Molecular characterization of mitochondrial DNA of different subtypes of male-sterile cytoplasms of the sugar beet Beta vulgaris L.

Summary Mitochondrial (mt) DNA from eight cytoplasmic male-sterile (cms) lines of sugar beet from different breeding stations was investigated by restriction fragment analysis and Southern hybridization. All cms lines showed similar but not identical restriction and hybridization signal patterns, readily distinguishable from those of fertile (N) cytoplasm. Digestion of the mtDNA with BamHI, EcoRI, SalI, and XhoI revealed distinct differences between the sterile lines, and six subtypes of the S cytoplasm could be distinguished. Differences between the sterile lines were confirmed by hybridization with a gene probe revealing minor, line-specific hybridization signals. The data presented provide evidence for the existence of considerable variation within the only commercially used source of cms in the sugar beet, the Owen's type of cytoplasm.     

Cytoplasmic male sterility in hybrids of sterile wild beet (Beta vulgaris ssp. maritima) and O-type fertile sugar beet (Beta vulgaris L.): molecular analysis of mitochondrial and nuclear genomes

The organization of the mitochondrial genome of B3, B4 and B5generations of hybrids created by backcrossing sterile wild beet Betamaritima with a fertile O-type sugar beet line was studied usingrestriction fragment length polymorphism (RFLP) analysis. Random amplifiedpolymorphic DNA (RAPD) analysis was used to study restoration of the fertile(O-type) sugar beet genotype in hybrids after multiple backcrossings.Restriction of mtDNAs from the cytoplasm of B. maritimaandhybrids revealed BamHI, EcoRI andXhoI restriction patterns different from those for sterileand fertile sugar beet lines. The most conspicuous feature of our accession ofsterile wild beet mtDNA was the absence of the 10.7-kbEcoRI fragment detected in the cytoplasm of S-type sterileB. maritima and sugar beet. The hybridization of digestedmtDNAs with coxII, atpA andatp6 homologous probes revealed alterations within thesegene loci that distinguished wild beet and hybrids from sugar beets.Characteristic hybridization profiles for the wild beet and B3, B4 and B5hybrids were observed for all probes regardless of the restrictase used todigest mtDNA. Notable changes in atpA andatp6 genes resulted when probes that comprised the5flanking sequences of these genes and a small part of the coding sequences wereused. RFLP analysis of the sterile B. maritimamitochondrial genome further supported the unique character of this source ofwild beet sterility. The genotypic differences between hybrids and parentalaccessions were determined by scoring PCR-RAPD reaction products for nineselected primers. The diversity of the B. maritimagenotyperesulted in a lower genetic similarity index in comparison with hybrids,sterileand fertile lines of sugar beet. The dendrogram obtained after cluster analysisdistinguished hybrids as a group that differed from wild beet and themaintainersugar beet line used for backcrossing. These results may indicate incompleterestoration of the fertile sugar beet genotype in hybrids.     

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.     

Cytoplasmic male sterility inBeta is associated with structural rearrangements of the mitochondrial DNA and is not due to interspecific organelle transfer

Summary Chloroplast (ct) and mitochondrial (mt) DNAs from four cytoplasmic male sterile (cms) and 22 normal fertile sugar beet lines and accessions of wild beets from the genusBeta have been compared with restriction analyses and Southern hybridizations. We have used restriction analyses of ctDNA as a phylogenetic marker to confirm the taxonomic relationships between the different cytoplasms. According to the ctDNA data, all four cms cytoplasms belong to the same taxonomic section,Beta. Restriction patterns of ct and mtDNA from fertile accessions produced analogous trees of similarity and showed a close correlation between the organellar DNA diversity and the accepted taxonomic classification of the species studied. However, the mtDNA restriction profiles of the four cms types differed dramatically from each other and from those of all fertile accessions from the genus. No indication of cytoplasmic introgression was found in any of the four investigated cms types. Southern hybridization to mtDNA revealed variant genomic arrangements in the different fertile and cms cytoplasms, indicating that rearrangement of the mitochondrial genome is a common denominator to the different cms systems inBeta. It may, indeed, be a common property to spontaneously occurring cms in all or most species.     

DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.     

