Screening the Sargasso Sea metagenome for data to investigate genome evolution in Ostreococcus (Prasinophyceae, Chlorophyta)

The Sargasso Sea water shotgun sequencing unveiled an unprecedented glimpse of marine prokaryotic diversity and gene content. The sequence data was gathered from 0.8 microm filtered surface water extracts, and revealed picoeukaryotic (cell size<2 microm) sequences alongside the prokaryotic data. We used the available genome sequence of the picoeukaryote Ostreococcus tauri (Prasinophyceae, Chlorophyta) as a benchmark for the eukaryotic sequence content of the Sargasso Sea metagenome. Sequence data from at least two new Ostreococcus strains were identified and analyzed, and showed a bias towards higher coverage of the AT-rich organellar genomes. The Ostreococcus nuclear sequence data retrieved from the Sargasso metagenome is divided onto 731 scaffolds of average size 3917 bp, and covers 23% of the complete nuclear genome and 14% of the total number of protein coding genes in O. tauri. We used this environmental Ostreococcus sequence data to estimate the level of constraint on intronic and intergenic sequences in this compact genome.     

The complete chloroplast and mitochondrial genome sequences of Boea hygrometrica: insights into the evolution of plant organellar genomes

The complete nucleotide sequences of the chloroplast (cp) and mitochondrial (mt) genomes of resurrection plant Boea hygrometrica (Bh, Gesneriaceae) have been determined with the lengths of 153,493 bp and 510,519 bp, respectively. The smaller chloroplast genome contains more genes (147) with a 72% coding sequence, and the larger mitochondrial genome have less genes (65) with a coding faction of 12%. Similar to other seed plants, the Bh cp genome has a typical quadripartite organization with a conserved gene in each region. The Bh mt genome has three recombinant sequence repeats of 222 bp, 843 bp, and 1474 bp in length, which divide the genome into a single master circle (MC) and four isomeric molecules. Compared to other angiosperms, one remarkable feature of the Bh mt genome is the frequent transfer of genetic material from the cp genome during recent Bh evolution. We also analyzed organellar genome evolution in general regarding genome features as well as compositional dynamics of sequence and gene structure/organization, providing clues for the understanding of the evolution of organellar genomes in plants. The cp-derived sequences including tRNAs found in angiosperm mt genomes support the conclusion that frequent gene transfer events may have begun early in the land plant lineage.     

Ostreococcus tauri: seeing through the genes to the genome

The marine green alga Ostreococcus tauri is the smallest-known free-living eukaryote. The recent sequencing of its genome extends this distinction, because it also has one of the smallest and most compact nuclear genomes. For other highly compacted genomes (e.g. those of microsporidian parasites and relic endosymbiont nucleomorphs), compaction is associated with severe gene loss. By contrast, O. tauri has retained a large complement of genes. Studying O. tauri should shed light on forces, other than parasitism and endosymbiosis, that result in densely packed genomes.     

Genome analysis and its significance in four unicellular algae, Cyanidioshyzon merolae, Ostreococcus tauri, Chlamydomonas reinhardtii, and Thalassiosira pseudonana

Algae play a more important role than land plants in the maintenance of the global environment and productivity. Progress in genome analyses of these organisms means that we can now obtain information on algal genomes, global annotation and gene expression. The full genome information for several algae has already been analyzed. Whole genomes of the red alga Cyanidioshyzon merolae, the green algae Ostreococcus tauri and Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana have been sequenced. Genome composition and the features of cells among the four algae were compared. Each alga maintains basic genes as photosynthetic eukaryotes and possesses additional gene groups to represent their particular characteristics. This review discusses and introduces the latest research that makes the best use of the particular features of each organism and the significance of genome analysis to study biological phenomena. In particular, examples of post-genome studies of organelle multiplication in C. merolae based on analyzed genome information are presented.     

