Complete chloroplast genome sequence of Elodea canadensis and comparative analyses with other monocot plastid genomes

Elodea canadensis is an aquatic angiosperm native to North America. It has attracted great attention due to its invasive nature when transported to new areas in its non-native range. We have determined the complete nucleotide sequence of the chloroplast (cp) genome of Elodea. Taxonomically Elodea is a basal monocot, and only few monocot cp genomes representing early lineages of monocots have been sequenced so far. The genome is a circular double-stranded DNA molecule 156,700bp in length, and has a typical structure with large (LSC 86,194bp) and small (SSC 17,810bp) single-copy regions separated by a pair of inverted repeats (IRs 26,348bp each). The Elodea cp genome contains 113 unique genes and 16 duplicated genes in the IR regions. A comparative analysis showed that the gene order and organization of the Elodea cp genome is almost identical to that of Amborella trichopoda, a basal angiosperm. The structure of IRs in Elodea is unique among monocot species with the whole cp genome sequenced. In Elodea and another monocot Lemna minor the borders between IRs and LSC are located upstream of rps19 gene and downstream of trnH-GUG gene, while in most monocots, IR has extended to include both trnH and rps19 genes. A phylogenetic analysis conducted using Bayesian method, based on the DNA sequences of 81 chloroplast genes from 17 monocot taxa provided support for the placement of Elodea together with Lemna as a basal monocot and the next diverging lineage of monocots after Acorales. In comparison with other monocots, the Elodea cp genome has gone through only few rearrangements or gene losses. IR of Elodea has a unique structure among the monocot species studied so far as its structure is similar to that of a basal angiosperm Amborella. This result together with phylogenetic analyses supports the placement of Elodea as a basal monocot to the next diverging lineage of monocots after Acorales. So far, only few cp genomes representing early lineages of monocots have been sequenced and, therefore, this study provides valuable information about the course of evolution in divergence of monocot lineages.     

The complete nucleotide sequence of the coffee (Coffea arabica L.) chloroplast genome: organization and implications for biotechnology and phylogenetic relationships amongst angiosperms

The chloroplast genome sequence of Coffea arabica L., the first sequenced member of the fourth largest family of angiosperms, Rubiaceae, is reported. The genome is 155 189 bp in length, including a pair of inverted repeats of 25 943 bp. Of the 130 genes present, 112 are distinct and 18 are duplicated in the inverted repeat. The coding region comprises 79 protein genes, 29 transfer RNA genes, four ribosomal RNA genes and 18 genes containing introns (three with three exons). Repeat analysis revealed five direct and three inverted repeats of 30 bp or longer with a sequence identity of 90% or more. Comparisons of the coffee chloroplast genome with sequenced genomes of the closely related family Solanaceae indicated that coffee has a portion of rps19 duplicated in the inverted repeat and an intact copy of infA . Furthermore, whole-genome comparisons identified large indels (> 500 bp) in several intergenic spacer regions and introns in the Solanaceae, including trnE (UUC)– trnT (GGU) spacer, ycf4 – cemA spacer, trnI (GAU) intron and rrn5 – trnR (ACG) spacer. Phylogenetic analyses based on the DNA sequences of 61 protein-coding genes for 35 taxa, performed using both maximum parsimony and maximum likelihood methods, strongly supported the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids, asterids, eurosids II, and euasterids I and II. Coffea (Rubiaceae, Gentianales) is only the second order sampled from the euasterid I clade. The availability of the complete chloroplast genome of coffee provides regulatory and intergenic spacer sequences for utilization in chloroplast genetic engineering to improve this important crop.     

Comparative genomics of four Liliales families inferred from the complete chloroplast genome sequence of Veratrum patulum O. Loes. (Melanthiaceae)

