Complete nucleotide sequence of the Oenothera elata plastid chromosome, representing plastome I of the five distinguishable euoenothera plastomes

We describe the 159,4443-bp sequence of the plastid chromosome of Oenothera elata (evening primrose). The Oe. elata plastid chromosome represents type I of the five genetically distinguishable basic plastomes found in the subsection Euoenothera. The genus Oenothera provides an ideal system in which to address fundamental questions regarding the functional integration of the compartmentalised genetic system characteristic of the eukaryotic cell. Its highly developed taxonomy and genetics, together with a favourable combination of features in its genetic structure (interspecific fertility, stable heterozygous progeny, biparental transmission of organelles, and the phenomenon of complex heterozygosity), allow facile exchanges of nuclei, plastids and mitochondria, as well as individual chromosome pairs, between species. The resulting hybrids or cybrids are usually viable and fertile, but can display various forms of developmental disturbance. Received: 9 January 2000 / Accepted: 29 January 2000     

Gene-dependent male sterility and plastomes in Oenothera

The plastids in the cells of the tapetum in anther of Oenothera are involved in the development of male sterility (mst). We combined nuclear homozygosity for each of the two mst genes with the four different plastomes of Oenothera and demonstrated that in both cases the sterile anther phenotype is independent of the plastome. The experiments provide additional information on competition between megaspores and embryo sacs in the ovule.     

Molecular marker systems for Oenothera genetics

The genus Oenothera has an outstanding scientific tradition. It has been a model for studying aspects of chromosome evolution and speciation, including the impact of plastid nuclear co-evolution. A large collection of strains analyzed during a century of experimental work and unique genetic possibilities allow the exchange of genetically definable plastids, individual or multiple chromosomes, and/or entire haploid genomes (Renner complexes) between species. However, molecular genetic approaches for the genus are largely lacking. In this study, we describe the development of efficient PCR-based marker systems for both the nuclear genome and the plastome. They allow distinguishing individual chromosomes, Renner complexes, plastomes, and subplastomes. We demonstrate their application by monitoring interspecific exchanges of genomes, chromosome pairs, and/or plastids during crossing programs, e.g., to produce plastome-genome incompatible hybrids. Using an appropriate partial permanent translocation heterozygous hybrid, linkage group 7 of the molecular map could be assigned to chromosome 9.8 of the classical Oenothera map. Finally, we provide the first direct molecular evidence that homologous recombination and free segregation of chromosomes in permanent translocation heterozygous strains is suppressed.     

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.     

The complete nucleotide sequences of the 5 genetically distinct plastid genomes of Oenothera, subsection Oenothera: II. A microevolutionary view using bioinformatics and formal genetic data

A unique combination of genetic features and a rich stock of information make the flowering plant genus Oenothera an appealing model to explore the molecular basis of speciation processes including nucleus-organelle coevolution. From representative species, we have recently reported complete nucleotide sequences of the 5 basic and genetically distinguishable plastid chromosomes of subsection Oenothera (I-V). In nature, Oenothera plastid genomes are associated with 6 distinct, either homozygous or heterozygous, diploid nuclear genotypes of the 3 basic genomes A, B, or C. Artificially produced plastome-genome combinations that do not occur naturally often display interspecific plastome-genome incompatibility (PGI). In this study, we compare formal genetic data available from all 30 plastome-genome combinations with sequence differences between the plastomes to uncover potential determinants for interspecific PGI. Consistent with an active role in speciation, a remarkable number of genes have high Ka/Ks ratios. Different from the Solanacean cybrid model Atropa/tobacco, RNA editing seems not to be relevant for PGIs in Oenothera. However, predominantly sequence polymorphisms in intergenic segments are proposed as possible sources for PGI. A single locus, the bidirectional promoter region between psbB and clpP, is suggested to contribute to compartmental PGI in the interspecific AB hybrid containing plastome I (AB-I), consistent with its perturbed photosystem II activity.     

Inactivation of pollen and other effects of genome-plastome incompatibility inOenothera

A series of strains of the homozygous speciesOenothera grandiflora (characterized by the genome BB and plastome III) were combined with plastome IV fromO. parviflora (BC-IV) by means of appropriate crosses. An incompatibility between genome B and plastome IV is expressed in the haplo- and diplophase: (1) B-IV pollen, though normally developed, is largely inactive. The extent of the inactivation varies between different strains and shows a seasonal fluctuation as determined by seed set in outcrossing and selfing experiments. (2) In most of the strains lethality of BB-IV embryos is the rule, leading to empty seeds. This can be ameliorated by including another plastome in the zygotes and developing embryos on account of the biparental plastid transmission inOenothera. It can best be demonstrated in crosses with a seed parent having normal green plastids of plastome IV and mutated chlorophyll deficient plastids from a different plastome in the pollen parent, leading to variegated progeny as well as a remainder of empty seeds. (3) In about one-half of the strains the BB-IV plants exhibit a temporary bleaching of thevirescens type. The incompatibily between genome B and plastome IV does not support the earlier assumption that plastome IV is the ancestor of plastomes II, III, and V. Instead, a precursor plastome is postulated from which plastomes II, III, and IV are descended. While plastome I can be derived from II, only plastome V can be descended from plastome IV.Deceased August 28, 1998.     

Sequence variation in the putative replication origins of the five genetically distinct basicEuoenothera plastid chromosomes (plastomes)

Approximately 4200 nucleotides of the 16S/23S rDNA spacer and the 5′ region flanking therrn operon of the plastid chromosomes representing the five basic, phylogenetically relatedEuoenothera plastomes were sequenced and compared. The sequences that harbor the putative replication origins are almost identical except for a 785-bp intercistronic segment between the genes for the 16S rRNA andtrnI. Differences are mainly caused by insertions/deletions and duplications; the predicted potential for formation of quite extensive secondary structure differs among the plastomes. Unexpected intraplastome variation has also been noted. Furthermore, the sequence-based and published genetically deduced plastome pedigrees differ significantly.     

The first complete plastome sequence of the basal asterid family Styracaceae (Ericales) reveals a large inversion

Plastome sequences are rich sources of information for resolving difficult phylogenetic relationships and provide genomic data for conservation studies. Here, the complete plastome sequence of Alniphyllum eberhardtii Guillaumin is reported, representing the first plastome of the basal asterid family Styracaceae (Ericales). The plastome is 155,384 bp in length and contains 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes, totaling 113 unique genes with 19 genes in the inverted repeat region. Unusual features of the plastome include the presence a large 20-kb inversion in the Large Single-Copy region, the pseudogenization of the accD gene, and the loss of the second intron from clpP. The 20-kb inversion includes 14 genes and has not been previously reported in other Ericales plastomes. Thirty-nine plastid simple sequence repeats (SSRs) that may provide genetic resources for the conservation of this economically import timber plant are characterized. Phylogenetic results inferred from ML and MP analyses of 66 plastid genes and 26 taxa reveal that the Styracaceae are sister to a clade including Actinidiaceae and Ericaceae and suggest that complete plastomes are likely to be very helpful in resolving the basal relationships among Ericales families, which have resisted resolution in smaller phylogenetic data sets.     

A Five-Base-Pair-Deletion in the Gene for the Large Subunit Causes the Lesion in the Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Plastome Mutant Sigma of Oenothera hookeri

The gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) has been mapped on the Oenothera hookeri plastid chromosome. It is located close to the gene for the herbicide-binding “32 kd” protein of the photosystem II reaction center (psbA), at a position different from that found in the ancestral angiosperm type of plastid chromosomes, due to an inversion in the large single-copy region. The gene codes for a polypeptide of 475 amino acid residues corresponding to a molecular mass of 52.7 kd. The deduced amino acid composition diverges by 4.8% from the amino acid sequence of the spinach protein and by 8.2% from that of maize. The corresponding nucleotide sequences differ by 8.5 % and 15 % from each other. The rbcL gene of the RuBPcase/oase-deficient Oenothera plastome mutant sigma contains a TTAAC deletion at amino acid residues 270/271 which introduces a frame shift and an amber stop codon seven triplets later. This lesion which probably arose by slipped mispairing is consistent with the previously observed, virtually full-length mRNA that is decoded into a truncated large subunit polypeptide of approximately 30 kd in vitro and in vivo.     

Passiflora plastome sequencing reveals widespread genomic rearrangements

Although past studies have included Passiflora among angiosperm lineages with highly rearranged plastid genomes (plastomes), knowledge about plastome organization in the genus is limited. So far only one draft and one complete plastome have been published. Expanded sampling of Passiflora plastomes is needed to understand the extent of the genomic rearrangement in the genus, which is also unusual in having biparental plastid inheritance and plastome‐genome incompatibility. We sequenced 15 Passiflora plastomes using either Illumina paired‐end or shotgun cloning and Sanger sequencing approaches. Assembled plastomes were annotated using Dual Organellar GenoMe Annotator (DOGMA) and tRNAscan‐SE. The Populus trichocarpa plastome was used as a reference to estimate genomic rearrangements in Passiflora by performing whole genome alignment in progressiveMauve. The phylogenetic distribution of rearrangements was plotted on the maximum likelihood tree generated from 64 plastid encoded protein genes. Inverted repeat (IR) expansion/contraction and loss of the two largest hypothetical open reading frames, ycf1 and ycf2, account for most plastome size variation, which ranges from 139 262 base pairs (bp) in P. biflora to 161 494 bp in P. pittieri. Passiflora plastomes have experienced numerous inversions, gene and intron losses along with multiple independent IR expansions and contractions resulting in a distinct organization in each of the three subgenera examined. Each Passiflora subgenus has a unique plastome structure in terms of gene content, order and size. The phylogenetic distribution of rearrangements shows that Passiflora has experienced widespread genomic changes, suggesting that such events may not be reliable phylogenetic markers.     

Plastome-Genome Interactions Affect Plastid Transmission in Oenothera

Plastids of Oenothera, the evening primrose, can be transmitted to the progeny from both parents. In a constant nuclear background, the frequency of biparental plastid transmission is determined by the types of plastid genomes (plastomes) involved in the crosses. In this study, the impact of nuclear genomes on plastid inheritance was analyzed. In general, the transmission efficiency of each plastome correlated strongly with its compatibility with the nuclear genome of the progeny, suggesting that plastome-genome interactions can influence plastid transmission by affecting the efficiency of plastid multiplication after fertilization. Lower frequencies of plastid transmission from the paternal side were observed when the pollen had poor vigor due to an incompatible plastome-genome combination, indicating that plastome-genome interactions may also affect the input of plastids at fertilization. Parental traits that affect the process of fertilization can also have an impact on plastid transmission. Crosses using maternal parents with long styles or pollen with relatively low growth capacity resulted in reduced frequencies of paternal plastid transmission. These observations suggest that degeneration of pollen plastids may occur as the time interval between pollination and fertilization is lengthened.     

Genetic markers in Andean Puya species (Bromeliaceae) with implications on plastome evolution and phylogeny

The Andean plant endemic Puya is a striking example of recent and rapid diversification from central Chile to the northern Andes, tracking mountain uplift. This study generated 12 complete plastomes representing nine Puya species and compared them to five published plastomes for their features, genomic evolution, and phylogeny. The total size of the Puya plastomes ranged from 159,542 to 159,839 bp with 37.3%–37.4% GC content. The Puya plastomes were highly conserved in organization and structure with a typical quadripartite genome structure. Each of the 17 consensus plastomes harbored 133 genes, including 87 protein‐coding genes, 38 tRNA (transfer RNA) genes, and eight rRNA (ribosomal RNA) genes; we found 69–78 tandem repeats, 45–60 SSRs (simple sequence repeats), and 8–22 repeat structures among 13 species. Four protein‐coding genes were identified under positive site‐specific selection in Puya. The complete plastomes and hypervariable regions collectively provided pronounced species discrimination in Puya and a practical tool for future phylogenetic studies. The reconstructed phylogeny and estimated divergence time for the lineage suggest that the diversification of Puya is related to Andean orogeny and Pleistocene climatic oscillations. This study provides plastome resources for species delimitation and novel phylogenetic and biogeographic studies.     

Comparative analysis of plastomes in Oxalidaceae: Phylogenetic relationships and potential molecular markers

The wood sorrel family, Oxalidaceae, is mainly composed of annual or perennial herbs, a few shrubs, and trees distributed from temperate to tropical zones. Members of Oxalidaceae are of high medicinal, ornamental, and economic value. Despite the rich diversity and value of Oxalidaceae, few molecular markers or plastomes are available for phylogenetic analysis of the family. Here, we reported four new whole plastomes of Oxalidaceae and compared them with plastomes of three species in the family, as well as the plastome of Rourea microphylla in the closely related family Connaraceae. The eight plastomes ranged in length from 150,673 bp (Biophytum sensitivum) to 156,609 bp (R. microphylla). Genome annotations revealed a total of 129–131 genes, including 83–84 protein-coding genes, eight rRNA genes, 37 tRNA genes, and two to three pseudogenes. Comparative analyses showed that the plastomes of these species have minor variations at the gene level. The smaller plastomes of herbs B. sensitivum and three Oxalis species are associated with variations in IR region sizes, intergenic region variation, and gene or intron loss. We identified sequences with high variation that may serve as molecular markers in taxonomic studies of Oxalidaceae. The phylogenetic trees of selected superrosid representatives based on 76 protein-coding genes corroborated the Oxalidaceae position in Oxalidales and supported it as a sister to Connaraceae. Our research also supported the monophyly of the COM (Celastrales, Oxalidales, and Malpighiales) clade.     

Identification of plastid genotypes in Oenothera subsect. Munzia by restriction fragment length polymorphisms (RFLPs)

 Five discrete plastid genotypes (plastomes), designated I–V and typified by Oenothera Hookeri, biennis, Lamarckiana, parviflora and argillicola respectively, have been previously characterized within the European subsect. Euoenothera. The evolutionarily more-derived plastome types (I, II and V) are generally less tolerant of new hybridization events than the ancestral types (III and IV), and were first identified based on their incompatibility reactions with standard hybrid nuclei. Restriction maps for all five plastomes are available for the enzymes PvuII, SalI, KpnI and PstI (Gordon et al. 1982). The present study employs PvuII and KpnI restriction digests to compare 28 of the 45 species of subsect. Munzia with Euoenothera plastomes I–V. The results of plastome RFLP fingerprinting show uniform divergence of the South American taxa from their European congeners; all share the previously documented 45-kb inversion in the large single-copy region reported by Hachtel et al. (1991). However, at least six new plastome types have evolved within subsect. Munzia, giving rise to small-fragment size differences of 0.1–0.7 kb. In two of these cases (Oe. featherstonei and Oe. longiflora) unique fragments occurred. For Oe. featherstonei the unique KpnI fragment resulted from a novel 2.2 kb insertion, whereas in Oe. longiflora an additional PvuII restriction site has been created. Received: 2 June 1998 / Accepted: 14 July 1998     

Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication

The spacer between the 16S and 23S rRNA genes of the chloroplast DNA has been implicated as an origin of replication in several species of plants. In the evening primrose, Oenothera, this site was found to vary greatly in size, with plastid genomes (plastomes) being readily distinguished. To determine whether plastome "strength" in transmission could be correlated with variation at oriB, the 16S rRNA-trnI spacer was sequenced from five plastomes. The size variation was found to be due to differential amplification (and deletion) of combinations of sequences belonging to seven families of direct repeats. From these comparisons, one short series of direct repeats and one region capable of forming a hairpin structure were identified as candidates for the factor that could be responsible for the differences between strong and weak plastome types. Ample sequence variation allowed phylogenetic inferences to be made about the relationships among the plastomes. Phylogenetic trees also could be constructed for most of the families of direct repeats. The amplifications and deletions of repeats that account for the size variation at oriB are proposed to have occurred through extensive replication slippage at this site.      

Complete Plastid Genome Sequence of the Basal Asterid Ardisia polysticta Miq. and Comparative Analyses of Asterid Plastid Genomes

Ardisia is a basal asterid genus well known for its medicinal values and has the potential for development of novel phytopharmaceuticals. In this genus of nearly 500 species, many ornamental species are commonly grown worldwide and some have become invasive species that caused ecological problems. As there is no completed plastid genome (plastome) sequence in related taxa, we sequenced and characterized the plastome of Ardisia polysticta to find plastid markers of potential utility for phylogenetic analyses at low taxonomic levels. The complete A. polysticta plastome is 156,506 bp in length and has gene content and organization typical of most asterids and other angiosperms. We identified seven intergenic regions as potentially informative markers with resolution for interspecific relationships. Additionally, we characterized the diversity of asterid plastomes with respect to GC content, plastome organization, gene content, and repetitive sequences through comparative analyses. The results demonstrated that the genome organizations near the boundaries between inverted repeats (IRs) and single-copy regions (SCs) are polymorphic. The boundary organization found in Ardisia appears to be the most common type among asterids, while six other types are also found in various asterid lineages. In general, the repetitive sequences in genic regions tend to be more conserved, whereas those in noncoding regions are usually lineage-specific. Finally, we inferred the whole-plastome phylogeny with the available asterid sequences. With the improvement in taxon sampling of asterid orders and families, our result highlights the uncertainty of the position of Gentianales within euasterids I.     

The complete plastome of Panax stipuleanatus: Comparative and phylogenetic analyses of the genus Panax (Araliaceae)

Panax stipuleanatus (Araliaceae) is an endangered and medicinally important plant endemic to China. However, phylogenetic relationships within the genus Panax have remained unclear. In this study, we sequenced the complete plastome of P. stipuleanatus and included previously reported Panax plastomes to better understand the relationships between species and plastome evolution within the genus Panax. The plastome of P. stipuleanatus is 156,069 base pairs (bp) in length, consisting of a pair of inverted repeats (IRs, each 25,887 bp) that divide the plastome into a large single copy region (LSC, 86,126 bp) and a small single copy region (SSC, 8169 bp). The plastome contains 114 unigenes (80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes). Comparative analyses indicated that the plastome gene content and order, as well as the expansion/contraction of the IR regions, are all highly conserved within Panax. No significant positive selection in the plastid protein-coding genes was observed across the eight Panax species, suggesting the Panax plastomes may have undergone a strong purifying selection. Our phylogenomic analyses resulted in a phylogeny with high resolution and supports for Panax. Nine proteincoding genes and 10 non-coding regions presented high sequence divergence, which could be useful for identifying different Panax species.     

Exploring the plastid genome disparity of liverworts

Sequencing the plastid genomes of land plants provides crucial improvements to our understanding of the plastome evolution of land plants. Although the number of available complete plastid genome sequences has rapidly increased in the recent years, only a few sequences have been yet released for the three bryophyte lineages, namely hornworts, liverworts, and mosses. Here, we explore the disparity of the plastome structure of liverworts by increasing the number of sequenced liverwort plastomes from five to 18. The expanded sampling included representatives of all major lineages of liverworts including the genus Haplomitrium. The disparity of the liverwort genomes was compared with other 2386 land plant plastomes with emphasis on genome size and GC‐content. We found evidence for structural conservatism of the plastid genomes in liverworts and a trend towards reduced plastome sequence length in liverworts and derived mosses compared to other land plants, including hornworts and basal lineages of mosses. Furthermore, Aneura and Haplomitrium were distinct from other liverworts by an increased GC content, with the one found in Haplomitrium only second to the lycophyte Selaginella. The results suggest the hypothesis that liverworts and other land plants inherited and conserved the plastome structure of their most recent algal ancestors.