The physical map of the chloroplast DNA from Asparagus officinalis L.

The genus Asparagus consists of 100–300 species of both dioecious and hermaphrodite plants. Since there are diploid, tetraploid, and hexaploid plants in this genus, RFLP (restriction fragment length polymorphism) analysis of chloroplast DNA (ctDNA) is suitable for examining the phylogenetic relationships. We have constructed a physical map of the ctDNA of garden asparagus (A. officinalis L. cv Mary Washington 500 W) using five restriction endonucleases, namely, BamHI, PstI, SalI, HindIII, and XhoI. Asparagus ctDNA was digested with restriction enzymes and cloned into plasmid and phage vectors, and a clone bank was constructed that covered 70% of the genome. A physical map was constructed by Southern hybridization of total DNA from asparagus with homologous and heterologous probes. The asparagus ctDNA was about 155 kb long and it contained two inverted repeats (23kb each) separated by a large single-copy region (90kb) and a small single-copy region (19kb). Fifteen genes, encoding photosynthesis-related proteins, rDNAs, and tRNAs, were localized on the physical map of asparagus ctDNA. Comparing the length and the gene order of asparagus ctDNA with that of other plants, we found that asparagus ctDNA was similar to tobacco ctDNA but different from rice ctDNA. The restriction patterns of the ctDNAs from several varieties of A. officinalis and three species of Asparagus were analyzed. The restriction patterns of the varieties of A. officinalis were very similar, but polymorphisms were detected among the three species of Asparagus.     

The Cucumis plastome: physical map,intrageneric variation and phylogenetic relationships

Summary A restriction map of the Cucumis melo L. (melon) plastome was constructed by using several mapping approaches: single and double digestions of the chloroplast DNA (chlDNA) with endonucleases (XhoI, SmaI, SacI and PvuII) and hybridization to heterologous chlDNA probes and to isolated melon chlDNA fragments. Four plastome-coded genes were located using heterologous probes. The overall organization and gene position of the melon plastome was found to be similar to that of tobacco and other angiosperm species. Restriction patterns based on digestion of the chlDNA with nine endonucleases were obtained in over 20 wild species and cultivated varieties of Cucumis. These led to mutational analysis of the restiction sites yielding the most parsimonious phylogenetic tree of the Cucumis plastome. Most African species from a compact group (Anguria group) which is distant from the melon, the cucumber and a few other species (C. sagittatus, C. metuliferus and C. humifructus). All of these are also far apart from each other. The distribution of polymorphic restriction sites along the Cucumis plastome is described and conservative regions as well as hot spots are suggested.     

Homologies to plastid DNA in the nuclear and mitochondrial genomes of potato

Summary Potato plastid DNA clones, representing onefourth of the potato plastome complexity and containing sequences of the 16SrRNA, rps16, atpA, atpE, psaA, psaB, trnK, trnV, and trnG genes, were used as hybridization probes on nuclear- and mitochondrial-enriched DNAs. Each probe hybridized to multiple nuclear restriction fragments distinct from the plastid cleavage products generated by the same endonucleases. The nuclear hybridizable fragments are highly methylated at their Hpall target sequences (C/CGG). In some instances, the transfer seemed to involve plastid regions of several kilobase pairs, as reflected by the co-integration in the nucleus of restriction sites that are distant in the plastome. Three clones hybridized additionally to distinct mitochondrial fragments. These results indicate that extensive DNA transfers did occur between plastids and other organelles in potato.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

Variations of chloroplast DNAs in the genus Pelargonium and their biparental inheritance

Summary The comparison of EcoRI patterns of chloroplast DNAs (ctDNAs) from five species of the genus Pelargonium and from 16 cultivars and varieties of Pelargonium zonale hort. demonstrates a remarkable inter- and intraspecific ctDNA (plastome) variation. The plastome of the P. zonale varieties could be differentiated into groups I, II and III. Reasons for this variation seem to be: occurrence of numerous spontaneous plastome mutations, intense hybridisation by gardeners and breeders, and biparental plastid inheritance.Crosses of P. zonale varieties with different ctDNA types lead to the direct evidence on the molecular level of biparental plastid inheritance and plastid sorting-out in F₁-hybrids.     

