Organ/tissue-specific changes in the mitochondrial genome organization of in-vitro cultures derived from different explants of a single wheat variety

Summary We have previously shown that the mitochondrial genome of long-term tissue cultures prepared from immature embryos of several varieties of cultivated wheat underwent variety-specific rearrangements resulting from either changes in the relative amounts of subgenomic components or from the appearance of novel genomic configurations. In the present work, both categories of rearrangements were studied in long-term tissue cultures initiated from other explants (shoot meristem, young leaf base, young root tip, immature inflorescence) of the same wheat variety (Chinese Spring) and were compared to those previously obtained with immature embryo cultures. Two main patterns of reorganization were found in a region of the mitochondrial genome known to be hypervariable in structure. In addition, some of the novel subgenomic configurations were obviously organ/tissue-specific whereas others were present in more than one type of organ. In several instances, the age of culture was found to determine the degree of mitochondrial DNA rearrangement. The data presented in this study strengthen the hypothesis of an association between a particular organization of the mitochondrial genome in tissue culture and its regeneration capacity.     

Unusual inheritance of the mitochondrial genome organization in the progeny of reciprocal crosses between alloplasmic hexaploid wheat regenerants

The transmission of a structurally-hypervariable fraction of the mitochondrial genome has been studied in 42 F₁ progenies obtained from reciprocal crosses between self-pollinated alloplasmic wheat plants regenerated after long-term somatic embryogenesis. This fraction of the genome is maternally and stoichiometrically inherited. In contrast, some additional restriction fragments specific to regenerated plants display a more complex mode of sexual transmission: one of the additional fragments was stoichiometrically and systematically inherited whereas two others were detected only in certain F₁ hybrids. Assuming that the detection, by Southern analysis, of such a fragment in regenerated plants is due to the amplification of a pre-existing substoichiometric molecule generated by the activation of a rare recombination event, our results suggest that the probability of detecting a novel fragment in the F₁ hybrids could be determined by the length of the repeated sequence at which recombination occurs.     

Identification of new mitochondrial genome organizations in wheat plants regenerated from somatic tissue cultures

Summary Plants have been regenerated from short-and long-term in vitro somatic tissue cultures made from immature embryos of the hexaploid wheat cultivar Chinese Spring. The mitochondrial genome organization of each regenerated plantlet was studied, after one selfing, by probing Sal I-restricted total DNA with cloned Sal I fragments of wheat mitochondrial DNA derived from a segment of the genome, which displays marked structural changes in response to in vitro culture. Short-term in vitro cultures give rise to regenerated plants whose mitochondrial genome organization is either close to that of the parental cultivar or to that of embryogenic callus cultures, except for a single plant which has an organization resembling that of short-term non-embryogenic cultures. In contrast, all but one of the plants regenerated from long-term cultures exhibited a mitochondrial genome organization similar to that of long-term nonembryogenic cultures. In addition, extra labelled bands were detected in some of the regenerated plants with two of the probes used. These results emphasize the importance of the duration of the in vitro step preceding the regeneration process: the longer it is, the higher the probability is of obtaining mitochondrial DNA variability in regenerated plants. Furthermore, since increasing the duration of the in vitro stetp results in the production of regenerated plants with a mitochondrial genome organization resembling that of non-embryogenic tissue cultures, the question is thus raised as to whether regeneration from long-term cultures is suitable for use in plant breeding.     

Multiple patterns of mtDNA reorganization in plants regenerated from different in vitro cultured explants of a single wheat variety

Summary We have previously shown that tissue cultures derived from various explants of the wheat variety Chinese Spring exhibit organ/tissue-specific changes in the organization of their mitochondrial genome. The aim of this work was to study the influence of passage out of in-vitro culture, and subsequent plant regeneration, on the in vitro induced reorganization of this genome. In all cases but one, subgenomic configurations present in both the donor parent and the tissue culture were evident, in corresponding regenerated plants. The presence, in regenerated plants, of subgenomic configurations found in tissue culture but undetectable in the donor parent appeared to be both timeand organ/tissue-dependent. Moreover, when present, these novel organizations were not systematically found in all regenerated plants. Finally, novel subgenomic configurations were specifically detected after passage out of in-vitro culture. As these results were obtained from a single plant variety, they clearly confirm the extreme plasticity of mitochondrial genome structure in response to in-vitro culture.Accepted by D. R. Pring     

