Cloning of mtDNA fragments homologous to mitochondrial S2 plasmid-like DNA in maize

Summary Mitochondria from S-type cytoplasmic male-sterile maize contain two small DNA species, S1 and S2, which are absent from other fertile and male-sterile cytoplasms. These species have been cloned in plasmid pBR322 by the homopolymer extension method. Probes made with these recombinant plasmids have been used to establish the homology between high molecular weight mitochondrial DNAs of fertile and male-sterile cytoplasms, and small mitochondrial plasmid-like molecules. Hybridization and mapping data show that S2 DNA copies are homologuous with sequences of the normal mitochondrial genome. A comparison of physical maps of different isolated mtDNA fragments indicates a heterogeneous arrangement of S2 sequences in the mtDNA population of normal fertile maize cytoplasm. The origin of this heterogeneity is discussed.     

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.     

Variations in mitochondrial DNA organisation between normal and male-sterile cytoplasms of maize.

Mitochondrial DNA from male-sterile lines of maize carrying S cytoplasm contains two small DNA species which are absent from N (fertile) and other male-sterile cytoplasms. Portions of these species have been purified and amplified by constructing recombinant plasmids in vitro. Probes made with these plasmids have been used to demonstrate; i) a homologous region in the N mitochondrial genome, which may indicate the origin of the S specific DNA species. ii) two other DNA species present in low amounts in S cytoplasm only. iii) the absence of strong homology to the S specific DNA species in mitochondria from C and T male-sterile cytoplasms.     

Cytoplasmic male sterility in sorghum: Organization and expression of mitochondrial genes in Indian CMS cytoplasms

Cytoplasmic male sterility in sorghum has been reported in a number of varieties originating in different geographical regions (India, Africa and America). We have attempted to characterize three male sterile cytoplasms of Indian origin designated as Maldandi, Guntur and Vizianagaram by studying restriction fragment length polymorphisms (RFLPs) and expression patterns of 14 mitochondrial genes. Our results indicate that the cytoplasms, classified tentatively as Indian A₄ types, are distinct from the American A₄ and A₁ types. Although they are identical to each other with respect to the location of 10 of the mitochondrial genes selected, they can be distinguished from each other on the basis of RFLPs inatp6, atp9 andrrn18. Further the three cytoplasms differ from their maintainers in the location ofnad3, rpsl2 andatpA. Differences are also observed in the pattern of expression ofatpA between all the sterile lines and their respective maintainers.     

Classification of normal and male-sterile cytoplasms in maize. I. Electrophoretic analysis of variation in mitochondrially synthesized proteins

Male-sterile cytoplasms of maize have previously been classified into three groups (T, S and C) according to their fertility ratings in various inbred backgrounds. In earlier studies, mitochondria from three male-sterile cytoplasms, representing each of these three groups, have been found to synthesize characteristic variant polypeptides that distinguish them from each other and from those of normal (N) cytoplasm. In order to determine the extent of cytoplasmic variation, we have now analyzed the translation products of mitochondria from 28 additional cytoplasmic sources. The results show that on this basis 18 of the cytoplasms are identical to the USDA (S) cytoplasm, three are identical to the Texas (T) cytoplasm and two are identical to the C cytoplasm. The five remaining cytoplasms are indistinguishable from normal, male-fertile (N) cytoplasm. Our classification of the cytoplasms is in general agreement with those based on fertility restoration. However, of three cytoplasms that have previously remained unclassified, two (B and D) have now been assigned to the S group and one (LF) to the N group. No heterogeneity in mitochondrial translation products was detected within the normal or any of the three male-sterile groups. The usefulness of the analysis of mitochondrial translation products as a method for classifying normal and male-sterile cytoplasms is discussed.     

A protein specific to mitochondria from S-type male-sterile cytoplasm of maize is encoded by an episomal DNA

Mitochondria of S-type cytoplasmic male sterile maize contain two linear double-stranded DNA molecules, S1 and S2. Two open reading frames (ORF1 and ORF2) are present in S2 DNA. Fragments from ORF1 were inserted into plasmids to achieve expression in Escherichia coli. Cells transformed with recombinant plasmids produced mRNA which hybridized with ORF1 and corresponding polypeptides were synthesized by in vivo and in vitro systems. Antiserum against a lacZ/S2 fusion protein precipitated the anticipated polypeptides from transformed E. coli cells and was therefore used to detect homologous peptide sequences in protein preparations of mitochondria from different maize cytoplasms. The antiserum detected a protein of 125 000 M_r present in mitochondria from male sterile B73S but absent from the fertile B73N cytoplasm.     