From small hosts come big viruses: the complete genome of a second Ostreococcus tauri virus, OtV-1

Ostreococcus tauri virus (OtV-1) is a large double-stranded DNA virus and a prospective member of the family Phycodnaviridae , genus Prasinovirus . OtV-1 infects the unicellular marine green alga O. tauri , the smallest known free-living eukaryote. Here we present the 191 761 base pair genome sequence of OtV-1, which has 232 putative protein-encoding and 4 tRNA-encoding genes. Approximately 31% of the viral gene products exhibit a similarity to proteins of known functions in public databases. These include a variety of unexpected genes, for example, a PhoH-like protein, a N -myristoyltransferase, a 3-dehydroquinate synthase, a number of glycosyltransferases and methyltransferases, a prolyl 4-hydroxylase, 6-phosphofructokinase and a total of 8 capsid proteins. A total of 11 predicted genes share homology with genes found in the Ostreococcus host genome. In addition, an intein was identified in the DNA polymerase gene of OtV-1. This is the first report of an intein in the genome of a virus that infects O. tauri. Fifteen core genes common to nuclear-cytoplasmic large dsDNA virus (NCLDV) genomes were identified in the OtV-1 genome. This new sequence data may help to redefine the classification of the core genes of these viruses and shed new light on their evolutionary history.     

The chloroplast protein translocation complexes of Chlamydomonas reinhardtii: a bioinformatic comparison of Toc and Tic components in plants, green algae and red algae

The recently completed genome of Chlamydomonas reinhardtii was surveyed for components of the chloroplast protein translocation complexes. Putative components were identified using reciprocal BlastP searches with the protein sequences of Arabidopsis thaliana as queries. As a comparison, we also surveyed the new genomes of the bryophyte Physcomitrella patens, two prasinophyte green algae (Ostreococcus lucimarinus and Ostreococcus tauri), the red alga Cyanidioschizon merolae, and several cyanobacteria. Overall, we found that the components of the import pathway are remarkably well conserved, particularly among the Viridiplantae lineages. Specifically, C. reinhardtii contained almost all the components found in A. thaliana, with two exceptions. Missing from C. reinhardtii are the C-terminal ferredoxin-NADPH-reductase (FNR) binding domain of Tic62 and a full-length, TPR-bearing Toc64. Further, the N-terminal domain of C. reinhardtii Toc34 is highly acidic, whereas the analogous region in C. reinhardtii Toc159 is not. This reversal of the vascular plant model may explain the similarity of C. reinhardtii chloroplast transit peptides to mitochondrial-targeting peptides. Other findings from our genome survey include the absence of Tic22 in both Ostreococcus genomes; the presence of only one Toc75 homolog in C. merolae; and, finally, a distinctive propensity for gene duplication in P. patens.     

Unique Regulation of the Calvin Cycle in the Ultrasmall Green Alga <Emphasis Type="Italic">Ostreococcus</Emphasis>

Glyceraldehyde-3-phosphate dehydrogenase (GapAB) and CP12 are two major players in controlling the inactivation of the Calvin cycle in land plants at night. GapB originated from a GapA gene duplication and differs from GapA by the presence of a specific C-terminal extension that was recruited from CP12. While GapA and CP12 are assumed to be generally present in the Plantae (glaucophytes, red and green algae, and plants), up to now GapB was exclusively found in Streptophyta, including the enigmatic green alga Mesostigma viride. However, here we show that two closely related prasinophycean green algae, Ostreococcus tauri and Ostreococcus lucimarinus, also possess a GapB gene, while CP12 is missing. This remarkable finding either antedates the GapA/B gene duplication or indicates a lateral recruitment. Moreover, Ostreococcus is the first case where the crucial CP12 function may be completely replaced by GapB-mediated GapA/B aggregation.     

Decoding and analysis of organelle genomes of Indian tea (Camellia assamica) for phylogenetic confirmation

The NCBI database has >15 chloroplast (cp) genome sequences available for different Camellia species but none for C. assamica. There is no report of any mitochondrial (mt) genome in the Camellia genus or Theaceae family. With the strong believes that these organelle genomes can play a great tool for taxonomic and phylogenetic analysis, we successfully assembled and analyzed cp and mt genome of C. assamica. We assembled the complete mt genome of C. assamica in a single circular contig of 707,441 bp length comprising of a total of 66 annotated genes, including 35 protein-coding genes, 29 tRNAs and two rRNAs. The first ever cp genome of C. assamica resulted in a circular contig of 157,353 bp length with a typical quadripartite structure. Phylogenetic analysis based on these organelle genomes showed that C. assamica was closely related to C. sinensis and C. leptophylla. It also supports Caryophyllales as Superasterids.     