The use of mitochondrial DNA polymorphism in the classification of individual onion plants by cytoplasmic genotypes

Summary Individual plants of a Japanese onion variety Sapporo-ki, which is characterized by the occasional occurrence of male-sterile plants, have been investigated for mitochondrial (mt) DNA polymorphism. Male-fertile and the Jones' cytoplasmic male-sterile (CMS) onions were also included for comparison. Southern blot hybridization with rrn26, cox-I, cox-II, cob, atpA and atp9 genes as probes revealed the two classes of mtDNA variation within a population of Sapporo-ki: Out of the 41 plants examined 19 contained mtDNA typical of malefertile plants, and 22 individuals contained mtDNA typical of the Jones' CMS genotype. Our results thus indcate that the use of the mitochondrial gene probes may greatly facilitate the classification of individual plants by cytoplasmic genotypes.     

Heterogeneity of Maize Cytoplasmic Genomes among Male-Sterile Cytoplasms

Maize mitochondrial and chloroplast DNA's were prepared from normal (fertile) lines or single crosses and from members of the T, C, and S groups of male-sterile cytoplasms. Restriction endonucleases HindIII, BamI, EcoRI, and SalI were used to restrict the DNA, and the resultant fragments were electrophoresed in agarose gels. The results show that the N (fertile), T, C, and S cytoplasms each contained distinct mitochondrial DNA (mtDNA). These distinctive patterns were unaffected by nuclear genotype. No evidence of paternal inheritance of mtDNA was observed. Chloroplast DNA (ctDNA) from the N, C, and T cytoplasms was indistinguishable by HindIII, SalI, or EcoRI endonuclease digestion. The S cytoplasm ctDNA, however, was slightly different from that of other cytoplasms, as indicated by a slight displacement of one band in HindIII digests. The molecular weight of maize ctDNA was estimated to be as high as 88 x 10⁶. Estimates of the minimum molecular weight of maize mtDNA ranged from 116–131 x 10⁶, but the patterns were to complex for an unambiguous determination. Based on HindIII data, a comparison of the molecular weight of mtDNA bands common to the N, T. C, and S cytoplasms suggests that C cytoplasm most closely resembles N cytoplasm. The T and S sources are more divergent from the C and N cytoplasms. These results indicate a possible gradation of relatedness among male-sterile cytoplasms. The marked variation in mtDNA, with apparently less variation in ctDNA, represents circumstantial, but compelling, evidence that mtDNA may be involved in the male sterility and disease susceptibility traits in maize.     

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.     

Variation in mitochondrial translation products in fertile and cytoplasmic male-sterile sugar beets

Summary Intact and functional mitochondria were isolated from sugar beet plants (Beta vulgaris L.) containing normal fertile (F) or cytoplasmic male-sterile (S₁–S₄) cytoplasms. Incorporation of ³⁵S-methionine by mitochondria isolated from both roots and leaves showed approximately 20 major and ten minor translation products. Comparison of the polypeptide synthesis patterns produced by leaf mitochondria from fertile plants of three different species within the genus Beta revealed several taxonomically related differences. Contrary to this, the patterns of polypeptides synthesized by mitochondria from roots and leaves of sugar beet plants containing the F and S₁–S₄ cytoplasms were very similar; in the S₁ and S₂ cytoplasms no qualitative, and only a few quantitative, differences from the F cytoplasm were observed. Thus, in these cases, cytoplasmic male sterility in sugar beet is not correlated with the constitutive expression of variant polypeptides. In the S₃ cytoplasm, however, an additional 6 kDa polypeptide was synthesized and in the S₄ cytoplasm an additional 10 kDa polypeptide was observed when compared with the F cytoplasm. The expression of cytoplasmic male sterility in sugar beet may be associated with these variant polypeptides. The mitochondrial polypeptides synthesized were identical in plants with different nuclear backgrounds but with identical S₁ cytoplasms. Mitochondria from plants with variants of the S₄ cytoplasm in the same nuclear genotype also showed identical patterns of polypeptide synthesis, including the synthesis of the 10 kDa S₄-specific polypeptide. Pulse-chase experiments did not affect the synthesis of this polypeptide.     

