Homeologous Plastid DNA Transformation in Tobacco Is Mediated by Multiple Recombination Events

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids

Scorzonera sensu lato (Asteraceae, Cichorieae) – taxonomic reassessment in the light of new molecular phylogenetic and carpological analyses

Scorzonera comprises 180–190 species and belongs to the subtribe Scorzonerinae. Its circumscription has long been the subject of debate and available molecular phylogenetic analyses affirmed the polyphyly of Scorzonera in its wide sense. We provide a re-evaluation of Scorzonera and other related genera, based on carpological (including anatomical) and extended molecular phylogenetic analyses. We present, for the first time, a comprehensive sampling, including Scorzonera in its widest sense and all other genera recognised in the Scorzonerinae. We conducted phylogenetic analyses using Maximum Parsimony, Maximum Likelihood and Bayesian analyses, based on sequences of the nuclear ribosomal ITS and of two plastid markers (partial rbcL and matK) and Maximum Parsimony for reconstructing the carpological character states at ancestral nodes. Achene characters, especially related to pericarp anatomy, such as general topography of the tissue types, disposition of the mechanical tissue and direction of its fibres, presence or absence of air cavities, provide, in certain cases, support for the phylogenetic lineages revealed. Confirming the polyphyly of Scorzonera, we propose a revised classification of the subtribe, accepting the genera Scorzonera (including four major clades: Scorzonera s. str., S. purpurea, S. albicaulis and Podospermum), Gelasia, Lipschitzia gen. nov. (for the Scorzonera divaricata clade), Pseudopodospermum, Pterachaenia (also including Scorzonera codringtonii), Ramaliella gen. nov. (for the S. polyclada clade) and Takhtajaniantha. A key to the revised genera and a characterisation of the genera and major clades are provided

The Toxoplasma gondii plastid replication and repair enzyme complex, PREX

A plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein to be active. Consistent with the reported absence of a plastid in Cryptosporidium species, in silico analysis of their genomes failed to demonstrate an orthologue of prex. These studies indicate that prex is conserved across the plastid-bearing apicomplexans and may play an important role in the replication of the plastid genome

Evolutionary relationships in Panicoid grasses based on plastome phylogenomics (Panicoideae; Poaceae)

Background: Panicoideae are the second largest subfamily in Poaceae (grass family), with 212 genera and approximately 3316 species. Previous studies have begun to reveal relationships within the subfamily, but largely lack resolution and/or robust support for certain tribal and subtribal groups. This study aims to resolve these relationships, as well as characterize a putative mitochondrial insert in one linage. Results: 35 newly sequenced Panicoideae plastomes were combined in a phylogenomic study with 37 other species: 15 Panicoideae and 22 from outgroups. A robust Panicoideae topology largely congruent with previous studies was obtained, but with some incongruences with previously reported subtribal relationships. A mitochondrial DNA (mtDNA) to plastid DNA (ptDNA) transfer was discovered in the Paspalum lineage. Conclusions: The phylogenomic analysis returned a topology that largely supports previous studies. Five previously recognized subtribes appear on the topology to be non-monophyletic. Additionally, evidence for mtDNA to ptDNA transfer was identified in both Paspalum fimbriatum and P. dilatatum, and suggests a single rare event that took place in a common progenitor. Finally, the framework from this study can guide larger whole plastome sampling to discern the relationships in Cyperochloeae, Steyermarkochloeae, Gynerieae, and other incertae sedis taxa that are weakly supported or unresolved.Fil: Burke, Sean V.. Northern Illinois University; Estados UnidosFil: Wysocki, William P.. Northern Illinois University; Estados UnidosFil: Zuloaga, Fernando Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion. Academia Nacional de Ciencias Exactas, Físicas y Naturales. Instituto de Botánica Darwinion; ArgentinaFil: Craine, Joseph M.. Jonah Ventures; Estados UnidosFil: Pires, J. Chris. University of Missouri; Estados UnidosFil: Edger, Patrick P.. Michigan State University; Estados UnidosFil: Mayfield Jones, Dustin. Donald Danforth Plant Science Center; Estados UnidosFil: Clark, Lynn G.. Iowa State University; Estados UnidosFil: Kelchner, Scot A.. University of Idaho; Estados UnidosFil: Duvall, Melvin R.. Northern Illinois University; Estados Unido

Genetic diversity in Cypripedium calceolus (Orchidaceae) with a focus on north-western Europe, as revealed by plastid DNA length polymorphisms

Background and Aims Cypripedium calceolus, although widespread in Eurasia, is rare in many countries in which it occurs. Population genetics studies with nuclear DNA markers on this species have been hampered by its large nuclear genome size. Plastid DNA markers are used here to gain an understanding of variation within and between populations and of biogeographical patterns. Methods Thirteen length-variable regions (microsatellites and insertions/deletions) were identified in non-coding plastid DNA. These and a previously identified complex microsatellite in the trnL-trnF intergenic spacer were used to identify plastid DNA haplotypes for European samples, with sampling focused on England, Denmark and Sweden. Key Results The 13 additional length-variable regions identified were two homopolymer (polyA) repeats in the rps16 intron and a homopolymer (polyA) repeat and ten indels in the accD-psa1 intergenic spacer. In accD-psa1, most of these were in an extremely AT-rich region, and it was not possible to design primers in the flanking regions; therefore, the whole intergenic spacer was sequenced. Together, these new regions and the trnL-trnF complex microsatellite allowed 23 haplotypes to be characterized. Many were found in only one or a few samples (probably due to low sampling density), but some commoner haplotypes were widespread. Most of the genetic variation was found within rather than between populations (83 vs. 18%, respectively). Two haplotypes occurred from the Spanish Pyrenees to Sweden. Conclusions Plastid DNA data can be used to gain an understanding of patterns of genetic variation and seed-mediated gene flow in orchids. Although these data are less information-rich than those for nuclear DNA, they present a useful option for studying species with large genomes. Here they support the hypothesis of long-distance seed dispersal often proposed for orchid