The sequence of the chloroplast genome, which is inherited maternally, contains useful information for many scientific fields such as plant systematics, biogeography and biotechnology because its characteristics are highly conserved among species. There is an increase in chloroplast genomes of angiosperms that have been sequenced in recent years. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Veratrum patulum Loes. (Melanthiaceae, Liliales) was analyzed completely. The circular double-stranded DNA of 153,699 bp consists of two inverted repeat (IR) regions of 26,360 bp each, a large single copy of 83,372 bp, and a small single copy of 17,607 bp. This plastome contains 81 protein-coding genes, 30 distinct tRNA and four genes of rRNA. In addition, there are six hypothetical coding regions (ycf1, ycf2, ycf3, ycf4, ycf15 and ycf68) and two open reading frames (ORF42 and ORF56), which are also found in the chloroplast genomes of the other species. The gene orders and gene contents of the V. patulum plastid genome are similar to that of Smilax china, Lilium longiflorum and Alstroemeria aurea, members of the Smilacaceae, Liliaceae and Alstroemeriaceae (Liliales), respectively. However, the loss rps16 exon 2 in V. patulum results in the difference in the large single copy regions in comparison with other species. The base substitution rate is quite similar among genes of these species. Additionally, the base substitution rate of inverted repeat region was smaller than that of single copy regions in all observed species of Liliales. The IR regions were expanded to trnH_GUG in V. patulum, a part of rps19 in L. longiflorum and A. aurea, and whole sequence of rps19 in S. china. Furthermore, the IGS lengths of rbcL-accD-psaI region were variable among Liliales species, suggesting that this region might be a hotspot of indel events and the informative site for phylogenetic studies in Liliales. In general, the whole chloroplast genome of V. patulum, a potential medicinal plant, will contribute to research on the genetic applications of this genus.     

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 complete structure of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) chloroplast genome: Its composition and comparative analysis

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.     

Phylogenetic and evolutionary implications of complete chloroplast genome sequences of four early-diverging angiosperms: Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae)

We have determined the complete chloroplast genome sequences of four early-diverging lineages of angiosperms, Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae), to examine the organization and evolution of plastid genomes and to estimate phylogenetic relationships among angiosperms. For the most part, the organization of these plastid genomes is quite similar to the ancestral angiosperm plastid genome with a few notable exceptions. Dioscorea has lost one protein-coding gene, rps16; this gene loss has also happened independently in four other land plant lineages, liverworts, conifers, Populus, and legumes. There has also been a small expansion of the inverted repeat (IR) in Dioscorea that has duplicated trnH-GUG. This event has also occurred multiple times in angiosperms, including in monocots, and in the two basal angiosperms Nuphar and Drimys. The Illicium chloroplast genome is unusual by having a 10 kb contraction of the IR. The four taxa sequenced represent key groups in resolving phylogenetic relationships among angiosperms. Illicium is one of the basal angiosperms in the Austrobaileyales, Chloranthus (Chloranthales) remains unplaced in angiosperm classifications, and Buxus and Dioscorea are early-diverging eudicots and monocots, respectively. We have used sequences for 61 shared protein-coding genes from these four genomes and combined them with sequences from 35 other genomes to estimate phylogenetic relationships using parsimony, likelihood, and Bayesian methods. There is strong congruence among the trees generated by the three methods, and most nodes have high levels of support. The results indicate that Amborella alone is sister to the remaining angiosperms; the Nymphaeales represent the next-diverging clade followed by Illicium; Chloranthus is sister to the magnoliids and together this group is sister to a large clade that includes eudicots and monocots; and Dioscorea represents an early-diverging lineage of monocots just internal to Acorus.     

The chloroplast genome of Phalaenopsis aphrodite (Orchidaceae): comparative analysis of evolutionary rate with that of grasses and its phylogenetic implications

Whether the Amborella/Amborella-Nymphaeales or the grass lineage diverged first within the angiosperms has recently been debated. Central to this issue has been focused on the artifacts that might result from sampling only grasses within the monocots. We therefore sequenced the entire chloroplast genome (cpDNA) of Phalaenopsis aphrodite, Taiwan moth orchid. The cpDNA is a circular molecule of 148,964 bp with a comparatively short single-copy region (11,543 bp) due to the unusual loss and truncation/scattered deletion of certain ndh subunits. An open reading frame, orf91, located in the complementary strand of the rrn23 was reported for the first time. A comparison of nucleotide substitutions between P. aphrodite and the grasses indicates that only the plastid expression genes have a strong positive correlation between nonsynonymous (Ka) and synonymous (Ks) substitutions per site, providing evidence for a generation time effect, mainly across these genes. Among the intron-containing protein-coding genes of the sampled monocots, the Ks of the genes are significantly correlated to transitional substitutions of their introns. We compiled a concatenated 61 protein-coding gene alignment for the available 20 cpDNAs of vascular plants and analyzed the data set using Bayesian inference, maximum parsimony, and neighbor-joining (NJ) methods. The analyses yielded robust support for the Amborella/Amborella-Nymphaeales-basal hypothesis and for the orchid and grasses together being a monophyletic group nested within the remaining angiosperms. However, the NJ analysis using Ka, the first two codon positions, or amino acid sequences, respectively, supports the monocots-basal hypothesis. We demonstrated that these conflicting angiosperm phylogenies are most probably linked to the transitional sites at all codon positions, especially at the third one where the strong base-composition bias and saturation effect take place.     

