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     

Phylogenetic relationships among fertile and petaloid male-sterile accessions of carrot, Daucus carota L.

 Mitochondrial (mt) DNA variation for six petaloid cytoplasmic male-sterile (CMS) and three fertile maintainer lines of carrot was assessed to establish genetic relationships. Total DNA was digested with restriction enzymes and probed with six homologous mtDNA cosmid probes. The six CMS accessions derived from wild carrot, four from Guelph, Ontario, one from Orleans, Massachusetts, and one from Madison, Wisconsin, were more closely related with each other (F=0.91) than with fertile maintainer lines derived from cultivated germplasm (F=0.62). The fertile maintainer lines were likewise found to be more similar to each other (F=0.78) than to the sterile lines. Three sterile lines, originating from wild carrot populations within 1 km of each other in Guelph, Ontario, were most closely related (F=0.96). The high degree of similarity among the six petaloid CMS lines which originated from individual wild carrot plants, some from geographically diverse regions, suggests that the cytoplasm responsible for this trait was imported to, or else evolved, only once in North America. Received: 1 December 1997 / Accepted: 12 December 1997     

A fertile revertant from petaloid cytoplasmic male-sterile carrot has a rearranged mitochondrial genome

 A spontaneously derived fertile plant was recovered from a petaloid cytoplasmic male-sterile (CMS) carrot inbred line. Genetic analysis indicated a single nuclear gene was responsible for the restoration to fertility. Within a family segregating for the nuclear restorer in combination with the sterility-inducing cytoplasm, fertile plants were recovered that could not restore fertility when crossed to sterile genotypes. Genetic analysis indicated cytoplasmic reversion for fertility, and Southern analysis, comparing mtDNA organization of the fertile revertant and its CMS progenitor, identified mitochondrial genome rearrangements. Hybridization of cosmids representing a 108-kb subgenomic circle of the sterile line to DNA of a fertile maintainer and fertile revertant lines indicated a similar mtDNA organization for these genotypes that was distinct from that of the sterile line. Six restriction fragments totalling 43.2 kb were common to the fertile maintainer and revertant and absent in the sterile; other restriction fragments totalling 38.2 kb were present only for the sterile line. Unique fragments of low stoichiometry, two for the fertile maintainer and three for the revertant, distinguished these lines. The reversion to fertility in the sterile line could have resulted from the amplification of a mitochondrial submolar genome highly homologous to that found in the fertile maintainer line. Received: 4 October 1997/Accepted: 12 December 1997     

Mitochondrial and total DNA RAPD patterns can distinguish restorers of CMS lines in sorghum

 Seven sorghum restorer lines that differentially restore (or maintain) the A1 and A2 cytoplasmic male-sterile (CMS) cytoplasms were studied by RFLP analyses of their mtDNAs and RAPD analyses of their mitochondrial DNA (mtDNA) and total DNA to understand nuclear mitochondrial combinations that are present in these lines. RFLP data from 11 mitochondrial gene probes were inadequate to classify these seven lines. However, the analysis of RAPD profiles of total DNA could distinguish these lines on the basis of their ability to restore completely or partially the fertility in the A1/A2 CMS cytoplasms. Interestingly, RAPD profiles of mtDNAs of these lines also followed the same pattern as that of the total DNA. These results indicate that the different restorer lines possess specific nuclear-cytoplasm combinations. Further, the results also show that the RAPD technique can be used to identify markers for different cytoplasms used in CMS. Received: 26 August 1997 / Accepted: 9 October 1997     

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.     

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     

Organizational differences between cytoplasmic male sterile and male fertile Brassica mitochondrial genomes are confined to a single transposed locus.

Comparison of the physical maps of male fertile (cam) and male sterile (pol) mitochondrial genomes of Brassica napus indicates that structural differences between the two mtDNAs are confined to a region immediately upstream of the atp6 gene. Relative to cam mtDNA, pol mtDNA possesses a 4.5 kb segment at this locus that includes a chimeric gene that is cotranscribed with atp6 and lacks an approximately 1kb region located upstream of the cam atp6 gene. The 4.5 kb pol segment is present and similarly organized in the mitochondrial genome of the common nap B.napus cytoplasm; however, the nap and pol DNA regions flanking this segment are different and the nap sequences are not expressed. The 4.5 kb CMS-associated pol segment has thus apparently undergone transposition during the evolution of the nap and pol cytoplasms and has been lost in the cam genome subsequent to the pol-cam divergence. This 4.5 kb segment comprises the single DNA region that is expressed differently in fertile, pol CMS and fertility restored pol cytoplasm plants. The finding that this locus is part of the single mtDNA region organized differently in the fertile and male sterile mitochondrial genomes provides strong support for the view that it specifies the pol CMS trait.     