A comparative study on variability and phylogeny of Triticum species

Summary Interspecific relationship studies between A, S and D genome diploid species, and between AAGG and AABB allotetraploid species of the genus Triticum were conducted using isoenzymatic characters located in dry mature seeds. Data was analyzed by the factorial analysis of correspondences, and dendograms were obtained by two different genetic distances. The discussion of results was based on the limitations of the study, intraspecific variability differences, isoperoxidase frequency differences and chromosomal location of peroxidases in T. aestivum cv. Chinese Spring. The closest relationship was found between dicoccoides and carthlicum. Relationships found between T. turgidum L. and T. timopheevvi Zhuk., both allotetraploid species and boeoticum; this species and speltoides; tauschi and searsii, and the last two species with bicorne, are discussed at the phylogenetic level.     

The presence of the enhanced K/Na discrimination trait in diploid Triticum species

Summary A number of accessions of the three species of diploid wheat, Triticum boeoticum, T. monococcum, and T. urartu, were grown in 50 mol m^-3 NaCl+2.5 mol m^-3 CaCl₂. Sodium accumulation in the leaves was low and potassium concentrations remained high. This was not the case in T. durum grown under the same conditions, and indicates the presence in diploid wheats of the enhanced K/Na discrimination character which has previously been found in Aegilops squarrosa and hexaploid wheat. None of the accessions of diploid wheat showed poor K/Na discrimination, which suggests that if the A genome of modern tetraploid wheats was derived from a diploid Triticum species, then the enhanced K/Na discrimination character became altered after the formation of the original allopolyploid. Another possibility is that a diploid wheat that did not have the enhanced K/Na discrimination character was involved in the hybridization event which produced tetraploid wheat, and that this diploid is now extinct or has not yet been discovered.     

Mitochondrial DNA diversity in the genera of Triticum and Aegilops revealed by southern blot hybridization

Summary Southern blot hybridization of total DNA to defined mitochondrial DNA sequences provides a sensitive assay for mtDNA variation in the genera of Triticum and Aegilops. A clear distinction between cytoplasms of tetraploid species sharing the AG haploid genome is reported for the first time. The Sitopsis section of the genus Aegilops showed the most extensive intra- and inter-specific variation, whereas no variation could be detected among the cytoplasms of polyploid Triticum species (wheats) sharing the AB haploid genome. Extensive cytoplasmic intraspecific diversity was revealed in Ae. speltoides.     

Nuclear genotype affects mitochondrial genome organization of CMS-S maize

Summary A WF9 strain of maize with the RD subtype of the S male-sterile cytoplasm (CMS-S) was converted to the inbred M825 nuclear background by recurrent backcrossing. The organization of the mitochondrial genomes of the F₁ and succeeding backcross progenies was analyzed and compared with the progenitor RD-WF9 using probes derived from the S1 and S2 mitochondrial episomes, and probes containing the genes for cytochrome c oxidase subunit I (coxI), cytochrome c oxidase subunit II (coxII) and apocytochrome b (cob). Changes in mitochondrial DNA (mtDNA) organization were observed for S1-, S2-, and coxI-homologous sequences that involve loss of homologous restriction enzyme fragments present in the RD-WF9 progenitor. With the coxI probe, the loss of certain fragments was accompanied by the appearance of a fragment not detectable in the progenitor. The changes observed indicate the effect of the nuclear genome on the differential replication of specific mitochondrial subgenomic entities.     

Variation of genome size and organization within hexaploid Festuca arundinacea

Summary Cytophotometric, karyological, and biochemical analyses were carried out in the meristems of seedlings obtained from seeds collected from 35 natural populations of hexaploid Festuca amndinacea in Italy. Highly significant differences between populations were observed in the amount of nuclear DNA (up to 32.3%). These changes are linked to variations in the amount of heterochromatin and in the frequency of repeated DNA sequences, and particularly of a fraction of them. Differences between populations in the arm ratios and total length of the chromosomes were also observed. The genome sizes of the populations are correlated positively with the mean temperature during the year and with that of the coldest month at the stations, and correlate negatively with their latitudes. The intraspecific genome changes observed are discussed in relation to other pertinent data to be found in the literature and in relation to their possible role in environmental adaptation.     

