A Solanum (+) Potentilla interfamily protoplast regenerant with Solanum characteristics but with a Potentilla plastome fraction

A Potentilla -specific plastome fraction in a predominantly Solanum -type regenerant from a protoplast co-culture was detected by RFLP. Fifty variegated lines from mixed protoplast populations of wildtype Potentilla caulescens and a Solanum hybrid containing albino-mutant potato plastids were studied. The nuclear genome of all lines was uniparental Solanum , as indicated by morphology, isozyme patterns and chromosome number. Heterofamily plastid DNA was detected in one line by using 6 plastid DNA probes of barley. All of the Solanum restriction fragments were recovered. Additionally, probes of the psb A gene and 16Sr DNA revealed Potentllia -specific signals, and an ndh E+G gene probe detected a nonparental fragment. Only Solanum -type fragments hybridized with the psh A gene probe in albino sublines. Protoplast-derived clones were either albinic or variegated. It is proposed that the line represents a cybrid with recombinant Solanum (+) Potentilla plastid DNA transmitted in combination with the entire Solanum plastid DNA.     

Homologies between nuclear and plastid DNA in spinach

Summary Homologies between spinach nuclear (n) DNA and Chloroplast (pt) DNA, have been detected with a clone bank of spinach ptDNA as hybridization probes to restriction fragments of nDNA prepared from purified root nuclei. Every cloned fragment of ptDNA showed homologies to discrete restriction fragments of nDNA, different from those of ptDNA, indicating integration of these homologies into nDNA. While most ptDNA clones were relatively large and probably contained several genes, sequence homologies were also found to the cloned plastid gene for RuBP carboxylase and the subunit of ptATPase. Many of the homologies in nDNA occur in regions of the genome that are highly methylated and are not digested by the methylation sensitive restriction endonucleases HpaII and MspI. In contrast these enzymes cleave ptDNA into small fragments which allows the nDNA homologies to be distinguished in total root DNA. The sequence homologies observed were not due to contaminating non nuclear sequences as shown by hybridization to mitochondrial (mt) and bacterial DNAs. The total amount of homology to ptDNA in nDNA is equivalent to about five copies of the plastome per haploid nuclear genome. The homologies generally appear to be in individual segments of less than 2 kbp in length, integrated into several different places in the genome.On sabbatical leave from Department of Botany, University College, Dublin, Ireland     

The plastome of a brown alga,Dictyota dichotoma

Summary Plastids of the brown algaDictyota dichotoma contain a single homogeneous DNA species which bands at a buoyant density of 1.693 g/cm³ in neutral CsCl equilibrium density gradients. The corresponding nuclear DNA has a density of 1.715 g/cm³. The molecular size of the plastid DNA is 123 kbp as calculated by both electron microscopy of spread intact circular molecules and gel electrophoresis following single and double digestions with various restriction enzymes. A restriction map has been constructed using the endonucleases Sal I, Bam HI, and Bgl II which cleave theDictyota plastome into 6, 12, and 17 fragments, respectively. No large repeated regions, as found in chlorophycean andEuglena plastid DNAs, were detected.Dictyota dichotoma is the first member from the chlorophyll c-line of the algal pedigree for which a physical map of plastid DNA has been established. Dedicated to Professor Dr. W. Stubbe on the occasion of his 65th birthday.     

Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis

Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.     

Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

Testing and using complete plastomes and ribosomal DNA sequences as the next generation DNA barcodes in Panax (Araliaceae)

Complete plastid genome (plastome) sequences and nuclear ribosomal DNA (nrDNA) regions have been proposed as candidates for the next generation of DNA barcodes for plant species discrimination. However, the efficacy of this approach still lacks comprehensive evaluation. We carried out a case study in the economically important but phylogenetically and taxonomically difficult genus Panax (Araliaceae). We generated a large data set of plastomes and nrDNA sequences from multiple accessions per species. Our data improved the phylogenetic resolution and levels of species discrimination in Panax, compared to any previous studies using standard DNA barcodes. This provides new insights into the speciation, lineage diversification and biogeography of the genus. However, both plastome and nrDNA failed to completely resolve the phylogenetic relationships in the Panax bipinnatifidus species complex, and only half of the species within it were recovered as monophyletic units. The results suggest that complete plastome and ribosomal DNA sequences can substantially increase species discriminatory power in plants, but they are not powerful enough to fully resolve phylogenetic relationships and discriminate all species, particularly in evolutionarily young and complex plant groups. To gain further resolving power for closely related species, the addition of substantial numbers of nuclear markers is likely to be required.     