Phylogeny of <i>Potamogeton</i> (Potamogetonaceae) revisited: implications for hybridization and introgression in Argentina

Potamogeton is a cosmopolitan genus of 90–95 species in which numerous hybridization events have occurred worldwide. A plant recently collected from Argentina exhibited ambiguous morphology that does not match any species of the genus. We aimed to assess if the plant coexisting with another Potamogeton species is a product of reticulate evolution. A concatenated plastid DNA (psbA-trnH, trnL intron, and trnL-trnF) and nuclear ribosomal DNA (5S-NTS) data sets, primarily based on previous studies sample set mainly consisting of American and Asian species, were analyzed using Bayesian inference. Nuclear ribosomal ITS sequences were also obtained from five Argentina materials. We recovered similar topologies from both the plastid DNA and nuclear ribosomal 5S-NTS analyses in which most specimens are consistently placed. The specimen of primal interest from Argentina strongly clustered with co-occurring linear-leaved species in the 5S-NTS tree, but was genetically identical to broad-leaved ones in the plastid DNA analysis. The ITS sequence of the specimen was the same as that of the linear-leaved species and no polymorphisms were observed. Considering the discrepant phylogenetic positions between the trees and lack of ITS infra-individual variations, the origin of the specimen from Argentina is better explained by hybridization and subsequent introgression.Facultad de Ciencias Naturales y Muse

Phylogeny of <i>Potamogeton</i> (Potamogetonaceae) revisited: implications for hybridization and introgression in Argentina

Potamogeton is a cosmopolitan genus of 90–95 species in which numerous hybridization events have occurred worldwide. A plant recently collected from Argentina exhibited ambiguous morphology that does not match any species of the genus. We aimed to assess if the plant coexisting with another Potamogeton species is a product of reticulate evolution. A concatenated plastid DNA (psbA-trnH, trnL intron, and trnL-trnF) and nuclear ribosomal DNA (5S-NTS) data sets, primarily based on previous studies sample set mainly consisting of American and Asian species, were analyzed using Bayesian inference. Nuclear ribosomal ITS sequences were also obtained from five Argentina materials. We recovered similar topologies from both the plastid DNA and nuclear ribosomal 5S-NTS analyses in which most specimens are consistently placed. The specimen of primal interest from Argentina strongly clustered with co-occurring linear-leaved species in the 5S-NTS tree, but was genetically identical to broad-leaved ones in the plastid DNA analysis. The ITS sequence of the specimen was the same as that of the linear-leaved species and no polymorphisms were observed. Considering the discrepant phylogenetic positions between the trees and lack of ITS infra-individual variations, the origin of the specimen from Argentina is better explained by hybridization and subsequent introgression.Facultad de Ciencias Naturales y Muse

Phylogenetics of Olea (Oleaceae) based on plastid and nuclear ribosomal DNA sequences: Tertiary climatic shifts and lineage differentiation times

Background and Aims The genus Olea (Oleaceae) includes approx. 40 taxa of evergreen shrubs and trees classified in three subgenera, Olea, Paniculatae and Tetrapilus, the first of which has two sections (Olea and Ligustroides). Olive trees (the O. europaea complex) have been the subject of intensive research, whereas little is known about the phylogenetic relationships among the other species. To clarify the biogeographical history of this group, a molecular analysis of Olea and related genera of Oleaceae is thus necessary. Methods A phylogeny was built of Olea and related genera based on sequences of the nuclear ribosomal internal transcribed spacer-1 and four plastid regions. Lineage divergence and the evolution of abaxial peltate scales, the latter character linked to drought adaptation, were dated using a Bayesian method. Key Results Olea is polyphyletic, with O. ambrensis and subgenus Tetrapilus not sharing a most recent common ancestor with the main Olea clade. Partial incongruence between nuclear and plastid phylogenetic reconstructions suggests a reticulation process in the evolution of subgenus Olea. Estimates of divergence times for major groups of Olea during the Tertiary were obtained. Conclusions This study indicates the necessity of revising current taxonomic boundaries in Olea. The results also suggest that main lines of evolution were promoted by major Tertiary climatic shifts: (1) the split between subgenera Olea and Paniculatae appears to have taken place at the Miocene-Oligocene boundary; (2) the separation of sections Ligustroides and Olea may have occurred during the Early Miocene following the Mi-1 glaciation; and (3) the diversification within these sections (and the origin of dense abaxial indumentum in section Olea) was concomitant with the aridification of Africa in the Late Miocen