A simple protocol for micropropagating diploid and tetraploid watermelon using shoot-tip explants

Shoot-tip explants from 21-day-old aseptically-germinated watermelon seedlings were incubated on solidified MS medium containing test concentrations of benzyladenine (BA) and kinetin (each at 0, 1, 5 or 10 µM), and thidiazuron (TDZ; 0, 0.1, 1 or 5 µM) for 8 weeks. Approximately 1.5x–2.8x more axillary shoots formed at the optimum BA level (1 µM) compared to the best TDZ (0.1 µM) or kinetin (10 µM) concentration. The ability of various diploid and tetraploid genotypes to undergo prolonged axillary shoot proliferation on medium with 1 µM BA was examined. Among the genotypes tested, the number of axillary shoots per explant was greater for Bush Jubilee and Jubilee II than for Minilee, Dixielee, and the tetraploid genotypes. For a majority of the genotypes tested, the number of shoots per explant was low (2.7–4.0) during the first month of culture, peaked (5.3–12.5) at 2 to 3 months, and then declined (3.7–7.7) at 6 months. In contrast, the number of shoots per explant was greatest (11.7) for Bush Jubilee during the first month of culture and declined to 7.7 by the sixth subculture. The percentage of rooted shoots varied from 60% to 100% and the percentage of acclimatized plants ranged from 21% to 96% depending on the genotype and the length of time in culture. Using this procedure, 13,200 finished plants could be produced in 3 months from 250 seedlings.This process is protected by United States patent No. 5,007,198. Those interested in using this technology must secure a license from the University of Florida.     

The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants

Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and rps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann     

Characterization of diploid,tetraploid and hexaploid Helianthus species by chromosome banding and FISH with 45S rDNA probe

Comparative karyotype analyses of five diploid, two tetraploid, and three hexaploid species of Helianthuswere performed using Feulgen staining, Giemsa C and CMA₃ (C-CMA) staining, and FISH with 45S rDNA probe. The karyotypes are composed by a basic number of x=17 with a predominance of meta- and submetacentric chromosome types. A polyploid series is associated with the basic number. Giemsa C- and C-CMA banding revealed terminal or interstitial heterochromatin according to the species, suggesting the existence of a mechanism that may be acting in the dispersion of heterochromatic segments in Helianthus. The nucleolar organizer regions were located at terminal chromosome positions by FISH with 45S rDNA probe. Diploid species presented four, six, and eight rDNA sites, tetraploid species showed eight sites and hexaploid species presented 12 rDNA sites. Karyomorphological differences include variation in number, size and chromosome morphology, suggesting that rearrangements involving small heterochromatic and rDNA segments played a major role in karyotype evolution.     

Rearrangements in the mitochondrial genome of somatic hybrid cell lines of Pennisetum americanum (L.) K. Schum. + Panicum maximum Jacq.

Summary Mitochondrial DNA from three somatic hybrid cell lines of Pennisetum americanum + Panicum maximum was compared with mitochondrial DNA of the parents. Gel electrophoresis of BamHI-restricted mitochondrial DNA indicated that extensive rearrangements had occurred in each of the three hybrid lines. The hybrid restriction patterns showed a combination of some bands from each parent plus novel fragments not present in either parent. Additional evidence for rearrangements was obtained by hybridization of eight DNA probes, carrying sequences of known coding regions, to Southern blots. Each of the somatic hybrids exhibited a partial combination of the parental mitochondrial genomes. These data suggest recombination or amplification of the mitochondrial genomes in the somatic hybrids.     

Complete elimination of maternal mitochondrial DNA during meiosis resulting in the paternal inheritance of the mitochondrial genome in Chlamydomonas species

Summary. The non-Mendelian inheritance of organellar DNA is common in most plants and animals. In the isogamous green alga Chlamydomonas species, progeny inherit chloroplast genes from the maternal parent, as paternal chloroplast genes are selectively eliminated in young zygotes. Mitochondrial genes are inherited from the paternal parent. Analogically, maternal mitochondrial DNA (mtDNA) is thought to be selectively eliminated. Nevertheless, it is unclear when this selective elimination occurs. Here, we examined the behaviors of maternal and paternal mtDNAs by various methods during the period between the beginning of zygote formation and zoospore formation. First, we observed the behavior of the organelle nucleoids of living cells by specifically staining DNA with the fluorochrome SYBR Green I and staining mitochondria with 3,3′-dihexyloxacarbocyanine iodide. We also examined the fate of mtDNA of male and female parental origin by real-time PCR, nested PCR with single zygotes, and fluorescence in situ hybridization analysis. The mtDNA of maternal origin was completely eliminated before the first cell nuclear division, probably just before mtDNA synthesis, during meiosis. Therefore, the progeny inherit the remaining paternal mtDNA. We suggest that the complete elimination of maternal mtDNA during meiosis is the primary cause of paternal mitochondrial inheritance. Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 901-0213, Japan.     