In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501

Summary The tobacco (Nicotiana tabacum) nuclear genome contains long tracts of DNA (i.e. in excess of 18 kb) with high sequence homology to the tobacco plastid genome. Five lambda clones containing these nuclear DNA sequences encompass more than one-third of the tobacco plastid genome. The absolute size of these five integrants is unknown but potentially includes uninterrupted sequences that are as large as the plastid genome itself. An additional sequence was cloned consisting of both nuclear and plastid-derived DNA sequences. The nuclear component of the clone is part of a family of repeats, which are present in about 400 locations in the nuclear genome. The homologous sequences present in chromosomal DNA were very similar to those of the corresponding sequences in the plastid genome. However significant sequence divergence, including base substitutions, insertions and deletions of up to 41 bp, was observed between these nuclear sequences and the plastid genome. Associated with the larger deletions were sequence motifs suggesting that processes such as DNA replication slippage and excision of hairpin loops may have been involved in deletion formation.     

Newly evolved highly repeated DNA sequences ofTupaia glis (Tupaiidae,Scandentia)

We have studied highly repeated DNA sequences ofTupaia glis (Tupaiidae, Scandentia) with restriction endonucleases and Southern blotting techniques. Five highly repeated DNA fragments have been isolated fromT. glis and hybridized with genomic DNAs (cleaved by different restriction enzymes) of several non-human primate species and one insectivore (E. europaeus), in order to highlight eventual differences or similarities of their highly repeated DNA sequences. Our first preliminary findings suggest that the newly isolated highly repeated DNA fragments ofT. glis are distinct from both non-human primates and insectivore, the two taxonomic groups considered most similar to the Tupaiidae.     

Clone bank and physical and genetic map of potato chloroplast DNA

Summary Clone banks of PvuII, BamHI and XhoI fragments were generated of the Solanum tuberosum cv Katahdin plastome. These clone banks, in conjunction with molecular hybridization to tobacco ctDNA probes, were used to construct a physical map of potato ctDNA. The potato plastome was found to be a circular molecule of 155–156 Kbp containing two inverted repeat regions of 23–27 Kbp. The arrangement of restriction sites is very similar to that of other Solanaceae plastomes. Heterologous hybridization to known ctDNA encoded gene probes from tobacco allowed us to establish a genetic map of the potato chloroplast genome. The arrangement of these genes on the potato plastome resembles that on most higher plant ctDNAs.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Genomic organisation of DNA segments containing foldback sequences

DNA clones containing foldback sequences, derived from Physarum polycephalum nuclear DNA, can be classified according to their pattern of hydridisation to Southern blots of genomic DNA. One group of DNA clones map to unique DNA loci when used as a probe to restriction digests of Physarum nuclear DNA. These cloned segments appear to contain dispersed repetitive sequence elements located at many hundreds of sites in the genome. Similar patterns of hybridisation are generated when these cloned DNA probes are annealed to DNA restriction fragments of genomic DNA obtained from a number of different Physarum strains, indicating that no detectable alteration has occurred at these genomic loci subsequent to the divergence of the strains as a result of the introduction or deletion of mobile genetic elements. However, deletion of segments of some cloned DNA fragments occurs following their propagation in Escherichia coli. A second, distinct group of clones are shown to be derived from highly methylated segments of Physarum DNA which contain very abundant repetitive sequences with regular, though complex, arrangements of restriction sites at their various genomic locations. It is suggested that these DNA segments contain clustered repetitive sequence elements. The results lead to the conclusion that foldback elements in Physarum DNA are located in segments of the genome which display markedly different patterns of sequence organisation and degree of DNA methylation.     