Sequencing and analysis of the complete mitochondrial genome of the giant dobsonfly Acanthacorydalis orientalis(McLachlan)(Insecta:Megaloptera:Corydalidae)

The complete mitochondrial genome of Acanthacorydalis orientalis(McLachlan)was determined and analyzed(GenBank accession number:KF840564).This paper represents the first mitochondrial(mt) genome of the dobsonfly genus Acanthacorydalis.The mt genome is a typical circular DNA of 15 753 bp composed of 37 genes with an A+T content of 76.7%.It has an ancestral gene arrangement of the insect mt genomes.Eleven of the 13 PCGs start with codon ATT and ATG,while several exceptions such as ATA and TTG respectively for atpS and nad\ are also present.Five protein-coding genes end with a single T,while others have a termination codon of TAA or TAG.Most tRNAs are folded into the typical clover-leaf structure except for the trnS 1 whose dihydrouridine arm was a simple loop.The secondary structure of rrnl consists of five structural domains and 50 helices,while the rrns includes three domains and 34 helices.The control region has a stretches of Ts with a length of 22 bp but lacks obvious tandem repeat region.Both Bayesian inference and maximum likelihood(ML) analyses,based on all 13 protein-coding genes and two rRNA genes of the mt genomes,confirm the monophyly of Corydalinae and suggest that Acanthacorydalis,together with Corydalus,which is an endemic dobsonfly genus from the New World,belong to a monophyletic lineage.     

青藏高原特有种-藏扇穗茅叶绿体基因组测序及序列分析

藏扇穗茅（Littledalea tibetica）是禾本科（Poaceae）雀麦族（Bromeae）中一个具有重要生态价值的多年生高山特有种,主要分布于青藏高原及其毗邻地区。本文采用基于第二代高通量测序平台的Illumina MiSeq技术,对青藏高原特有种—藏扇穗茅进行了叶绿体基因组测序,首次建立了雀麦族物种的标准测序流程;同时,以其近缘物种—黑麦草（Lolium perenne）的叶绿体基因组序列作为参考,组装获得它的叶绿体基因组序列。结果表明,藏扇穗茅叶绿体基因组序列全长136 852 bp,GC含量为38.5%,呈典型的四段式结构,其中大（LSC）、小（SSC）单拷贝区大小分别为80 970和12 876 bp,反向互补重复区（IR）大小为21 503 bp,共注释得到141个基因,包含95个蛋白编码基因、38个tRNA基因和8个rRNA基因,主要分布于大单拷贝区和小单拷贝区。同时,基于藏扇穗茅和其它30种禾本科植物叶绿体基因全序列构建的系统发育树显示,藏扇穗茅与早熟禾亚科中小麦族植物亲缘关系较近。     

Molecular characterization of a cloned dolphin mitochondrial genome

Summary DNA clones have been isolated that span the complete mitochondrial (mt) genome of the dolphin,Cephalorhynchus commersonii. Hybridization experiments with purified primate mtDNA probes have established that there is close resemblance in the general organization of the dolphin mt genome and the terrestrial mammalian mt genomes. Sequences covering 2381 bp of the dolphin mt genome from the major noncoding region, three tRNA genes, and parts of the genes encoding cytochrome b, NADH dehydrogenase subunit 3 (ND3), and 16S rRNA have been compared with corresponding regions from other mammalian genomes. There is a general tendency throughout the sequenced regions for greater similarity between dolphin and bovine mt genomes than between dolphin and rodent or human mt genomes.     

