Diversity of Vicia faba circular mtDNA in whole plants and suspension cultures

Summary A comparative analysis of the Vicia faba mitochondrial genome in whole plants and in longterm suspension culture has been conducted. Restriction fragment patterns of the mtDNA isolated from these two sources were notably different. Electronmicroscopic analysis also revealed significant differences. Large circular mtDNA patterns shifted from a 37–80 kb subpopulation, which was predominant in whole plants, to 18–34 kb subpopulations although in both classes notable quantities of circular molecules of 80 to 120 kb and more were also found. Both in whole plant and suspension culture cells very large circular DNAs were observed. Some of them had lengths nearly 290 kb and could be considered as evidence of the existence of master chromosomes. The minicircular DNA population was also altered. In the suspension culture we observed a notable increase of percentage of minicircles with sizes near 1 kb. Simultaneously, the percentage of minicircles with sizes near 3.5–10 kb significantly increased in suspension culture cells. In addition, a new peak (10–12 kb) of minicircles appeared. Copy number alterations for some sequences homologous to CCC1A, CCC1B and CCC2 (Negruk et al. 1982, 1985) were shown. Southern hybridization revealed the existence of a family of minicircles having sizes 1.4–2 kb with predominance of CCC1A, CCC1B and CCC2. The copy numbers of CCC1B and some minor minicircles was changed in the suspension culture when compared with the whole plants.     

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.     

Restoration of normal stamen development and pollen formation by fusion of different cytoplasmic male-sterile cultivars of Nicotiana tabacum

Summary Fusion of two cytoplasmic male-sterile cultivars of Nicotiana tabacum, one with N. bigelovii cytoplasm and one with N. undulata cytoplasm, resulted in the restoration of male fertility in cybrid plants. All male-fertile cybrids exhibited fused corollas, which is characteristic for the cultivar with N. undulata cytoplasm, while their stamen structures varied from cybrid to cybrid, some producing stamens with anthers fused to petal-like appendages and one producing stamens of a normal appearance for N. tabacum. Restriction enzyme digestion and agarose gel electrophoresis of mitochondrial DNA showed that mitochondrial DNA of the fertile cybrids was more similar to the male-sterile cultivar with the cytoplasm of N. undulata than to the cultivar with N. bigelovii cytoplasm. Some restriction fragments were unique to the male-fertile cybrids. Comparisons between stamen structure and mitochondrial DNA for eight fertile progeny from one cybrid plant led to the identification of several restriction fragments that appeared at enhanced levels in connection with normal stamen development.     

Intraspecific heterogeneity of the Vicia faba mitochondrial genome: evidence for multiregional rearrangements in the mitochondrial chromosome associated with coxII-orf192 chimeric gene formation

Summary Previous RFLP-analysis of mtDNA isolated from different lines and cultivars of Vicia faba with respect to variability of the coxII gene revealed two types of mitochondrial genome: one with a normal coxII gene and the other with both normal coxII and chimeric coxII-orf192 genes. In this study we analyzed other regions of these two types of mitochondrial genome and found significant differences in the arrangement of regions around the coxII, coxIII, cob, rrn26 and atpA genes. More detailed analysis of the rrn26 and atpA gene regions showed that these genes are associated with recombinationally active repeats. Restriction maps of the rrn26 and atpA gene regions in different recombinative variants are presented.     

Mitochondrial plasmids in related cultivars of crookneck and stable and unstable butternut squash

Summary Two series of two to four plasmids are contained in mitochondria of butternut squash (Cucurbita moschatd). The plasmids are composed of DNA, and are consistent in number within a cultivar but vary in number among cultivars. Plasmid patterns and homology suggest that the faster migrating series of plasmids are the supercoiled form of the slower running plasmids. Plasmid 1 was present in all cultivars except New Hampshire Butternut. Plasmid 2 was only observed in the original crookneck cultivar, Canada Crookneck. Plasmids 3 and 4 were present in all accessions tested. The presence of plasmid 1 in a cultivar does not follow a maternal pattern of inheritance; this may be due to the ability of the plasmid to insert into and excise from the main mitochondrial DNA, or the recombination of some of the smaller plasmids to create the larger plasmid 1. There is some homology between plasmid 1 and plasmids 3 and 4, and between plasmid 1 and main band mitochondrial DNA. The equal presence of plasmid 1 in mitochondria of F₁ seedlings from the pollination of New Hampshire Butternut by Ponca Butternut to that in F₁ seedlings of the reciprocal cross indicates that there is probably a dominant nuclear effect on the ability to produce plasmid 1 either by release from the mitochondrial genome or by recombination of the smaller plasmids. There is no obvious relationship between the presence or number of the mitochondrial plasmids and the butternut fruit shape or stability of the butternut trait of the cultivars.     

