Methylation of mitochondrial DNA in carrot (Daucus carota L.)

The methylation status of carrot (Daucus carota L.) mitochondrial DNA (mtDNA) was studied using isoschizomeric restriction enzymes MspI/HpaII (CCGG) and MvaI/EcoRII [CC(A/T)GG]. Southern hybridisations with probes for mitochondrial genes coxII and atpA were performed. MtDNAs isolated from non-embryogenic cell suspensions and roots were analysed. No differences were found using MspI/HpaII but after digesting the mtDNA with MvaI and EcoRII, some qualitative and quantitative differences between the restriction patterns appeared. Distinction was also revealed after Southern hybridisation with the coxII probe. These data indicate that the mtDNA of carrot is methylated in CNG trinucleotides and unmethylated in CG dinucleotides in CCGG sequences. The results were reproducible for cell suspensions of various genotypes and even cultivars but the extent of methylation was different in the root. The possible role of methylation in the mitochondrial genome of higher plants is discussed. Received: 16 April 1997 / Revision received: 4 July 1997 / Accepted: 30 July 1997     

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     

Inheritance of chloroplast and mitochondrial DNA in alloplasmic forms of the genus Daucus

The inheritance of mitochondrial (mt) and chloroplast (ct) DNA in the progeny from interspecific crosses between the cultivated carrot (Daucus carota sativus) and wild forms of the genus Daucus was investigated by analysis of mt and ct RFLPs in single plants of the parental and filial generations. We observed a strict maternal inheritance of the organellar DNAs in all interspecific crosses examined. Previous studies on putative F₂ plants from a cross between Daucus muricatus x D. carota sativus suggested paternal inheritance of ctDNA. Our reinvestigation of this material revealed that the mtDNA of the putative F₂ plants differed from the mtDNA of both putative parents. Therefore, our data suggest that the investigated material originated from other, not yet identified, parents. Consequently, the analysis of this material cannot provide evidence for a paternal inheritance of ctDNA.     

Quantitative variation in components of the maize mitochondrial genome between tissues and between plants with different male-sterile cytoplasms

Summary The amounts of a 1.9 kb mitochondrial plasmid relative to sequences in another mitochondrial DNA replicon and also to nuclear ribosomal DNA sequences have been compared in maize leaves and anthers. Similar comparisons have been made between plants with the same nuclear genotype but containing normal, S, or T cytoplasms. The ratio of 1.9 kb plasmid to nuclear rDNA is lower in plants with normal cytoplasm than in plants with S or T cytoplasm. It also differs between leaves and anthers. Furthermore, the relative concentration of the mitochondrial DNA sequences belonging to different replicons differs between leaves and anthers. It is concluded that components of different mitochondrial replicons are not maintained in fixed ratios during development and that the concentration of the 1.9 kb plasmid is regulated, in part, by cytoplasmically-inherited determinants. The 1.9 kb plasmid is absent from lines with the Vg cytoplasm, but related sequences are found in the maize nuclear genome.     

Molecular and genetic analysis of an embryonic gene, DC 8, from Daucus carota L

Summary To understand the morphogenetic and physiological processes occurring during plant embryogenesis, we isolated cDNA clones homologous to genes preferentially expressed during somatic embryogenesis. One of these cDNA clones detected an embryo-specific mRNA species with a corresponding protein of 66 kDa. The expression pattern of the mRNA is similar between somatic and zygotic embryos of carrots. To characterize the gene encoding this mRNA, we isolated the corresponding genomic clones. Molecular analysis of the DNA from several haploid and diploid carrots showed that the mRNA was encoded by a single copy gene, named DC 8. DNA sequence analysis showed that the gene consisted of three exons and coded for a hydrophilic protein with a central region composed of 17 repeats. At the NH₂-terminus no typical signal sequence was found. Immunocytochemical analysis localized the protein primarily in the vacuoles and protein bodies of zygotic embryos; the cytoplasm showed some antibody staining. The protein was also found in cell walls of endosperm tissue. The amount of DC 8 protein was too low for it to be categorized as a seed storage protein; its role in embryogenesis remains to be determined.     

Fertile revertants from S-type male-sterile maize grown in vitro

Summary Plants were regenerated from callus cultures of maize inbred W182BN with the S(USDA) type of cytoplasmic male sterility (cms). Some regenerates from 16 of 18 separate cultures had fertile tassels. Many other regenerates, whose fertility could not be scored accurately because of abnormal plant morphology, produced fertile progeny after pollination with N cytoplasm W182BN. Revertant plants and/or progeny were obtained from all 18 cultures, which included the CA, D, LBN, and S sources of cmsS. More revertants were recovered from cultures maintained as callus for 12 months than from 3–4 month old cultures. Several types of evidence (absence of segregation for fertility after selfing or pollination of revertants with standard W182BN, pollen viability counts, failure of revertants to restore sterile cmsS lines to fertility, mitochondrial DNA analyses) indicated that the reversion to fertility involved cytoplasmic rather than nuclear alterations. All revertants examined lacked the S1 and S2 plasmid-like DNAs characteristic of the mitochondrial genome of sterile cmsS lines. Most callus cultures lost S1 and S2 after 13–20 months in vitro. No revertants were seen among thousands of W182BN cmsS plants grown from seed in the field or among plants from tissue cultures of W182BN with the C or T types of cms. The cytoplasmic revertants recovered from culture may be useful for the molecular analysis of cmsS.     