Development of SCAR markers for early identification of cytoplasmic male sterility genotype in chili pepper (Capsicum annuum L.)

We previously used Southern blot analysis to detect restriction-length polymorphisms between male fertile and cytoplasmic male sterile (CMS) cytoplasms at the coxII and atp6 loci of the mtDNA of Capsicum annuum L. Two copies of atp6 were found in each male fertile and CMS pepper lines. Interestingly, one of the copies of atp6 in CMS pepper was a 3'-truncated pseudogene. The open reading frame of the coxII gene was the same in the fertile (N-) and CMS (S-) lines. However, the nucleotide sequence in the S-cytoplasm diverged from that in the N-cytoplasm 41 bp downstream of the stop codon. To develop CMS-specific sequence-characterized amplified region (SCAR) markers, inverse PCR was performed to characterize the nucleotide sequences of the 5' and 3' flanking regions of mitochondrial atp6 and coxII from the cytoplasms of male fertile (N-) and CMS (S-) pepper plants. Based on these data, two CMS-specific SCAR markers, 607 and 708 bp long, were developed to distinguish N-cytoplasm from S-cytoplasm by PCR. The CMS-specific PCR bands were verified for 20 cultivars containing either N- or S-cytoplasm. PCR amplification of CMS-specific mitochondrial nucleotide sequences will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F1 seed lots. The strategy used in this report for identifying CMS-specific markers could be adopted for many other crops where CMS is used for F1 seed production.     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

Structural and Expressional Variations of the Mitochondrial Genome Conferring the Wild Abortive Type of Cytoplasmic Male Sterility in Rice

The so-called ＂wild abortive＂ （WA） type of cytoplasmic male sterility （CMS） derived from a wild rice species Oryza rufipogon has been extensively used for hybrid rice breeding. However, extensive analysis of the structure of the related mitochondrial genome has not been reported, and the CMS-associated gene（s） remain unknown. In this study, we exploited a mitochondrial genome-wide strategy to examine the structural and expressional variations in the mitochondrial genome conferring the CMS. The entire mitochondriai genomes of a CMS-WA line and two normal fertile rice lines were amplified by Long-polymerase chain reaction into tilling fragments of up to 15.2 kb. Restriction and DNA blotting analyses of these fragments revealed that structural variations occurred in several regions in the WA mitochondrial genome, as compared to those of the fertile lines. All of the amplified fragments covering the entire mitochondrial genome were used as RNA blot probes to examine the mitochondriai expression profile among the CMS-WA and fertile lines. As a result, only two mRNAs were found to be differentially expressed between the CMS-WA and the fertile lines, which were detected by a probe containing the nad5 and orf153 genes and the other having the ribosomal protein gene rpl5, respectively. These mRNAs are proposed to be the candidates for further identification and functional studies of the CMS gene.     

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.     

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.     

Stoichiometric differences in DNA molecules containing the atpA gene suggest mechanisms for the generation of mitochondrial genome diversity in maize

Four genomic arrangements of the maize mitochondrial atpA gene (encoding the α subunit of the F₁ ATPase), have been characterized. Most N (fertile) and S (male-sterile) cytoplasms contain two atpA arrangements of equal abundance. Prolonged exposure of blots of maize mitochondrial DNA probed with atpA-specific sequences show that cytoplasms previously reported to lack one of the atpA arrangements do contain the second arrangement but at low levels. Similarly, restriction fragments containing the atpA gene previously thought unique to male-sterile S and T cytoplasms are present in low abundance in fertile cytoplasms. These observations suggest that fertile and male-sterile cytoplasms of maize may be more closely related than previously thought, and suggest possible mechanisms to explain the observed mitochondrial genome diversity.