Chloroplast DNA differences between two species of Oenothera subsection Munzia: location in the chloroplast genome and relevance to possible interactions between nuclear and plastid genomes

Summary The chloroplast DNAs (cpDNAs) of Oenothera berteriana and Oe. odorata (subsection Munzia) were examined by restriction endonuclease analysis with Sal I, Pvu II, Kpn I, Pst I, Hind III, and Bam HI. The fragment patterns show that these cpDNAs have all 133 restriction sites in common as well as a lot of individual bands. Nevertheless the cpDNAs of the two species can be distinguished by distinct differences in size between a small number of fragments. The 42 cleavage sites produced by Sal I, Pvu II and Kpn I were mapped on the circular cpDNAs. This was achieved by an approach which combined experimental and mathematical procedures. The overall serial order of the fragments was found to be the same for both cpDNAs. The size differences of individual fragments in the Sal I, Pvu II and Kpn I patterns between Oe. berteriana and Oe. odorata cpDNA are located within five regions scattered along the plastid chromosome. Two of these regions have been localized in the larger and one in the smaller of the two single-copy parts of the cpDNA molecule. The remaining two overlap the borders between the large single-copy and each of the duplicated parts of the molecule. The positions of distinct restriction sites are altered among the two Oenothera plastome DNAs by 0.02–0.4 MDa (30–600 base pairs). These alterations probably result from insertions/deletions.Abbreviations cpDNA chloroplast, plastid DNA - Oe. Oenothera - MDa Megadalton - rRNA, rDNA ribosomal RNA, DNA Dedicated to Professor Berthold Schwemmle, Tübingen, on the occasion of his 60th birthday     

Evaluation of the Extent of Homologous Chloroplast DNA Sequences in the Mitochondrial Genome of Cowpea (Vigna unguiculata L.)

Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs.     

Recombinant DNA analysis indicates that the multiple chromosomes of maize mitochondria contain different sequences

Twenty-eight Bam H 1 restriction fragments were isolated from normal mitochondrial DNA of maize by recombinant DNA techniques to investigate the organization of the mitochondrial genome. Each cloned fragment was tested by molecular hybridization against a Bam digest of total mitochondrial DNA. Using Southern transfers, we identified the normal fragment of origin for d each clone. Twenty-three of the tested clones hybridized only to the fragment from which the clone was derived. In five cases, labeling of an additional band indicated some sequence repetition in the mitochondrial genome. Four clones from normal mitochondrial DNA were found which share sequences with the plasmid-like DNAs, S-1 and S-2, found in S male sterile cytoplasm. The total sequence complexity of the clones tested is 121×10⁶ d (daltons), which approximates two thirds of the total mitochondrial genome (estimated at 183×10⁶ d). Most fragments do not share homology with other fragments, and the total length of unique fragments exceeds that of the largest circular molecules observed. Therefore, the different size classes of circular molecules most likely represent genetically discrete chromosomes in a complex organelle genome. The variable abundance of different mitochondrial chromosomes is of special interest because it represents an unusual mechanism for the control of gene expression by regulation of gene copy number. This mechanism may play an important role in metabolism or biogenesis of mitochondria in the development of higher plants.     

Analysis of the plastid DNA in an Oenothera plastome mutant deficient in ribulose bisphosphate carboxylase

Summary Plastid DNA of the light green Oenothera plastome mutant sigma, from plastome I, which is deficient in ribulose bisphosphate carboxylase, has been compared with wild-type chloroplast DNA from plastome I and the related plastome IV. For this, double digestions with the restriction endonucleases Sal I, Pst I and Kpn I were used. Chloroplast DNA from plastomes I and IV differs in the sizes of several fragments, with the changes being from under 0.1 to about 0.6 Md in size. In the cleavage patterns of the mutant DNA compared to the wild-type DNA from plastome I, the only differences observed are two possible deletions of less than 0.1 Md from a fragment known to partly cover the genes for the ribosomal RNAs and from a fragment located in the small single-copy region of the molecule. It is concluded that the ribulose bisphosphate carboxylase deficiency in this mutant is not caused by a major deletion in the plastid DNA.     