Cultivated potato chloroplast DNA differs from the wild type by one deletion — evidence and implications

Summary The chloroplast DNA (ctDNA) of Solanum tuberosum ssp. tuberosum (T type) and S. chacoense (W type) yield five different restriction fragment patterns with five different restriction endonucleases. DNA-DNA hybridization tests revealed that these differences were all caused by one physical deletion (about 400 bp in size) in the ctDNA of ssp. tuberosum. This suggests that T type ctDNA of the common potato and of Chilean tuberosum originated from W type ctDNA. The deleted region of the T type ctDNA is probably not concerned with gene-cytoplasmic male sterility.Reference to a specific brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned     

Origin of chloroplast DNA diversity in the Andean potatoes

Summary Wide chloroplast DNA (ctDNA) diversity has been reported in the Andean cultivated tetraploid potato, Solanum tuberosum ssp. andigena. Andean diploid potatoes were analyzed in this study to elucidate the origin of the diverse ctDNA variation of the cultivated tetraploids. The ctDNA types of 58 cultivated diploid potatoes (S. stenotomum, S. goniocalyx and S. phureja), 35 accessions of S. sparsipilum, a diploid weed species, and 40 accessions of the wild or weed species, S. chacoense, were determined based on ctDNA restriction fragment patterns of BamHI, HindIII and PvuII. Several different ctDNA types were found in the cultivated potatoes as well as in weed and wild potato species; thus, intraspecific ctDNA variation may be common in both wild and cultivated potato species and perhaps in the higher plant kingdom as a whole. The ctDNA variation range of cultivated diploid potatoes was similar to that of the tetraploid potatoes, suggesting that the ctDNA diversity of the tetraploid potato could have been introduced from cultivated diploid potatoes. This provided further evidence that the Andean cultivated tetraploid potato, ssp. andigena, could have arisen many times from the cultivated diploid populations. The diverse but conserved ctDNA variation noted in the Andean potatoes may have occurred in the early stage of species differentiation of South American tuber-bearing Solanums.     

Restriction fragment map of sugar beet (Beta vulgaris L.) chloroplast DNA

Summary A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes SmaI, PstI and PvuII. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In common with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S+16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.     

Intraspecific variation of chloroplast DNA in Dioscorea bulbifera L.

Summary Restriction fragment length polymorphism (RFLP) analysis of chloroplast (ct) DNAs from 15 accessions of Dioscorea bulbifera collected from Africa and Asia was carried out using the Southern hybridization technique. Eight cloned ctDNA fragments of D. bulbifera and D. opposita, which cover 80% of the total chloroplast genome, were used as the probes to detect variation in ctDNA digested with nine restriction endonucleases. Ten variable sites, located in the large and small single-copy regions, were disclosed among the 15 accessions, of which six showed base substitution and four carried length mutation. Positions of the latter mutations were determined on the physical map of ctDNA. Based on these results, chloroplast genomes of the 15 accessions could be classified into nine types. Their phylogenetic relationships are assumed to be as follows: (1) African and Asian chloroplast genomes diverged from each other at the earliest point in time; (2) E-type chloroplast genome, occurring in the south-east edge of the Asian continent, appears to be the most ancient among all the Asian chloroplast genomes; and (3) four chloroplast genomes, found in Asian insular regions, are probably derived independently from the E-type genome. The discrepancy between the taxonomic relationship and the proposed chloroplast genome phylogeny of the present materials is noted.     

Chloroplast DNA evolution in potato (Solanum tuberosum L.)

Summary A deletion specific to chloroplast (ct) DNA of potato (Solanum tuberosum ssp. tuberosum) was determined by comparative sequence analysis. The deletion was 241 bp in size, and was not flanked by direct repeats. Five small, open reading frames were found in the corresponding regions of ctDNAs from wild potato (S. tuberosum ssp. andigena) and tomato (Lycopersicon esculentum). Comparison of the sequences of 1.35-kbp HaeIII ctDNA fragments from potato, tomato, and tobacco (Nicotiana tabacum) revealed the following: the locations of the 5 ends of both rubisco large subunit (rbcL) and ATPase beta subunit (atp) mRNAs were probably the same as those of spinach (Spinacia oleracea); the promoter regions of the two genes were highly conserved among the four species; and the 5 untranslated regions diverged at high rates. A phylogenetic tree for the three potato cultivars, one tomato cultivar, and one tobacco cultivar has been constructed by the maximum parsimony method from DNA sequence data, demonstrating that the rate of nucleotide substitution in potato ctDNA is much slower than that in tomato ctDNA. This fact might be due to the differences in the method of propagation between the two crops.     