Atypical regulation of a green lineage-specific B-type cyclin-dependent kinase

Cyclin-dependent kinases (CDKs) are the main regulators of cell cycle progression in eukaryotes. The role and regulation of canonical CDKs, such as the yeast (Saccharomyces cerevisiae) Cdc2 or plant CDKA, have been extensively characterized. However, the function of the plant-specific CDKB is not as well understood. Besides being involved in cell cycle control, Arabidopsis (Arabidopsis thaliana) CDKB would integrate developmental processes to cell cycle progression. We investigated the role of CDKB in Ostreococcus (Ostreococcus tauri), a unicellular green algae with a minimal set of cell cycle genes. In this primitive alga, at the basis of the green lineage, CDKB has integrated two levels of regulations: It is regulated by Tyr phosphorylation like cdc2/CDKA and at the level of synthesis-like B-type CDKs. Furthermore, Ostreococcus CDKB/cyclin B accounts for the main peak of mitotic activity, and CDKB is able to rescue a yeast cdc28(ts) mutant. By contrast, Ostreococcus CDKA is not regulated by Tyr phosphorylation, and it exhibits a low and steady-state activity from DNA replication to exit of mitosis. This suggests that from a major role in the control of mitosis in green algae, CDKB has evolved in higher plants to assume other functions outside the cell cycle.     

Life-cycle and genome of OtV5, a large DNA virus of the pelagic marine unicellular green alga Ostreococcus tauri

Large DNA viruses are ubiquitous, infecting diverse organisms ranging from algae to man, and have probably evolved from an ancient common ancestor. In aquatic environments, such algal viruses control blooms and shape the evolution of biodiversity in phytoplankton, but little is known about their biological functions. We show that Ostreococcus tauri, the smallest known marine photosynthetic eukaryote, whose genome is completely characterized, is a host for large DNA viruses, and present an analysis of the life-cycle and 186,234 bp long linear genome of OtV5. OtV5 is a lytic phycodnavirus which unexpectedly does not degrade its host chromosomes before the host cell bursts. Analysis of its complete genome sequence confirmed that it lacks expected site-specific endonucleases, and revealed the presence of 16 genes whose predicted functions are novel to this group of viruses. OtV5 carries at least one predicted gene whose protein closely resembles its host counterpart and several other host-like sequences, suggesting that horizontal gene transfers between host and viral genomes may occur frequently on an evolutionary scale. Fifty seven percent of the 268 predicted proteins present no similarities with any known protein in Genbank, underlining the wealth of undiscovered biological diversity present in oceanic viruses, which are estimated to harbour 200Mt of carbon.     

Shotgun proteomic analysis of the unicellular alga Ostreococcus tauri

Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living eukaryotes. The O. tauri genome sequence was determined in 2006. Molecular, physiological and taxonomic data that has been generated since then highlight its potential as a simple model species for algae and plants. However, its proteome remains largely unexplored. This paper describes the global proteomic study of O. tauri, using mass spectrometry-based approaches: phosphopeptide enrichment, cellular fractionation, label-free quantification and (15)N metabolic labeling. The O. tauri proteome was analyzed under the following conditions: sampling at different times during the circadian cycle, after 24h of illumination, after 24h of darkness and under various nitrogen source supply levels. Cell cycle related proteins such as dynamin and kinesin were significantly up-regulated during the daylight-to-darkness transition. This is reflected by their higher intensity at ZT13 and this transition phase coincides with the end of mitosis. Proteins involved in several metabolic mechanisms were found to be up-regulated under low nitrogen conditions, including carbon storage pathways, glycolysis, phosphate transport, and the synthesis of inorganic polyphosphates. Ostreococcus tauri responds to low nitrogen conditions by reducing its nitrogen assimilation machinery which suggests an atypical adaptation mechanism for coping with a nutrient-limited environment.     

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.     

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.     

PHYLOGENETIC ANALYSIS AND GENOME SIZE OF OSTREOCOCCUS TAURI (CHLOROPHYTA, PRASINOPHYCEAE)

Ostreococcus tauri Courties et Chrétiennot-Dinet is the smallest described autotrophic eukaryote dominating the phytoplanktonic assemblage of the marine Mediterranean Thau lagoon (France). Its taxonomic position was partly elucidated from ultrastructure and high-pressure liquid chromatography (HLPC) pigment analysis. The sequence analysis of the 18S rDNA gene of O. tauri measured here is available in EMBL Nucleotide Sequence Database (accession number: Y15814) and allowed to clarify its phylogenetic position. O. tauri belongs to the Prasinophyceae and appears very close to Mantoniella, a typical scaly Prasinophyceae, morphologically very different from the naked and coccoid Ostreococcus. An electrophoretic analysis of O. tauri shows that the nucleus contains 10.20 mbp. This small genome, fragmented into 14 chromosomes ranging in size from 300 to 1500 kbp, confirms the minimalist characteristics of Ostreococcus tauri.     