Comparative analysis of 343 plastid genomes of Solanum section Petota: Insights into potato diversity,phylogeny, and species discrimination

Common potato (Solanum tuberosum L.) and its wild relatives belong to Solanum section Petota. This section's phylogeny and species delimitation are complicated due to various ploidy levels, high heterozygosity, and frequent interspecific hybridization. Compared to the nuclear genome, the plastid genome is more conserved, has a haploid nature, and has a lower nucleotide substitution rate, providing informative alternative insights into the phylogenetic study of section Petota. Here, we analyzed 343 potato plastid genomes from 53 wild and four cultivated species. The diversity of sequences and genomes was comprehensively analyzed. A total of 24 species were placed in a phylogenetic tree based on genomic data for the first time. Overall, our results not only confirmed most existing clades and species boundaries inferred by nuclear evidence but also provided some distinctive species clade belonging and the maternally inherited evidence supporting the hybrid origin of some species. Furthermore, the divergence times between the major potato clades were estimated. In addition, the species discriminatory power of universal barcodes, nuclear ribosomal DNA, and whole and partial plastid genomes and their combinations were thoroughly evaluated; the plastid genome performed best but had limited discriminatory power for all survey species (40%). Overall, our study provided not only new insights into phylogeny and DNA barcoding of potato but also provided valuable genetic data resources for further systematical research of Petota.     

Abundance of plastid DNA insertions in nuclear genomes of rice and Arabidopsis

Pairwise comparison of whole plastid and draft nuclear genomic sequences of Arabidopsis thaliana and Oryza sativa L. ssp. indica shows that rice nuclear genomic sequences contain homologs of plastid DNA covering about 94 kb (83%) of plastid genome and including one or more full-length intact (without mutations resulting in premature stop codons) homologues of 26 known protein-coding (KPC) plastid genes. By contrast, only about 20 kb (16%) of chloroplast DNA, including a single intact plastid-derived KPC gene, is presented in the nucleus of A. thaliana. Sixteen rice plastid genes have at least one nuclear copy without any mutation or with only synonymous substitutions. Nuclear copies for other ten plastid genes contain both synonymous and non-synonymous substitutions. Multiple ESTs for 25 out of 26 KPC genes were also found, as well as putative promoters for some of them. The study of substitutions pattern shows that some of nuclear homologues of plastid genes may be functional and/or are under the pressure of the positive natural selection. The similar comparative analysis performed on rice chromosome 1 revealed 27 contigs containing plastid-derived sequences, totalling about 84 kb and covering two thirds of chloroplast DNA, with the intact nuclear copies of 26 different KPC genes. One of these contigs, AP003280, includes almost 57 kb (45%) of chloroplast genome with the intact copies of 22 KPC genes. At the same time, we observed that relative locations of homologues in plastid DNA and the nuclear genome are significantly different.     

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.     

Complex organization of zein genes in maize

We have examined the fragments of maize nuclear DNA that are homologous to three cloned cDNAs prepared from zein mRNA. Southern blots of high molecular weight ( > 40 kb) maize nuclear DNA cleaved with BamHI, HindIII or EcoRI were hybridized to three zein cDNA plasmid probes. Multiple restriction fragments in a wide range of size classes were observed to hybridize with all three probes. Our results indicate the occurrence of families of genes in the maize genome that are related by their sequences to a given zein mRNA sequence.     

Conservation of human Y chromosome sequences among male great apes: Implications for the evolution of Y chromosomes

Nine newly described single-copy and lowcopy-number genomic DNA sequences isolated from a flow-sorted human Y chromosome library were mapped to regions of the human Y chromosome and were hybridized to Southern blots of male and female great ape genomic DNAs (Gorilla gorilla, Pan troglodytes, Pongo pygmaeus). Eight of the nine sequences mapped to the euchromatic Y long arm (Yq) in humans, and the ninth mapped to the short arm or pericentromeric region. All nine of the newly identified sequences and two additional human Yq sequences hybridized to restriction fragments in male but not female genomic DNA from the great apes, indicating Y chromosome localization. Seven of these 11 human Yq sequences hybridized to similarly-sized restriction endonuclease fragments in all the great ape species analyzed. The five human sequences that mapped to the most distal subregion of Yq (deletion of which region is associated with spermatogenic failure in humans) were hybridized to Southern blots generated by pulsed-field gel electrophoresis. These sequences define a region of approximately 1 Mb on human Yq in which HpaII tiny fragment (HTF) islands appear to be absent. The conservation of these human Yq sequences on great ape Y chromosomes indicates a greater stability in this region of the Y than has been previously described for most anonymous human Y chromosomal sequences. The stability of these sequences on great ape Y chromosomes seems remarkable given that this region of the Y does not undergo meiotic recombination and the sequences do not appear to encode genes for which positive selection might occur. Correspondence to: B. Steele Allen