Molecular characterization of a new type of cytoplasmic male sterile sugar beet

A line (named Cl) of cytoplasmic sterility of sugar beet whose cytoplasm derived from Betacicla Turkey was obtained by interspecific hybrid. Its cytoplasm and a spontaneous male sterile cytoplasm from wild beet Beta maritima (named M) were compared with that of Owen's sterile line (S-cms) and a common maintainer of them named N was used as control. RFLP and RAPD methods were mainly used in our experiments. The restriction fragment patterns of mtDNAs were found to be likely but for a few of specific low-lighted electrophoresis bands in Cl. The results of Southern hybridization of six heterogeneous mitochondrial genes as probes to digests of mtDNAs by six restriction enzymes showed to be analogous between S and M lines. But the Cl mtDNA was sorted out by hybridization of atpA probe. Difference of low-molecular-weight mitochondrial DNAs was found among the three sterile lines. Three RNA molecules weighing about 4.2kb stably existed in Cl mitochondria. Our results of RAPD also supported that the Cl cytoplas     

Rearrangements in sugar beet mitochondrial DNA induced by cell suspension,callus cultures and regeneration

Structural alterations in mitochondrial DNAs (mtDNAs) from a plant of a sterile sugar beet line, callus derived from it, suspension-cultured cells and plants regenerated from the callus were studied. BamHI restriction analysis revealed that structural alterations between the mtDNAs of the callus and the control plant had occurred. Multiple rearrangements were also demonstrated in the mtDNA from the suspension culture, of which some were similar to those appearing in the callus, and others had arisen de novo. Rearrangements were also identified by means of blot hybridization of BamHI-digested mtDNA from suspension-cultured cells with the genes encoding subunit II of cytochrome oxidase (cox II) and subunit 1 of NADH-dehydrogenase (Nd1). No alterations were observed in the mitochondrial genome of the callus and regenerants. The location of the genes for the -subunit of F1-ATPase (atpA) and apocytochrome b (cob) in the mtDNA remained unchanged.Our salient finding was of a plant with an altered mitochondrial genome as judged by EcoRI and BamHI restriction analysis. This exceptional plant had retained the sterile phenotype like all of the other regenerants and the parent. The set of plasmid-like molecules of mtDNA remained the same as that in the control plant and in all of the regenerants, callus and suspension-cultured cells. The only type of plasmid-like molecule found in all of the DNAs was the 1.6-kbp minicircle, which is a feature of sterile cytoplasms. These structural changes in mtDNA were obviously a consequence of somaclonal variation during the in vitro cultivation of the sugar beet cells.     

Different CMS sources found in Beta vulgaris ssp maritima: mitochondrial variability in wild populations revealed by a rapid screening procedure

Summary Mitochondrial DNA (mtDNA) variation in natural Beta maritima populations has been characterized by way of Southern blot hybridizations of total DNA using non-radioactive probes and chemiluminescent detection. It was found that the previously described N (normal) mitochondrial type could be subdivided into three subtypes. A new mitochondrial genotype (type R) was distinguished in addition to the previously described type S. Both are male-sterile cytoplasms and can produce a. segregation of sexual phenotypes in their progenies depending on the nuclear background. The populations contained at least two to four different mitochondrial genotypes.     

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.     

RAPD analysis of isolated mitochondrial DNA reveals heterogeneity in elite wild abortive (WA) cytoplasm in rice

 RAPD profiles were generated using mitochondrial DNA (mtDNA) isolated from two cytoplasmic male-sterile lines, two restorer lines and four maintainer lines of rice. Of the 40 primers tested, 25 generated consistent and easily scoreable patterns that were used for the computation of pairwise similarities as well as UPGMA analyses. The different lines of rice, including lines IR58025A and IR62829A that contained the same wild abortive (WA) cytoplasm, were distinguishable on the basis of RAPD profiles. These latter two lines were not distinguishable from each other by mtDNA RFLP analyses with as many as 16 mtDNA probes. The data illustrate the utility of the RAPD technique as a powerful tool for distinguishing different cytoplasms that by other techniques appear to be similar. To our knowledge, this is the first report wherein RAPD profiles obtained with isolated mtDNA templates enable the distinction between two or more types of cytoplasms in rice. Received: 1 April 1997 / Accepted: 2 June 1997     

Mapping of a chloroplast RFLP marker associated with the CMS cytoplasm of sugar beet (Beta vulgaris)

The Owen cytoplasm of male-sterile sugar beet is associated with several alterations of mitochondrial DNA and one additional HindIII site of chloroplast DNA. The region of this HindIII site has been cloned and sequenced. The site maps in a small reading frame (orf32) close to the ycf7 (orf31) gene in the petG-psbE region of chloroplast DNA. Possible functional implications of the results are discussed. The chloroplast RFLP marker described could be useful for studies on chloroplast-mitochondrial interactions, CMS of sugar beet, and the origin of the Owen cytoplasm.