Variability and inheritance of histone genes H3 and H4 in Vicia faba

Summary We have compared copy numbers and blothybridization patterns of histone genes (H3 plus H4) between and within individuals of broad bean (Vicia faba). Copy number differences among individuals in the population of 200 individuals were as great as 27 fold, and as much as 3.2 fold among separate leaves of the same plant. Among F₂ progeny from genetic crosses, up to a 5.4-fold range was seen (mean=3.5 fold), and among F₁ progeny of self-pollinated plants, up to a 5.9-fold range was observed (mean=2.3 fold). Histone gene blot-hybridization patterns for EcoRI and HindIII were also variable among individuals and indicated that the genes are probably clustered in only a few chromosomal loci. The degree of variation in histone gene copy number per haploid genome (2–55 copies, or 27 fold) was similar to that found previously for ribosomal RNA genes (230–22000, or 95 fold) of V. faba. However, the two gene families change independently, since individuals with a high or low copy number for one gene can have either a high or low copy number for the other. The mechanisms(s) for rapid gene copy number change may be similar for these gene families.     

Measurements of genetic variability for symbiotic dinitrogen fixation in fieldgrwon fababean (Vicia faba L.) using a low level15N-tracer technique

Before starting a breeding program aimed at improving the nitrogen nutrition ofVicia faba, the authors tried an alternative technique to the acetylene reduction assay, to measure some genetic variability in the plant material. The quantity of dinitrogen fixed by several cultivars ofVicia faba was estimated using a low enrichment¹⁵N tracer method and high precision¹⁵N mass spectrometry. The fababeans were cultivated for two years in two different soils. The percentage of fixed dinitrogen in the seed varied between genotypes from 40 to 83% of the total nitrogen and was positively correlated with the total seed nitrogen (r=0.64 to 0.86). A highly significant positive correlation was also found between the total seed nitrogen and the quantity of fixed dinitrogen in the seed (r=0.95 to 0.99). The technique used to measure dinitrogen fixation proved to be useful and reliable enough to discriminate between various genotypes, grown over a period of two years in two different soils. However, several non-fixing control plants showed significant differences in their¹⁵N enrichment and the problem of choosing a good reference plant was raised and 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.     

Comparative electrophoretical analysis of plasmid-like mitochondrial DNAs in Vicia faba and in some other legumes

Minicircular DNAs found in mitochondria of 6-d-old etiolated seedlings of Vicia faba L. were also present in mitochondria isolated from cell suspension cultures and from green leaves of this plant. These results support the suggestion that the plasmid-like molecules found in mitochondria of V. faba are an essential component of the mitochondrial genome. The minicircular DNAs were, apparently, peculiar for the species V. faba since they were found in all three cultivars of this species which were studied. The distribution pattern of plasmid-like DNAs in Vicia villosa L. was completely different and mitochondria from Medicago sativa L. also contained specific minicircular DNAs. Thus, minicircular DNAs are typical for the mitochondrial genomes of several legumes and different plant species have their specific mitochondrial plasmid-like DNAs     

The metabolism of ethylene to ethylene oxide in cultivars of Vicia faba: Relationship with waterlogging resistance

The ability of cultivars of Vicia faba L. to metabolize ethylene and the resistance of such cultivars to waterlogging was assessed. Those cultivars showing the highest resistance to waterlogging also had the ability to rapidly metabolize ethylene. It is suggested that a causal relationship may exist between the two processes.     