Genetic mapping with dispersed repeated sequences in the rice blast fungus: mapping the SMO locus

Summary The SMO genetic locus in strains of the fungus Magnaporthe grisea that infect weeping lovegrass, directs the formation of correct cell shapes in asexual spores, infection structures, and asci. We have identified and characterized a Smo^– strain of M. grisea that infects rice. The smo mutation in this strain segregates as a single gene mutation and is allelic to previously identified smo alleles. A marked reduction in pathogenicity co-segregates with the Smo^– phenotype, suggesting that the SMO locus plays a role in rice pathogenicity. A family of dispersed repeated DNA sequences, called MGR, have been discovered in the nuclear DNA of M. grisea rice pathogens. Genetic crosses between Smo^– rice pathogens and Smo⁺ non-rice pathogens were used to follow the segregation of the SMO locus and individual MGR sequences. Using DNA blot analysis with cloned MGR hybridization probes, we mapped the SMO locus to a chromosomal region flanked by two closely linked MGR sequences. We demonstrated that the copy number of MGR sequences could be reduced in subsequent crosses to non-rice pathogens of M. grisea, and that new MGR sequences did not occur following meiosis indicating that these sequences are stable in the genome. We conclude that restriction fragment polymorphism mapping with cloned MGR sequences as hybridization probes is an effective way to map genes in the rice blast fungus.     

Homologies to chloroplast DNA in the nuclear DNA of a number of Chenopod species

Summary Sequences homologous to chloroplast (ct)DNA have been found in nuclear DNA in five species of the Chenopodiaceae, extending the earlier observations of promiscuous DNA in Spinacia oleracea (Timmis and Scott 1983). Using the 7.7 kbp spinach ctDNA Pst I fragment as a hybridization probe, several separately located homologies to ctDNA were resolved in the nuclear DNA of Beta vulgaris, Chenopodium quinoa, and Enchylaena tomentosa. In Chenopodium album and Atriplex cinerea the major region of homology was to a nuclear Eco RI fragment (6 kbp) indistinguishable from that in ctDNA. These homologies may therefore involve larger tracts of ctDNA because the same restriction sites are apparently retained in the nucleus. This suggests that in these latter two species there is a contrasting, more homogeneous arrangement of ctDNA transpositions in the nucleus.     

Cybrid production based on mutagenic innactivation of protoplasts and rescuing of mutant plastids in fusion products: Potato with a plastome fromS. bulbocastanum andS. pinnatisectum

A procedure for cybrid production, based on double treatment of donor protoplasts by physical and afterwards chemical mutagens at superlethal doses (-irradiation at a dose of 1000 Gy was applied for the inactivation of nuclei; 3–5 mMN-nitroso-N-methylurea was used for the efficient induction of plastome mutation) and the rescuing of mutant plastids after fusion with untreated recipient protoplasts, was developed. For identification of mutant donor-type plastids in fusion products a selection for streptomycin was performed. In two sets of experiments, in whichS. tuberosum served as the recipient of foreign cytoplasm with the wild tuber-bearing speciesS. bulbocastanum andS. pinnatisectum as donors, a total of about 40 streptomycin-resistant colonies was isolated. Eight regenerants from theS. tuberosum+S. bulbacastanum fusion combination and four fromS. tuberosum+S. pinnatisectum were further investigated using chromosome counting, analysis of esterase isoenzymes, restriction analysis of organelle DNA, and blot hybridization. All but one plant from both combinations were characterised as potato cybrids possessing exclusively foreign plastids and retaining a morphology typical of the recipient. Only in one line was rearranged mtDNA detected. The availability of potato cybrids facilitates the analysis of plastome-encoded breeding traits and the identification of the most valuable source of cytoplasm among the wild potato species. The described system for producing cybrids without genetic selectable markers in the parental material offers the possibility for the rescue of cytoplasmic mutations which are impossible to isolate by conventional approaches.     

Geographic partitioning of chloroplast DNA variation in the genusDatisca (Datiscaceae)

Datisca (Datiscaceae) is a ditypic genus with an intercontinentally disjunct distribution. Chloroplast DNA restriction site data was obtained from 23 populations and four 10–20 year old herbarium specimens ofD. glomerata and three populations ofD. cannabina from throughout their geographic ranges in western North America and southwest-central Asia, respectively. InD. glomerata, plastome diversity is partitioned geographically. All populations from southern California have a common plastome, while most populations north of this region share a relatively divergent plastome (0.49% sequence divergence). Likewise, these plastomes are highly divergent (0.87% mean sequence divergence) from those found inD. cannabina. Biogeographic processes dating to the Pleistocene and Late Miocene may be responsible for these intra- and interspecific patterns of chloroplast DNA divergence.     