The Organelle Genomes of Hassawi Rice (Oryza sativa L.) and Its Hybrid in Saudi Arabia: Genome Variation, Rearrangement, and Origins

Hassawi rice (Oryza sativa L.) is a landrace adapted to the climate of Saudi Arabia, characterized by its strong resistance to soil salinity and drought. Using high quality sequencing reads extracted from raw data of a whole genome sequencing project, we assembled both chloroplast (cp) and mitochondrial (mt) genomes of the wild-type Hassawi rice (Hassawi-1) and its dwarf hybrid (Hassawi-2). We discovered 16 InDels (insertions and deletions) but no SNP (single nucleotide polymorphism) is present between the two Hassawi cp genomes. We identified 48 InDels and 26 SNPs in the two Hassawi mt genomes and a new type of sequence variation, termed reverse complementary variation (RCV) in the rice cp genomes. There are two and four RCVs identified in Hassawi-1 when compared to 93-11 (indica) and Nipponbare (japonica), respectively. Microsatellite sequence analysis showed there are more SSRs in the genic regions of both cp and mt genomes in the Hassawi rice than in the other rice varieties. There are also large repeats in the Hassawi mt genomes, with the longest length of 96,168 bp and 96,165 bp in Hassawi-1 and Hassawi-2, respectively. We believe that frequent DNA rearrangement in the Hassawi mt and cp genomes indicate ongoing dynamic processes to reach genetic stability under strong environmental pressures. Based on sequence variation analysis and the breeding history, we suggest that both Hassawi-1 and Hassawi-2 originated from the Indonesian variety Peta since genetic diversity between the two Hassawi cultivars is very low albeit an unknown historic origin of the wild-type Hassawi rice.     

Prasinoviruses of the marine green alga Ostreococcus tauri are mainly species specific

Prasinoviruses infecting unicellular green algae in the order Mamiellales (class Mamiellophyceae) are commonly found in coastal marine waters where their host species frequently abound. We tested 40 Ostreococcus tauri viruses on 13 independently isolated wild-type O. tauri strains, 4 wild-type O. lucimarinus strains, 1 Ostreococcus sp. ("Ostreococcus mediterraneus") clade D strain, and 1 representative species of each of two other related species of Mamiellales, Bathycoccus prasinos and Micromonas pusilla. Thirty-four out of 40 viruses infected only O. tauri, 5 could infect one other species of the Ostreococcus genus, and 1 infected two other Ostreococcus spp., but none of them infected the other genera. We observed that the overall susceptibility pattern of Ostreococcus strains to viruses was related to the size of two host chromosomes known to show intraspecific size variations, that genetically related viruses tended to infect the same host strains, and that viruses carrying inteins were strictly strain specific. Comparison of two complete O. tauri virus proteomes revealed at least three predicted proteins to be candidate viral specificity determinants.     

Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent

The search for a nitric oxide synthase (NOS) sequence in the plant kingdom yielded two sequences from the recently published genomes of two green algae species of the Ostreococcus genus, O. tauri and O. lucimarinus. In this study, we characterized the sequence, protein structure, phylogeny, biochemistry, and expression of NOS from O. tauri. The amino acid sequence of O. tauri NOS was found to be 45% similar to that of human NOS. Folding assignment methods showed that O. tauri NOS can fold as the human endothelial NOS isoform. Phylogenetic analysis revealed that O. tauri NOS clusters together with putative NOS sequences of a Synechoccocus sp strain and Physarum polycephalum. This cluster appears as an outgroup of NOS representatives from metazoa. Purified recombinant O. tauri NOS has a K(m) for the substrate l-Arg of 12 ± 5 μM. Escherichia coli cells expressing recombinant O. tauri NOS have increased levels of NO and cell viability. O. tauri cultures in the exponential growth phase produce 3-fold more NOS-dependent NO than do those in the stationary phase. In O. tauri, NO production increases in high intensity light irradiation and upon addition of l-Arg, suggesting a link between NOS activity and microalgal physiology.