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 DNA diversity and male sterility in natural populations of Daucus carota ssp carota

Mitochondrial variability was investigated in natural populations of wild carrot (Daucus carota ssp carota) in different regions: South of France, Greece, and various sites in the Mediterranean Basin and Asia. Total DNA was digested with two restriction endonucleases (EcoRV and HindIII) and probed with three mitochondrial DMA-specific genes (coxI, atp6, and coxII). Twenty-five different mitochondrial types were found in 80 analyzed individuals. Thirteen mitotypes were found among the 7 French populations studied. On average, 4.4 different mitotypes were observed per population, and these mitotypes were well-distributed among the populations. All of the mitochondrial types were specific to a single region. However, the proportion of shared restriction fragments between 2 mitotypes from different regions was not particularly lower than that which occurred among mitotypes from a single region. On the basis of the sexual phenotype [male-sterile (MS) or hermaphrodite] of the plants studied in situ and that of their progeny, 2 mitotypes were found to be highly associated with male sterility. Eighty percent of the plants bearing these mitotypes were MS in situ, and all of these plants produced more than 30% MS plants in their progeny. This association with male sterility was consistent in several populations, suggesting an association with a cytoplasmic male-sterility system. Moreover, these two mitotypes had very similar mitochondrial DNA restriction patterns and were well-differentiated from the other mitotypes observed in wild plants and also from those observed in the two CMS types already known in the cultivated carrot. This suggests that they correspond to a third cytoplasmic sterility.     

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.     

Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea starPisaster ochraceus

Summary We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea starPisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA^glu and tRNA^thr are 3 to the 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.     

Analysis of chloroplast and mitochondrial DNAs in asymmetric somatic hybrids between tobacco and carrot

Summary Chloroplast and mitochondrial DNAs have been examined by comparison of restriction enzyme patterns in asymmetric hybrid plants, resulting from the fusion between leaf mesophyll protoplasts of Nicotiana tabacum (Solanaceae), and irradiated cell culture protoplasts of Daucus carota (Umbellifereae). These somatic hybrids with normal tobacco morphology were selected as a consequence of the transfer of methotrexate and 5-methyltryptophan resistance from carrot to tobacco. The restriction patterns of chloroplast DNAs in somatic hybrids were indistinguishable from the tobacco parent. However, we found somatic hybrids with mitochondrial DNA significantly different from either parent, as judged by analysis of fragment distribution after restriction enzyme digestion. The possible formation of altered mitochondrial DNA molecules as the result of parasexual hybrid production between two phylogenetically highly divergent plant species will be discussed.     

Mitochondrial genome diversity among cultivars of daucus carota (ssp. sativus) and their wild relatives

Summary Restriction fragment patterns of mitochondrial DNAs (mtDNAs) from 13 carrot cultivars (Daucus carota ssp. sativus), wild carrot (ssp. carota), ssp. gummifer, and D. capillifolius were compared with each other using four restriction endonucleases. The mtDNAs of the 13 carrot cultivars could be classified into three distinct types — I, II and III — and were also clearly distinguishable from the mtDNAs of wild carrot (type IV), gummifer (V) and D. capillifolius (VI). The proportions of common restriction fragments (F values) shared by two of the three mtDNA types (I, II and III) of carrot cultivars were approximately 0.5–0.6. The F values were 0.4–0.5 for mitochondrial genomes between wild carrot, ssp. gummifer and D. capillifolius. The mitochondrial genomes between wild carrot and the carrot cultivars showed closer homologies those between wild carrot, ssp. gummifer, and D. capillifolius. The diversity of the mitochondrial genomes among the carrot cultivars is too high to presume that it was generated from the cytoplasm of only one common ancestor during the relatively short history of carrot breeding. These results suggested that the three types of cytoplasms found in the carrot cultivars might have existed in a prototype of D. carota in pre-historical times.     

RFLP analysis of mitochondrial DNA from cytoplasmic male-sterile lines of pearl millet

Mitochondrial DNA (mtDNA) from 13 cytoplasmic male-sterile (cms) lines from diverse sources were characterized by Southern blot hybridization to pearl millet and maize mtDNA probes. Hybridization patterns of mtDNA digested with PstI, BamHI, SmaI or XhoI and probed with 13.6-, 10.9-, 9.7- or 4.7-kb pearl millet mtDNA clones revealed similarities among the cms lines 5141 A and ICMA 1 (classified as the S-A1 type of cytoplasm based on fertility restoration patterns), PMC 30A and ICMA 2. The remaining cms lines formed a distinct group, within which three subgroups were evident. Among the maize mitochondiral gene clones used, the coxI probe revealed two distinct groups of cytoplasms similar to the pearl millet mtDNA clones. The atp9 probe differentiated the cms line 81 A4, derived from P. glaucum subsp. monodii, while the coxII gene probe did not detect any polymorphism among the cms lines studied. MtDNA digested with BamHI, PstI or XhoI and hybridized to the atp6 probe revealed distinct differences among the cms lines. The maize atp6 gene clone identified four distinct cytoplasmic groups and four subgroups within a main group. The mtDNA fragments hybridized to the atp6 gene probe with differing intensities, suggesting the presence of more than one copy of the gene in different stoichiometries. Rearrangements involving the coxI and/or rrn18-rrn5 genes (mapped within the pearl millet clones) probably resulted in the S-A1 type of sterility. Rearrangements involving the atp6 gene (probably resulting in chimeric form) may be responsible for male sterility in other cms lines of pearl millet.