Cytological localization of fast renaturing and satellite DNA sequences inVicia faba

Summary DNA sequences reassociating within a Cot value of 1.8×10^–1 and those producing a light satellite in a CsCl density gradient were isolated fromVicia faba DNA and hybridizedin situ on squashes of roots of the same species. Silver grains were seen to be scattered over both the interphase nuclei and the metaphase chromosomes after hybridization with fast renaturing DNA sequences, indicating these are fairly regularly interspersed in theV. faba genome. Clustered labeling occurred after hybridization with satellite DNA sequences, indicating these are clustered in the genome. The localization of satellite DNA in chromosomes appeared to correspond closely to the position of the bright bands detectable after staining with quinacrine mustard. After hybridization with both DNA probes, labeling intensity over the nuclei of meristematic cells was higher than that over the nuclei of differentiating and/or differentiated cells. These results are discussed in relation to the structure of the cell nucleus, the mechanism of quinacrine banding and to previous data suggesting underrepresentation of nuclear repeated DNA sequences in differentiatingV. faba root cells.     

Deletion analysis of the Brassica napus cruciferin gene cru 1 promoter in transformed tobacco: promoter activity during early and late stages of embryogenesis is influenced by cis-acting elements in partially separate regions

To define sequences in the cruciferin gene cru1 promoter of importance for expression, tobacco (Nicotina tabacum L.) plants were transformed with constructs in which the cru1 promoter, in front of the intact cru1 structural gene, was truncated at –1216, –974, –736, –515, –306, –46 and –17 bp relative to the cap-site. Cru1 expression in tobacco seeds was studied by Northern analysis, Western analysis and in-situ hybridizations. Comparisons of the Northern analysis of RNA from tobacco seeds harvested at 18 d after pollination with the Western analysis of protein from mature seeds showed that the regions between –974 to –736 and –306 to –46 were important for the expression of cru1 at an early developmental stage, whereas the regions –736 to –515 and –515 to –306 were important for expression throughout embryogenesis. By investigating the mRNA levels in transgenic seeds at different stages of development, indications were obtained that the two latter regions exerted their effects during the later stages. The in-situ hybridization showed that cru1 mRNA was distributed in parenchyma cells throughout the embryo in seeds expressing constructs –974 and –736. Constructs –515 and –306 showed an expression restricted to the axis or axis and parts of the cotyledons. Sequence comparisons of the cru1 promoter with other storage-protein gene promoters, identified several motifs implicated in gene regulation. Gel retardation assays with synthetic oligonucleotides showed that a region present in both cru1 and BnC1 promoters, a CANNTG motif, an SEF3 motif, an abscisic-acid-responsive element and an RY-like motif interacted specifically in vitro with DNA-binding proteins present in nuclear extracts from seeds of Brassica napus L. harvested 40 d after pollination.Abbreviations ABA abscisic acid - DAP days after pollination This work was supported by grants from the Swedish Research Council for Forestry and Agriculture and from the Swedish Natural Science Research Council. Ms Ulla Pihlgren, Elisabeth Westergren and Elfi Öhren are acknowledged for expert technical assistance.     

Root bacteria from nematicidal plants and their biocontrol potential against trichodorid nematodes in potato

Trichodorid nematodes (Nematoda: Trichodoridae) are vectors of tobacco rattle virus (TRV), one of the causal agents of spraing disease in potato. Root bacteria from nematicidal plants and their control potential against Trichodoridae were the focus of this study. Bacteria isolated from the roots of 12 nematicidal plants and potato were characterized for their production of hydrolytic enzymes, hydrogen cyanide, phenol oxidation ability and antifungal activity towards the potato pathogen Rhizoctonia solani. Based on these functional traits, bacteria isolates were selected and tested in greenhouse conditions on potato (cv. Saturna) for their effect on plant growth, and screened for nematicidal activity against Paratrichodorus pachydermus and Trichodorus primitivus in naturally infested soil. Sixteen bacteria isolates out of 44 reduced nematode densities by 50–100%. Nine selected isolated were further tested by bacterizing potato tubers (cv. King Edward) which were planted in a trichodorid and TRV-infested soil. Four bacterial isolates consistently reduced nematode densities (by 56.7–74.4%) with no visual negative effect on plant growth. These isolates were tentatively identified, partly by fatty acid methyl ester (FAME) analysis as: Stenotrophomonas maltophilia, Bacillus mycoides, Pseudomonas sp., and one unidentified bacterium. The isolates originated from potato, Plantago major, Thymus vulgaris and Asparagus officinalis, respectively. Two Pseudomonas isolates obtained from Zinnia elegans and selected for their strong nematicidal activity in soil screening tests, did not reduce the nematode population when tested on potato. It is concluded that plants releasing nematicidal compounds may harbour nematode-antagonistic bacteria as well.     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Summary Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6–12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background.