Sequence analysis of Vicia faba highly repeated DNA: the BamHI repeated sequence families

Cleavage of Vicia faba nuclear DNA with the restriction endonuclease BamHI yielded discrete size classes of 250, 850, 900, 990, 1 150, 1 500 and 1 750 bp of highly repetitive DNA. Each of these sequence families comprised about 3% of the total genomic DNA. Some sequence members from each sequence family were cloned in pBR322 and their primary structures determined. Computer analyses of nucleotide sequences suggested the existence of about 60 bp sequence periodicity within the repeating unit of the 990 bp sequence family, though the extent of homology among the surmised shorter subrepeat units was very low. With other BamHI sequence families, however, the data did not show any clear internal sequence periodicity. The repeat units of the 850 bp and 1 750 bp sequence families contained nucleotide sequences homologous to the 250 bp family sequence. No sequence relationship between or among other sequence families was observed. There was 13–25% sequence variation among 6 cloned members of the 250 bp family and probably also among those of other BamHI repeat families. DNA sequences homologous to these V. faba BamHI repeat families were detected in Pisum sativum DNA by Southern blot hybridization. Furthermore, very weak cross-hybridization was observed with plant DNAs from Phaseolus vulgaris, Triticum aestivum, Cucumis sativus and Trillium kamtschaticum.     

On the toleration of duplications and deletions by the Vicia faba genome

Summary From eight pairs of crosses between differently reconstructed diploid karyotypes of Vicia faba, the progeny after selfing of plants heterozygous for both parental chromosome reconstructions were inspected for occurrence and transmission of duplications and deletions of defined chromosome segments, comprising together about one third of the metaphase genome length. The duplications and deletions studied involved either one or more chromosome segments of the respective karyotype (0.8%–9.1% of the metaphase length). They arose during meiosis in double heterozygotes by crossing over between partially homologous chromosomes or by mis-segregation from multivalents. While most duplications, provided they were not accompanied by deletions and in dependence on the segment involved, were viable and transmissible, even in homozygous state, deletions had lethal effects on gametes of both sexes.     

Explaining differences in flea beetle Phyllotreta cruciferae goeze densities in simple and mixed broccoli cropping systems as a function of individual behavior

Diversification of habitat has proved to be an efficient way to reduce insect pest levels in agroecosystems. Some general theories used to explain this fact, such as the natural enemies and the resource concentration hypotheses, do not always clearly apply because, in many cases, pest individuals and population response seem controlled by more specific insect-plant interactions. In non replicated plots, we found substantially lower flea beetle densities in mixed broccoli-Vicia cropping systems compared to broccoli monoculture. These results were consistent with those from controlled experiments reported in the literature. To investigate if beetle behavior was related to such population reduction, the movement behavior of marked individuals of Phyllotreta cruciferae Goeze released in plots composed solely of broccoli plants and of broccoli mixed with Vicia faba or Vicia sativa plants, was followed and analyzed. The mean tenure time of beetles was longer in simple than in mixed cultures. Also, more beetles tended to fly out and leave mixed cultures compared to monoculture. This resulted in faster reduction of artificially introduced flea beetle populations in the mixed systems.Flea beetles landing on cover crop plants spent considerable time entangled in Vicia sativa branches or attempting to reach the upper part of the tall Vicia faba plants from which they could fly away. It is possible that the beetles characteristic movement on these two species of cover crops increased their risk of predation and the time and energy expended before they reached suitable host plants. Nevertheless, it seems that the detected flea beetle emigration rates were more than sufficient to account for the population trends observed.     

Cytophotometrical measurement of nuclear DNA content in some coniferous and deciduous trees

Summary The DNA content of nuclei from meristematic root tip cells of five coniferous and one deciduous tree species and, for comparison, ofVicia faba was cytophotometrically determined. The DNA values of diploid nuclei fromGinkgo biloba are approximately a quarter lower than those fromVicia faba. The nuclear DNA values of the other tree species are merely a third to a ninth part of those ofVicia faba. In three tree species, as well as diploid, we have found nuclei of different polyploid level.The reliability of different cytochemical methods, which are used for determination of the nuclear DNA content, is critically analyzed. The DNA values of the investigated tree species are discussed in connection with the evolution of the DNA content in higher plants.Dedicated to Professor Dr. F. Mechelke in honour of his 60th birthday     

Differential fate of plastid and mitochondrial genomes in Petunia somatic hybrids