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.     

Plastid genome of <Emphasis Type="Italic">Seseli montanum</Emphasis>: Complete sequence and comparison with plastomes of other members of the Apiaceae family

This work reports the complete plastid (pt) DNA sequence of Seseli montanum L. of the Apiaceae family, determined using next-generation sequencing technology. The complete genome sequence has been deposited in GenBank with accession No. KM035851. The S. montanum plastome is 147,823 bp in length. The plastid genome has a typical structure for angiosperms and contains a large single-copy region (LSC) of 92,620 bp and a small single-copy region (SSC) of 17,481 bp separated by a pair of 18,861 bp inverted repeats (IRa and IRb). The composition, gene order, and AT-content in the S. montanum plastome are similar to that of a typical flowering plant pt DNA. One hundred fourteen unique genes have been identified, including 30 tRNA genes, four rRNA genes, and 80 protein genes. Of 18 intron-containing genes found, 16 genes have one intron, and two genes (ycf3, clpP) have two introns. Comparative analysis of Apiaceae plastomes reveals in the S. montanum plastome a LSC/IRb junction shift, so that the part of the ycf2 (4980 bp) gene is located in the LSC, but the other part of ycf2 (1301 bp) is within the inverted repeat. Thus, structural rearrangements in the plastid genome of S. montanum result in an enlargement of the LSC region by means of capture of a large part of ycf2, in contrast to eight Apiaceae plastomes where the complete ycf2 gene sequence is located in the inverted repeat.     

Characterization of the Complete Chloroplast Genome of Apple (Malus × domestica,Rosaceae)*

Apple (Malus × domestica) is one of the most important temperate fruits. To better understand the molecular basis of this species, we characterized the complete chloroplast (cp) genome sequence downloaded from Genome Database for Rosaceae. The cp genome of apple is a circular molecule of 160068bp in length with a typical quadripartite structure of two inverted repeats (IRs) of 26352bp, separated by a small single copy region of 19180bp (SSC) and a large single copy region (LSC) of 88184bp. A total of 135 predicted genes (115 unique genes, and another 20 genes were duplicated in the IR) were identified, including 81 protein coding genes, four rRNA genes and 30 tRNA genes. Three genes of ycf15, ycf68 and infA contain several internal stop codons, which were interpreted as pseudogenes. The genome structure, gene order, GC content and codon usage of apple are similar to the typical angiosperm cp genomes. Thirty repeat regions (≥30bp) were detected, twenty one of which are tandem, six are forward and three are inverted repeats. Two hundred thirty seven simple sequence repeat (SSR) loci were revealed and most of them are composed of A or T, contributing to a distinct bias in base composition. Additionally, average 10000bp non coding region contains 24 SSR sites, while protein coding region contains five SSR sites, indicating an uneven distribution of SSRs. The complete cp genome sequence of apple reported in this paper will facilitate the future studies of its population genetics, phylogenetics and chloroplast genetic engineering.     

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     

Junctions of the large single copy region and the inverted repeats in Spinacia oleracea and Nicotiana debneyi chloroplast DNA: sequence of the genes for tRNAHis and the ribosomal proteins S19 and L2.

This work describes the organization, at the nucleotide sequence level, of genes flanking the junctions of the large single copy regions and the inverted repeats of Spinacia oleracea (spinach) and Nicotiana debneyi chloroplast DNAs. In both genomes, trnH1, the gene for tRNA-His(GUG) is located at the extremity of the large single copy region 3' to psbA, the gene for the 35 kd Photosystem 2 protein. Both psbA and trnH1 are transcribed towards the inverted repeat. In spinach, the first 48 codons of rps19, the gene for the chloroplast ribosomal protein S19, lie in the inverted repeat and the last 44 codons lie in the large single copy region at the end opposite to that carrying trnH1. The gene for a protein homologous to the E. coli ribosomal protein L2, rp12, is in the inverted repeat immediately 5' to rps19 and, like rps19, is transcribed towards the large single copy region. In N. debneyi, but not in spinach, rp12 is interrupted by a 666 bp insertion. The gene for tRNA-lle(CAT), trnl1, is located in the inverted repeats of spinach and N. debneyi, 5' to rp12 and is transcribed in the same direction as rp12.     

Chloroplast genome (cpDNA) of Cycas taitungensis and 56 cp protein-coding genes of Gnetum parvifolium: insights into cpDNA evolution and phylogeny of extant seed plants

Phylogenetic relationships among the 5 groups of extant seed plants are presently unsettled. To reexamine this long-standing debate, we determine the complete chloroplast genome (cpDNA) of Cycas taitungensis and 56 protein-coding genes encoded in the cpDNA of Gnetum parvifolium. The cpDNA of Cycas is a circular molecule of 163,403 bp with 2 typical large inverted repeats (IRs) of 25,074 bp each. We inferred phylogenetic relationships among major seed plant lineages using concatenated 56 protein-coding genes in 37 land plants. Phylogenies, generated by the use of 3 independent methods, provide concordant and robust support for the monophylies of extant seed plants, gymnosperms, and angiosperms. Within the modern gymnosperms are 2 highly supported sister clades: Cycas-Ginkgo and Gnetum-Pinus. This result agrees with both the "gnetifer" and "gnepines" hypotheses. The sister relationships in Cycas-Ginkgo and Gnetum-Pinus clades are further reinforced by cpDNA structural evidence. Branch lengths of Cycas-Ginkgo and Gnetum were consistently the shortest and the longest, respectively, in all separate analyses. However, the Gnetum relative rate test revealed this tendency only for the 3rd codon positions and the transversional sites of the first 2 codon positions. A PsitufA located between psbE and petL genes is here first detected in Anthoceros (a hornwort), cycads, and Ginkgo. We demonstrate that the PsitufA is a footprint descended from the chloroplast tufA of green algae. The duplication of ycf2 genes and their shift into IRs should have taken place at least in the common ancestor of seed plants more than 300 MYA, and the tRNAPro-GGG gene was lost from the angiosperm lineage at least 150 MYA. Additionally, from cpDNA structural comparison, we propose an alternative model for the loss of large IR regions in black pine. More cpDNA data from non-Pinaceae conifers are necessary to justify whether the gnetifer or gnepines hypothesis is valid and to generate solid structural evidence for the monophyly of extant gymnosperms.     

苹果叶绿体基因组特征分析

苹果（Malus×domestica）是最重要的温带水果之一。为了能更好的了解本种的分子生物学基础．对已发布的苹果叶绿体全基因组序列进行了结构特征分析。结果显示苹果的叶绿体基因组全长为160068bp,具有典型的被子植物叶绿体基因组的环状四分体结构,包含大单拷贝区（LSC）,小单拷贝区（SSC）和两个反向互补重复区（IRs）,长度分别为88184bp,19180bp和26352bp。基因组共有135个基因（20个基因分布在反向互补重复区,因此整个基因组包含115个不同的基因）。按照功能进行分类,这115个基因包括81个蛋白质编码基因,4个rRNA编码基因和30个tRNA基因。其中,ycf15．ycf68和infA三个基因包含多个终止密码子,推测可能为假基因。苹果的基因组结构．基因顺序．GC含量和密码子使用偏好均与典型的被子植物叶绿体基因组类似。在苹果的叶绿体基因组中,共检测到30个大于30bp的重复序列,其中包括21串联重复,6个正向重复和3个反向重复序列;并检测到237个简单重复序列（SSR）位点,大部分的SSR位点都偏向于A或者T组成。此外,每10000bp非编码区平均分布有24个SSR位点,而编码区平均有5个SSR位点,表明SSRs在叶绿体基因组上的分布是不均匀的。本文对苹果叶绿体基因组序列特征的报道,将有助于促进该种的居群遗传学、系统发育和叶绿体基因工程的研究。     

The Chloroplast Genome Sequence of Mungbean (Vigna radiata) Determined by High-throughput Pyrosequencing: Structural Organization and Phylogenetic Relationships

Mungbean is an economically important crop which is grown principally for its protein-rich dry seeds. However, genomic research of mungbean has lagged behind other species in the Fabaceae family. Here, we reported the complete chloroplast (cp) genome sequence of mungbean obtained by the 454 pyrosequencing technology. The mungbean cp genome is 151 271 bp in length which includes a pair of inverted repeats (IRs) of 26 474 bp separated by a small single-copy region of 17 427 bp and a large single-copy region of 80 896 bp. The genome contains 108 unique genes and 19 of these genes are duplicated in the IR. Of these, 75 are predicted protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. Relative to other plant cp genomes, we observed two distinct rearrangements: a 50-kb inversion between accD/rps16 and rbcL/trnK-UUU, and a 78-kb rearrangement between trnH/rpl14 and rps19/rps8. We detected sequence length polymorphism in the cp homopolymeric regions at the intra- and inter-specific levels in the Vigna species. Phylogenetic analysis demonstrated a close relationship between Vigna and Phaseolus in the phaseolinae subtribe and provided a strong support for a monophyletic group of the eurosid I.     

The chloroplast genome of a chlorophylla+c-containing alga,Odontella sinensis

The chloroplast genome of a marine centric diatom,Odontella sinensis, was cloned and sequenced. The circular genome is 119,704 bp in length (AC=Z67753;). It contains an inverted repeat sequence of 7,725 bp separating two single-copy regions of 38,908 and 65,346 bp, respectively, and 174 genes and open reading frames, of which nine are duplicated within the inverted repeat segments.     

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.     

Complete Chloroplast Genome of Sedum sarmentosum and Chloroplast Genome Evolution in Saxifragales

Comparative chloroplast genome analyses are mostly carried out at lower taxonomic levels, such as the family and genus levels. At higher taxonomic levels, chloroplast genomes are generally used to reconstruct phylogenies. However, little attention has been paid to chloroplast genome evolution within orders. Here, we present the chloroplast genome of Sedum sarmentosum and take advantage of several available (or elucidated) chloroplast genomes to examine the evolution of chloroplast genomes in Saxifragales. The chloroplast genome of S. sarmentosum is 150,448 bp long and includes 82,212 bp of a large single-copy (LSC) region, 16.670 bp of a small single-copy (SSC) region, and a pair of 25,783 bp sequences of inverted repeats (IRs).The genome contains 131 unique genes, 18 of which are duplicated within the IRs. Based on a comparative analysis of chloroplast genomes from four representative Saxifragales families, we observed two gene losses and two pseudogenes in Paeonia obovata, and the loss of an intron was detected in the rps16 gene of Penthorum chinense. Comparisons among the 72 common protein-coding genes confirmed that the chloroplast genomes of S. sarmentosum and Paeonia obovata exhibit accelerated sequence evolution. Furthermore, a strong correlation was observed between the rates of genome evolution and genome size. The detected genome size variations are predominantly caused by the length of intergenic spacers, rather than losses of genes and introns, gene pseudogenization or IR expansion or contraction. The genome sizes of these species are negatively correlated with nucleotide substitution rates. Species with shorter duration of the life cycle tend to exhibit shorter chloroplast genomes than those with longer life cycles.     

The complete chloroplast genome sequence of Mahonia bealei (Berberidaceae) reveals a significant expansion of the inverted repeat and phylogenetic relationship with other angiosperms

Mahonia bealei (Berberidaceae) is a frequently-used traditional Chinese medicinal plant with efficient anti-inflammatory ability. This plant is one of the sources of berberine, a new cholesterol-lowering drug with anti-diabetic activity. We have sequenced the complete nucleotide sequence of the chloroplast (cp) genome of M. bealei. The complete cp genome of M. bealei is 164,792 bp in length, and has a typical structure with large (LSC 73,052 bp) and small (SSC 18,591 bp) single-copy regions separated by a pair of inverted repeats (IRs 36,501 bp) of large size. The Mahonia cp genome contains 111 unique genes and 39 genes are duplicated in the IR regions. The gene order and content of M. bealei are almost unarranged which is consistent with the hypothesis that large IRs stabilize cp genome and reduce gene loss-and-gain probabilities during evolutionary process. A large IR expansion of over 12 kb has occurred in M. bealei, 15 genes (rps19, rpl22, rps3, rpl16, rpl14, rps8, infA, rpl36, rps11, petD, petB, psbH, psbN, psbT and psbB) have expanded to have an additional copy in the IRs. The IR expansion rearrangement occurred via a double-strand DNA break and subsequence repair, which is different from the ordinary gene conversion mechanism. Repeat analysis identified 39 direct/inverted repeats 30 bp or longer with a sequence identity ≥ 90%. Analysis also revealed 75 simple sequence repeat (SSR) loci and almost all are composed of A or T, contributing to a distinct bias in base composition. Comparison of protein-coding sequences with ESTs reveals 9 putative RNA edits and 5 of them resulted in non-synonymous modifications in rpoC1, rps2, rps19 and ycf1. Phylogenetic analysis using maximum parsimony (MP) and maximum likelihood (ML) was performed on a dataset composed of 65 protein-coding genes from 25 taxa, which yields an identical tree topology as previous plastid-based trees, and provides strong support for the sister relationship between Ranunculaceae and Berberidaceae. Molecular dating analyses suggest that Ranunculaceae and Berberidaceae diverged between 90 and 84 mya, which is congruent with the fossil records and with recent estimates of the divergence time of these two taxa.     

Implications of the Plastid Genome Sequence of Typha (Typhaceae, Poales) for Understanding Genome Evolution in Poaceae

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.