The mitochondrial genome of the firefly, Pyrocoelia rufa: complete DNA sequence, genome organization, and phylogenetic analysis with other insects

The complete nucleotide sequences of the mt genome from the firefly, Pyrococelia rufa (Coeleoptera: Lampyridae) was determined. The circular genome is 17,739-bp long, and contains a typical gene complement, order, and arrangement identical to Drosophila yacuba. The presence of 1,724-bp long intergenic spacer in the P. rufa mt genome is unique. The putative initiation codon for ND1 gene appears to be TTG, instead of frequently found ATN. All tRNAs showed stable canonical clover-leaf structure of other mt tRNAs, except for tRNA(Ser) (AGN), DHU arm of which could not form stable stem-loop structure. Phylogenetic analysis among insect orders confirmed a monophyletic Endopterygota, a monophyletic Mecopterida, a monophyletic Diptera, a monophyletic Lepidoptera, and a monophyletic Coleoptera, suggesting that the complete insect mt genome sequence has a resolving power in the diversification events within Endopterygota. However, internal relationships among three coleopteran species are not clear, and the inclusion of some insect orders (i.e., apterygotan T. gertschi) in the analysis provided inconsistent results compared to other molecular studies.     

Phylogenetic relationships of Triticum tauschii,the D genome donor to hexaploid wheat

Summary Isoelectric focusing of seed esterase (Est-5) isozymes in 79 T. tauschii accessions from diverse sources revealed the presence of six different seed esterase phenotypes. In one of these phenotypes, exclusive to a var. meyeri accession (AUS 18989), no detectable enzymatic activity was observed. Segregation in crosses between T. tauschii (D^t) accessions confirmed three of the seed esterase phenotypes to be alleles of the designated Est-D ^t5 gene locus; the inheritance pattern of these isozymes was not affected by the subspecies differences between the parents. On the bases of variation in Est-5 and their Glu-1 and Gli-1 gene loci (in a previous study in this series), only three strangulata accessions showed consistent homology with their prevalent gene expression in the D genome of hexaploid wheat. The implications of these observations for further interpreting the phyletic nature of the D genome donor in natural hexaploid wheat synthesis are also reported.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Electrophoretic analysis of the high-molecular-weight glutenin subunits of Triticum monococcum,T. urartu,and the A genome of bread wheat (T. aestivum)

Summary The high molecular weight (HMW) subunit composition of glutenin was analysed by sodium dodecyl sulphate, polyacrylamide gel electrophoresis (SDS-PAGE) in the A genome of 497 diploid wheats and in 851 landraces of bread wheat. The material comprised 209 accessions of wild Triticum monococcum ssp. boeoticum from Greece, Turkey, Lebanon, Armenia, Iraq, and Iran; 132 accessions of the primitive domesticate T. monococcum ssp. monococcum from many different germplasm collections; one accession of free-threshing T. monococcum ssp. sinskajae; 155 accessions of wild T. urartu from Lebanon, Turkey, Armenia, Iraq, and Iran; and landraces of T. aestivum, mainly from the Mediterranean area and countries bordering on the Himalayan Mountains. Four novel HMW glutenin sub-units were discovered in the landraces of bread wheat, and the alleles that control them were designated Glu-Ald through Glu-Alg, respectively. The HMW subunits of T. monococcum ssp. boeoticum have a major, x subunit of slow mobility and several, less prominent, y subunits of greater mobility, all of which fall within the mobility range of HMW subunits reported for bread wheat. In T. monococcum ssp. monococcum the range of the banding patterns for HMW subunits was similar to that of ssp. boeoticum. However, two accessions, while containing y subunits were null for x subunits. The single accession of Triticum monococcum ssp. sinskajae had a banding pattern similar to that of most ssp. boeoticum and ssp. monococcum accessions. The HMW subunit banding patterns of T. urartu accessions were distinct from those of T. monococcum. All of them contained one major x and most contained one major y subunit. In the other accessions a y subunit was not expressed. The active genes for y subunits, if transferred to bread wheat, may be useful in improving bread-making quality.     

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.     

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang