Extensive Rearrangements in the Chloroplast Genome of <Emphasis Type="Italic">Trachelium caeruleum</Emphasis> Are Associated with Repeats and tRNA Genes

Chloroplast genome organization, gene order, and content are highly conserved among land plants. We sequenced the chloroplast genome of Trachelium caeruleum L. (Campanulaceae), a member of an angiosperm family known for highly rearranged genomes. The total genome size is 162,321 bp, with an inverted repeat (IR) of 27,273 bp, large single-copy (LSC) region of 100,114 bp, and small single-copy (SSC) region of 7,661 bp. The genome encodes 112 different genes, with 17 duplicated in the IR, a tRNA gene (trnI-cau) duplicated once in the LSC region, and a protein-coding gene (psbJ) with two duplicate copies, for a total of 132 putatively intact genes. ndhK may be a pseudogene with internal stop codons, and clpP, ycf1, and ycf2 are so highly diverged that they also may be pseudogenes. ycf15, rpl23, infA, and accD are truncated and likely nonfunctional. The most conspicuous feature of the Trachelium genome is the presence of 18 internally unrearranged blocks of genes inverted or relocated within the genome relative to the ancestral gene order of angiosperm chloroplast genomes. Recombination between repeats or tRNA genes has been suggested as a mechanism of chloroplast genome rearrangements. The Trachelium chloroplast genome shares with Pelargonium and Jasminum both a higher number of repeats and larger repeated sequences in comparison to eight other angiosperm chloroplast genomes, and these are concentrated near rearrangement endpoints. Genes for tRNAs occur at many but not all inversion endpoints, so some combination of repeats and tRNA genes may have mediated these rearrangements.     

The chloroplast genome from a lycophyte (microphyllophyte), <Emphasis Type="Italic">Selaginella uncinata</Emphasis>, has a unique inversion,transpositions and many gene losses

We determined the complete nucleotide sequence of the chloroplast genome of Selaginella uncinata, a lycophyte belonging to the basal lineage of the vascular plants. The circular double-stranded DNA is 144,170 bp, with an inverted repeat of 25,578 bp separated by a large single copy region (LSC) of 77,706 bp and a small single copy region (SSC) of 40,886 bp. We assigned 81 protein-coding genes including four pseudogenes, four rRNA genes and only 12 tRNA genes. Four genes, rps15, rps16, rpl32 and ycf10, found in most chloroplast genomes in land plants were not present in S. uncinata. While gene order and arrangement of the chloroplast genome of another lycophyte, Hupertzia lucidula, are almost the same as those of bryophytes, those of S. uncinata differ considerably from the typical structure of bryophytes with respect to the presence of a unique 20 kb inversion within the LSC, transposition of two segments from the LSC to the SSC and many gene losses. Thus, the organization of the S. uncinata chloroplast genome provides a new insight into the evolution of lycophytes, which were separated from euphyllophytes approximately 400 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis

The complete nucleotide sequence of mulberry (Morus indica cv. K2) chloroplast genome (158,484 bp) has been determined using a combination of long PCR and shotgun-based approaches. This is the third angiosperm tree species whose plastome sequence has been completely deciphered. The circular double-stranded molecule comprises of two identical inverted repeats (25,678 bp each) separating a large and a small single-copy region of 87,386 bp and 19,742 bp, respectively. A total of 83 protein-coding genes including five genes duplicated in the inverted repeat regions, eight ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids, were assigned on the basis of homology to predicted genes from other chloroplast genomes. The mulberry plastome lacks the genes infA, sprA, and rpl21 and contains two pseudogenes ycf15 and ycf68. Comparative analysis, based on sequence similarity, both at the gene and genome level, indicates Morus to be closer to Cucumis and Lotus, phylogenetically. However, at genome level, inclusion of non-coding regions brings it closer to Eucalyptus, followed by Cucumis. This may reflect differential selection pressure operating on the genic and intergenic regions of the chloroplast genome.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Communicated by Y. Tsumura     

The complete chloroplast genome sequences of Solanum tuberosum and comparative analysis with Solanaceae species identified the presence of a 241-bp deletion in cultivated potato chloroplast DNA sequence

The complete nucleotide sequence of the chloroplast genome of potato Solanum tuberosum L. cv. Desiree was determined. The circular double-stranded DNA, which consists of 155,312 bp, contains a pair of inverted repeat regions (IRa, IRb) of 25,595 bp each. The inverted repeat regions are separated by small and large single copy regions of 18,373 and 85,749 bp, respectively. The genome contains 79 proteins, 30 tRNAs, 4 rRNAs, and unidentified genes. A comparison of chloroplast genomes of seven Solanaceae species revealed that the gene content and their relative positions of S. tuberosum are similar to the other six Solanaceae species. However, undefined open reading frames (ORFs) in LSC region were highly diverged in Solanaceae species except N. sylvestris. Detailed comparison was identified by numerous indels in the intergenic regions that were mostly located in the LSC region. Among them, a single large 241-bp deletion, was not associated with direct repeats and found in only S. tuberosum, clearly discriminates a cultivated potato from wild potato species Solanum bulbocastanum. The extent of sequence divergence may provide the basis for evaluating genetic diversity within the Solanaceae species, and will be useful to examine the evolutionary processes in potato landraces.     

Probabilistic Analysis Indicates Discordant Gene Trees in Chloroplast Evolution

Abstract Analyses of whole-genome data often reveal that some genes have evolutionary histories that diverge from the majority phylogeny estimated for the entire genome. We present a probabilistic model that deals with heterogeneity among gene trees, implement it via the Gibbs sampler, and apply it to the plastid genome. Plastids and their genomes are transmitted as a single block without recombination, hence homogeneity among gene trees within this genome is expected. Nevertheless, previous work has revealed clear heterogeneity among plastid genes (e.g., Delwiche and Palmer 1996). Other studies, using whole plastid genomes of various algae and land plants, found little additional heterogeneity (Martin et al. 1998; Adachi et al. 2000). We augment the earlier studies by using a data set of 14 taxa: 6 land plants, 2 green algae, a diatom, 2 red algae and a cryptophyte, the cyanelle of the glaucocystophyte Cyanophora, and the blue–green alga Synechocystis as an outgroup. Contrary to the earlier analyses, we cannot find even a single, dominant consensus tree. Therefore, we formulate a probabilistic model that divides the genes into two sets: those that follow the consensus tree and those that have independent gene trees. No particular tree is supported by more than three-fourths of the genes. But the set of genes that follows a certain tree is fairly independent of data processing and the method of analysis. With one possible exception, we find no evidence for collinear or functionally related genes to follow similar trees. The phylogenetic pattern also seems independent of bias in amino acid composition. Among possible explanations for the observed phenomenon, the hypothesis that different genes have different covarion structures is difficult to assess. But gene duplication may be possible through the inverted or direct repeat regions, while horizontal gene transfer seems less likely. In contrast to green algae and land plants, inverted repeat regions in red algae and in Cyanophora show abundant differences among the copies. Thus, genes may get duplicated when they are recruited into the inverted repeat region and one of the two copies may be lost after leaving the inverted repeat region.     

Gene localization on the chloroplast DNA of the maiden hair fern;Adiantum capillus-veneris

Gene maps were constructed for the inverted repeat region and for the adjacent large single copy region of the chloroplast genome of the maiden hair fern,Adiantum capillus-veneris L. Gene order and organization was very different from the typical angiosperm chloroplast genome (e.g. tobacco). Elongation of inverted repeat and a minimum of two inversions must be postulated to account for the unusual genome structure.     

Achlya mitochondrial DNA: gene localization and analysis of inverted repeats

Summary Mitochondrial DNA from four strains of the oomycete Achlya has been compared and nine gene loci mapped, including that of the ribosomal protein gene, var1. Examination of the restriction enzyme site maps showed the presence of four insertions relative to a map common to all four strains. All the insertions were found in close proximity to genic regions. The four strains also cotained the inverted repeat first observed in A. ambisexualis (Hudspeth et al. 1983), allowing an examination by analysis of retained restriction sites of the evolutionary stability of repeated DNA sequences relative to single copy sequences. Although the inverted repeat is significantly more stable than single copy sequences, more detailed analysis indicated that this stability is limited to the portion encoding the ribosomal RNA genes. Thus, the apparent evolutionary stability of the repeat does not appear to derive from the inverted repeat structure per se.Abbreviations ATPase 6, 9 genes for ATPase subunits 6 and 9 - COI, II, III genes for cytochrome oxidase subunits 1, 2, and 3 - COB gene for apocytochrome b - L-, S-RNA genes for the mitochondrial large and small ribosomal RNAs - mtDNA mitochondrial DNA - var1 gene for the S. cerevisiae mitochondrially, encoded ribosomal protein - m.u. map units - bp base pairs - kb kilobase pairs     

ORGANIZATION OF THE CHLOROPLAST GENOME OF THE FRESHWATER CENTRIC DIATOM CYCLOTELLA MENEGHINIANA1

We constructed a complete physical map and a partial gene map of the chloroplast genome of Cyclotella meneghiniana Kützing clone 1020-1a (Bacillariophyceae). The 128-kb circular molecule contains a 17-kb inverted repeat, which divides the genome into single copy regions of65 kb and 29 kb. This is the largest genome and inverted repeat found in any diatom examined to date. In addition to the 16S and 23S ribosomal RNA genes, the inverted repeat contains both the ndhD gene (as yet unexamined in other diatoms) and the psbA gene (located similarly in one of two other examined diatoms). The Cyclotella chloroplast genome exists as two equimolar populations of inversion isomers that differ in the relative orientation of their single copy sequences. This inversion heterogeneity presumably results from intramolecular recombination within the inverted repeat. For the first time, we map the ndhD, psaC, rpofi, rpoCl, and rpoC2 genes to the chloroplast genome of a chlorophyll c-containing alga. While the Cyclotella chloroplast genome retains some prokaryotic and land plant gene clusters and operons, it contains a highly rearranged gene order in the large and small single copy regions compared to all other examined diatom, algal, and land plant chloroplast genomes.     

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.By hybridization with spinach chloroplast rRNAs it could be shown that each of the two repeat units contains one set of rRNA genes in the order: 16S rRNA gene — spacer — 23S rRNA gene — 5S rRNA gene.A spinach chloroplast mRNA preparation (14S RNA), which is predominantly translated into a 32 Kilodalton (Kd) protein [9], hybridized strongly to a DNA fragment in the large single copy region, immediately outside one of the inverted repeats. With another mRNA preparation (18S), which mainly directs the in vitro synthesis of a 55 Kd protein [9], hybridization was observed with two DNA regions, located between 211° and 233° and between 137° and 170°, respectively. Finally, with a spinach chloroplast genomic probe for the large subunit of ribulose 1,5-bisphosphate carboxylase [17], hybridization was found with a DNA fragment located between 137° and 158° on the map.     

Extensive Reorganization of the Plastid Genome of <Emphasis Type="Italic">Trifolium subterraneum</Emphasis> (Fabaceae) Is Associated with Numerous Repeated Sequences and Novel DNA Insertions

The plastid genome of Trifolium subterraneum is 144,763 bp, about 20 kb longer than those of closely related legumes, which also lost one copy of the large inverted repeat (IR). The genome has undergone extensive genomic reconfiguration, including the loss of six genes (accD, infA, rpl22, rps16, rps18, and ycf1) and two introns (clpP and rps12) and numerous gene order changes, attributable to 14–18 inversions. All endpoints of rearranged gene clusters are flanked by repeated sequences, tRNAs, or pseudogenes. One unusual feature of the Trifolium subterraneum genome is the large number of dispersed repeats, which comprise 19.5% (ca. 28 kb) of the genome (versus about 4% for other angiosperms) and account for part of the increase in genome size. Nine genes (psbT, rbcL, clpP, rps3, rpl23, atpB, psbN, trnI-cau, and ycf3) have also been duplicated either partially or completely. rpl23 is the most highly duplicated gene, with portions of this gene duplicated six times. Comparisons of the Trifolium plastid genome with the Plant Repeat Database and searches for flanking inverted repeats suggest that the high incidence of dispersed repeats and rearrangements is not likely the result of transposition. Trifolium has 19.5 kb of unique DNA distributed among 160 fragments ranging in size from 30 to 494 bp, greatly surpassing the other five sequenced legume plastid genomes in novel DNA content. At least some of this unique DNA may represent horizontal transfer from bacterial genomes. These unusual features provide direction for the development of more complex models of plastid genome evolution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chloroplast DNA variation among five species ofRanunculaceae: Structure,sequence divergence,and phylogenetic relationships

A restriction site map of the chloroplast genome ofCaltha palustris L. (Ranunculaceae) has been constructed for 13 restriction endonucleases using filter hybridization with cloned tobacco chloroplast DNA fragments. A size of 153.8 kb has been estimated for theCaltha chloroplast genome. Forty-six chloroplast genes and four open reading frames have been mapped using small tobacco chloroplast gene probes. Chloroplast DNA sequence divergence has been estimated for all pairs of five species ofRanunculaceae, Caltha palustris, Ranunculus bulbosus, R. fascicularis, R. recurvatus, andTrollius ledebourii, and ranges between 0.2% and 9.6% for the total genome. Divergence values are much higher in the small and large single copy regions than in the inverted repeat. Phylogenetic relationships between the five species have been hypothesized using chloroplast DNA restriction site mapping. One hundred and six informative restriction site mutations have been detected using eleven restriction endonucleases. Cladistic analyses of the restriction site mutations have been performed using Wagner and Dollo parsimony algorithms, and confidence intervals have been calculated for the resulting monophyletic groups using bootstrapping. It is demonstrated that restriction site comparisons are applicable to theRanunculaceae on intergeneric level, with the exception of groups having extensive genomic rearrangements. Moreover, sequence divergence is low enough at the interspecific level to allow phylogenetic analyses within genera such asRanunculus.     

Physical map of chloroplast DNA in sugi,Cryptomeria japonica

Summary To investigate the evolution of conifer species, we constructed a physical map of the chloroplast DNA of sugi, Cryptomeria japonica, with four restriction endonucleases, PstI, SalI, SacI and XhoI. The chloroplast genome of C. japonica was found to be a circular molecule with a total size of approximately 133 kb. This molecule lacked an inverted repeat. Twenty genes were localized on the physical map of C. japonica cpDNA by Southern hybridization. The chloroplast genome structure of C. japonica showed considerable rearrangements of the standard genome type found in vascular plants and differed markedly from that of tobacco. The difference was explicable by one deletion and five inversions. The chloroplast genome of C. japonica differed too from that of the genus Pinus which also lacks one of the inverted repeats. The results indicate that the conifer group originated monophyletically from an ancient lineage, and diverged independently after loss of an inverted repeat structure.     

Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed (DQ119058). The circular double-stranded DNA, consisting of 155,527 bp, contained a pair of inverted repeat regions (IRa and IRb) of 25,187 bp each, which were separated by small and large single copy regions of 86,879 and 18,274 bp, respectively. The presence and relative positions of 113 genes (76 peptide-encoding genes, 30 tRNA genes, four rRNA genes, and three conserved open reading frames) were identified. The major portion (55.76%) of the C. sativus chloroplast genome consisted of gene-coding regions (49.13% protein coding and 6.63% RNA regions; 27.81% LSC, 9.46% SSC and 18.49% IR regions), while intergenic spacers (including 20 introns) made up 44.24%. The overall G-C content of C. sativus chloroplast genome was 36.95%. Sixteen genes contained one intron, while two genes had two introns. The expansion/contraction manner of IR at IRb/LSC and IR/SSC border in Cucumis was similar to that of Lotus and Arabidopsis, and the manner at IRa/LSC was similar to Lotus and Nicotiana. In total, 56 simple sequence repeats (more than 10 bases) were identified in the C. sativus chloroplast genome.     

The chloroplast genomes of conifers lack one of the rRNA-encoding inverted repeats

Summary Chloroplast DNA from species of five different conifer genera was extracted and studied by Southern blot analysis. For all these species, hybridization with heterologous probes specific for 16 S and 23 S rDNA detected only one chloroplast DNA fragment per enzyme digest. This observation suggests that the 16 S and 23 S rRNA genes are not duplicated in these genomes. The unique 16 S rDNA-containing BamHI fragment from Pinus contorta Dougl. was cloned and restriction mapped. Apart from the 16 S rRNA gene, this fragment also contained the psbC and psbD genes. It is concluded that the chloroplast genomes of a wide taxonomic range of conifers lack one of the inverted repeat elements and that a dislocation of the psbDC gene cluster has occurred in P. contorta.     

滇黄精叶绿体全基因组序列及其密码子使用偏性分析

为探究滇黄精(Polygonatum kingianum)叶绿体全基因组特征和密码子使用偏性,利用第二代测序技术对滇黄精嫩叶进行测序,再经组装与注释后得到其叶绿体基因组全序列,通过MISA、EMBOSS和CodonW等软件对滇黄精叶绿体全基因组的SSR位点、系统发育及密码子偏好性进行分析。结果表明,滇黄精完整叶绿体基因组长度为155 852 bp,基因组平均GC含量为37.7%,其大、小单拷贝区(LSC)长度分别为84 633和185 25 bp,反向重复区长度为26 347 bp,注释了132个基因,包括86个蛋白编码基因、38个tRNA基因和8个核糖rRNA基因。叶绿体基因组中共有69个SSR位点,绝大多数属于单碱基重复的A/T类型。系统发育分析表明滇黄精与格脉黄精(P. tessellatum)亲缘关系近,可能与分布地域有关。密码子偏好性分析表明,滇黄精叶绿体基因组密码子使用模式受到自然选择影响大于突变因素,最终确定9个最优密码子。因此, 滇黄精叶绿体基因组遗传结构和系统发育位置及其密码子偏倚的分析,为叶绿体基因工程研究提供理论依据。     

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.     

红边龙血树叶绿体基因组特征及其系统发育分析

红边龙血树(Dracaena marginata)是一种在全球广泛种植的龙血树属园艺植物,具有较高的观赏价值和药用价值。本研究首次利用高通量测序技术对红边龙血树叶片进行全基因组测序,组装得到完整的叶绿体基因组序列,并进行注释、序列特征比较和系统发育分析。结果表明,红边龙血树叶绿体基因组包含一个典型的四分体结构,长度为154926 bp,是目前已报道的龙血树属中叶绿体基因组最小的物种;共拥有132个基因,包含86个编码蛋白基因、38个转运RNA基因和8个核糖体RNA基因;密码子偏好性分析发现存在偏好使用A/U碱基结尾的现象,整体上密码子偏好性较低;共鉴定出46个简单重复序列位点和54个长重复序列,分别在大单拷贝区与反向重复区有最大检出率;种间边界分析发现边界区域基因存在相对位置差异,扩张收缩情况总体较为相似;与近缘种进行系统发育分析,红边龙血树与细枝龙血树聚为一类,关系最近,符合形态学分类特征。对红边龙血树叶绿体基因组的解析为龙血树属植物的物种鉴定、遗传多样性和叶绿体转基因工程等提供了重要数据基础。     

Complete Sequence of the Duckweed (<Emphasis Type="Italic">Lemna minor</Emphasis>) Chloroplast Genome: Structural Organization and Phylogenetic Relationships to Other Angiosperms

The complete nucleotide sequence of the duckweed (Lemna minor) chloroplast genome (cpDNA) was determined. The cpDNA is a circular molecule of 165,955 bp containing a pair of 31,223-bp inverted repeat regions (IRs), which are separated by small and large single-copy regions of 89,906 and 13,603 bp, respectively. The entire gene pool and relative positions of 112 genes (78 protein-encoding genes, 30 tRNA genes, and 4 rRNA genes) are almost identical to those of Amborella trichopoda cpDNA; the minor difference is the absence of infA and ycf15 genes in the duckweed cpDNA. The inverted repeat is expanded to include ycf1 and rps15 genes; this pattern is unique and does not occur in any other sequenced cpDNA of land plants. As in basal angiosperms and eudicots, but not in other monocots, the borders between IRs and a large single-copy region are located upstream of rps19 and downstream of trnH, so that trnH is not included in IRs. The model of rearrangements of the chloroplast genome during the evolution of monocots is proposed as the result of the comparison of cpDNA structures in duckweed and other monocots. The phylogenetic analyses of 61 protein-coding genes from 38 plastid genome sequences provided strong support for the monophyly of monocots and position of Lemna as the next diverging lineage of monocots after Acorales. Our analyses also provided support for Amborella as a sister to all other angiosperms, but in the bayesian phylogeny inference based on the first two codon positions Amborella united with Nymphaeales.