Diversity among male-sterility-inducing and male-fertile cytoplasms of onion

Hybrid-onion (Allium cepa) seed is produced using systems of cytoplasmic-genic male sterility (CMS). Two different sources of CMS (S and T cytoplasms) have been genetically characterized. Testcrosses of N-cytoplasmic maintaining and restoring genotypes to S and T cytoplasmic lines demonstrated that different alleles, or loci, restore male fertility for these two male-sterile cytoplasms. Other sources of CMS have been used or reported in Europe, Japan and India, and their relationships to S and T cytoplasms are not clear. Restriction fragment length polymorphisms were identified in the organellar genomes among commercially used male-sterile cytoplasms from Holland, Japan and India, and were compared to S and T cytoplasms. Mitochondrial DNA diversity among 58 non-S-cytoplasmic open-pollinated onion populations was also assessed. All five putative CMS lines selected from the Indian population Nasik White Globe were identical to S cytoplasm for all polymorphisms in the chloroplast genome, and always possessed the same-sized mitochondrial fragments as S cytoplasm. T cytoplasm, the male-sterile cytoplasm used to produce the Dutch hybrid Hygro F₁, and two sources of CMS from Japan, were similar and showed numbers of mitochondrial polymorphisms similar to those observed among the 58 non-S-cytoplasmic open-pollinated populations. This research demonstrates that the same, or very similar, male-sterile cytoplasms have been independently isolated and exploited for hybrid-seed production in onion. Received: 27 October 1999 / Accepted: 12 February 2000     

Analyses of mitochondrial DNA structure and expression in three cytoplasmic male-sterile chicories originating from somatic hybridisation between fertile chicory and CMS sunflower protoplasts

Cytoplasmic male-sterile (CMS) chicories have been previously obtained by somatic hybridisation between fertile industrial chicory protoplasts and CMS sunflower protoplasts. In this study, we compared three different CMS chicory cybrids that originated from three different fusion events. The cybrids were backcrossed with different witloof chicories in order to transfer the three male-sterile cytoplasms from an industrial chicory nuclear environment to a witloof chicory nuclear context. Southern hybridisation, using different mitochondrial genes as probes, revealed that the three cybrid mitochondrial genomes were different and that they were stable throughout backcrossing generations regardless of the pollinator. However, pollinators were found to influence floral morphologies – with one being able to restore fertility – showing that nuclear context can affect the sterility of the cybrids. PCR and RFLP analyses revealed that the orf522 sequence, responsiblefor CMS in PET1 sunflower, was present in two out of the three cytoplasms studied, namely 411 and 523, but was absent from the other cytoplasm, 524. We thus concluded that orf522 is not responsible for CMS in the 524 cybrid. Although the orf522 gene is present in the 411 and 523 cytoplasms, it is probably not responsible for the sterile phenotype of these cybrids. Received: 3 June 1998 / Accepted: 30 April 1999     

Recombination events across the atpA-associated repeated sequences in the mitochondrial genomes of beets

 The mitochondrial atpA gene sequence of the normal fertile sugarbeet (cv ‘TK81-0’) exists in one full-length version and one truncated version, both of which are present in normal stoichiometry and have a 406-bp segment in common. The PCR approach as well as prolonged exposure of Southern blots indicates that the products of the recombination across the 406-bp repeat are present in substoichiometric amounts in the ‘TK81-0’ genome. Intriguingly, one of these substoichiometric sequence arrangements was revealed to be preferentially amplified in an evolutionary lineage that led to a cytoplasmic male-sterile variant [I-12CMS(2)] in wild beets. We also found the 406-bp repeat to be part of a 6.5-kb repeat in the mitochondrial genome of I-12CMS(2). This 6.5-kb duplication is likely to involve recombination between two sets of repeats (the above-mentioned 406-bp repeat and a 7-bp repeat) in an ancestral beet mitochondria. Received: 4 October 1997 / Accepted: 31 October 1997     

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.     

Morphological changes in homeotic cytoplasmic male-sterile carrots combined with fertile cytoplasm by asymmetrical cell fusion

 The cytoplasmic male-sterile (CMS) carrot (Daucus carota ssp. sativus) with the petaloid phenotype was asymmetrically fused with eight different fertile cytoplasms to convert the CMS to a fertile state. Restoration to the fertile phenotype was successful with an over 20% efficiency. Cybrids with brown anther sterile, incomplete petaloid sterile, or "combined flower" fused on the same axis were also observed. Restricted DNA fragment patterns revealed that the mitochondrial genome organizations of the cybrids were not identical to those of their parents but were of an intermediate type. Repeated cell fusion to introduce two different foreign cytoplasms into the CMS cytoplasm was effective for obtaining fertile plants. The role of mitochondrial factors which regulate flower organ morphogenesis was demonstrated. Received: 14 December 1998 / Revision received: 7 March 1999 · Accepted: 3 June 1999