Summary The chloroplast (cp) and mitochondrial (mt) DNAs of Petunia somatic hybrid plants, which were derived from the fusion of wild-type P. parodii protoplasts with albino P. inflata protoplasts, were analyzed by endonuclease restriction and Southern blot hybridization. Using ³²P-labelled probes that distinguished the two parental cpDNAs at a BamH1 site and at a HpaII site, only the P. parodii chloroplast genome was detected in the 10 somatic hybrid plants analyzed. To examine whether cytoplasmic mixing had resulted in rearrangement of the mitochondrial genome in the somatic hybrids, restriction patterns of purified somatic hybrid and parental mtDNAs were analyzed. Approximately 87% of those restriction fragments which distinguish the two parental genomes are P. inflata-specific. Restriction patterns of the somatic hybrid mtDNAs differ both from the parental patterns and from each other, suggesting that an interaction occurred between the parental mitochondrial genomes in the somatic fusion products which resulted in generation of the novel mtDNA patterns. Southern blot hybridization substantiates this conclusion. In addition, somatic hybrid lines derived from the same fusion product were observed to differ in mtDNA restriction pattern, reflecting a differential sorting-out of mitochondrial genomes at the time the plants were regenerated.     

Stable inheritance and expression of the CMS traits introduced by asymmetric protoplast fusion

The donor-recipient protoplast fusion method was used to produce cybrid plants and to transfer cytoplasmic male sterility (CMS) from two cytoplasmic male-sterile lines MTC-5A and MTC-9A into a fertile japonica cultivar, Sasanishiki. The CMS was expressed in the cybrid plants and was stably transmitted to their progenies. Only cytoplasmic traits of the male-sterile lines, especially the mitochondrial DNAs, were introduced into the cells of the fertile rice cultivar. More than 80% of the cybrid plants did not set any seeds upon selfing. Sterile cybrid plants set seeds only when they were fertilized with normal pollen by hand and yielded only sterile progenies. This maternally inherited sterility of the cybrid plants showed that they were characterized by CMS. The CMS of cybrid plants could be restored completely by crossing with MTC-10R which had the single dominant gene Rf-1 for restoring fertility. These results indicated that CMS was caused by the mitochondrial genome introduced through protoplast fusion. The introduced CMS was stably transmitted to their progenies during at least eight backcross generations. These results demonstrate that cybrids generated by the donor-recipient protoplast fusion technique can be used in hybrid rice breeding for the creation of new cytoplasmic male-sterile rice lines.     

Comparison of chloroplast and mitochondrial DNA from five morphologically distinct Beta vulgaris cultivars: sugar beet,fodder beet,beet root,foliage beet,and Swiss chard

Summary Two cytoplasms, N and S, are used in the breeding of sugar beet, Beta vulgaris var. altissima. These cytoplasms can be distinguished by their mitochondrial DNA. In an attempt to detect new cytoplasms, we compared the restriction profiles of chloroplast and mitochondrial DNA from five different cultivars of Beta vulgaris. All restriction patterns of chloroplast DNA were identical. With the exception of sugar beet with S-cytoplasm, all cultivars studied showed the same restriction profile of mitochondrial DNA, indicating that these cultivars all contain the N-cytoplasm. These results are discussed with regard to the large morphological differences of the cultivars and the cytoplasmic variability found in natural populations of the wild beet, Beta maritima.     

Variant mitochondrial protein and DNA patterns associated with cytoplasmic male-sterile lines of Nicotiana

Summary Variation in mitochondrial protein synthesis and genome organization was investigated. Three different alloplasmic cytoplasmic male-sterile Nicotiana tabacum cultivars, carrying N. repanda, N. suaveolens or N. debneyi cytoplasm, were analysed together with corresponding male-fertile parental and restored material. Although several differences were detected in the proteins synthesized by isolated mitochondria from the male-sterile and male-fertile plants, most of these were related to the origin of the mitochondria. However, a 23 kD protein was synthesized in the male-sterile cultivar carrying N. debneyi mitochondria, but not in other lines containing this cytoplasm. This protein was also present in the male-fertile parent containing N. tabacum mitochondria. Only the enhanced production of a 30 kD protein in the lines carrying mitochondria from N. repanda or N. debneyi was exclusively correlated with CMS. This protein was not present in any of the corresponding male-fertile parental and restored lines. Restriction enzyme analysis of mitochondrial DNA revealed a difference in abundance of a 5.6 kb XhoI fragment between lines containing N. debneyi mitochondria. No rearrangements of mitochondrial DNA was found between male-fertile and male-sterile lines carrying N. repanda or N. suaveolens cytoplasm. These results might indicate that CMS in alloplasmic Nicotiana cultivars is caused by alterations in the expression of mitochondrial genes, rather than by induced changes in the genome.