetum remains in good condition but for an abnormally long period of time. Tapetal autolysis proceeds in the same manner in both male-steriles and -fertiles with only the stage at which it occurs differing between the types of plants. Mitochondria were prominent in the tapetal tissue of all onion types throughout all stages of microsporogenesis and were still visible during the last stages of tapetal autolysis. In a detailed study of type 2 behaviour, no differences in mitochondrial volumes were found until the tapetum hypertrophied.