Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.     

Plant plastid engineering

Genetic material in plants is distributed into nucleus, plastids and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is becoming more popular and an alternative to nuclear gene transformation because of various advantages like high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission. Recently, much progress in plastid engineering has been made. In addition to model plant tobacco, many transplastomic crop plants have been generated which possess higher resistance to biotic and abiotic stresses and molecular pharming. In this mini review, we will discuss the features of the plastid DNA and advantages of plastid transformation. We will also present some examples of transplastomic plants developed so far through plastid engineering, and the various applications of plastid transformation.     

基于质体基因组学方法研究广义玄参科的系统发育

该研究利用GenBank数据库已公开发表的玄参科及相关类群的107属129个物种的质体基因组数据对广义玄参科的系统发育关系进行了分析。该文利用蛋白质编码基因构建了矩阵，并采用最大似然法及贝叶斯推断重建系统发育树。基于两种分析方法获得的系统发育树的拓扑结构完全一致且分辨率及支持率较高。在ML树中，总分支数为129个，其中支持率≥70%的分支数目为123个。结果表明：(1)广义玄参科不是一个单系类群，隶属于广义玄参科的51个物种(37属)分散于列当科、泡桐科、美丽桐科、通泉草科、母草科、狭义玄参科和车前科。(2)狭义玄参科为单系类群，除原隶属于广义玄参科的Bontia、Calamphoreus、Diocirea、Eremophila、Glycocystis、Leucophyllum、玄参属和毛蕊花属外，还包括了原隶属于马钱科的醉鱼草属和原隶属于苦槛蓝科的苦槛蓝属。(3)唇形目为一个单系，目下共形成了14个支持率高的单系分支，对应于14个科(其中美丽桐科和胡麻科仅包括一个物种，不包括在内),科间关系得到较好的解决，木犀科为最早分化出来的类群，其余的类群共同组成核心唇形目。在核心唇形目中，类群...     

Strategies for complete plastid genome sequencing

Plastid sequencing is an essential tool in the study of plant evolution. This high‐copy organelle is one of the most technically accessible regions of the genome, and its sequence conservation makes it a valuable region for comparative genome evolution, phylogenetic analysis and population studies. Here, we discuss recent innovations and approaches for de novo plastid assembly that harness genomic tools. We focus on technical developments including low‐cost sequence library preparation approaches for genome skimming, enrichment via hybrid baits and methylation‐sensitive capture, sequence platforms with higher read outputs and longer read lengths, and automated tools for assembly. These developments allow for a much more streamlined assembly than via conventional short‐range PCR. Although newer methods make complete plastid sequencing possible for any land plant or green alga, there are still challenges for producing finished plastomes particularly from herbarium material or from structurally divergent plastids such as those of parasitic plants.     

The plastid genome of the red alga Laurencia

We present the 174,935 nt long plastid genome of the red alga Laurencia sp. JFC0032. It is the third plastid genome characterized for the largest order of red algae (Ceramiales). The circular‐mapping plastid genome is small compared to most florideophyte red algae, and our comparisons show a trend toward smaller plastid genome sizes in the family Rhodomelaceae, independent from a similar trend in Cyanidiophyceae. The Laurencia genome is densely packed with 200 annotated protein‐coding genes (188 widely conserved, 3 open reading frames shared with other red algae and 9 hypothetical coding regions). It has 29 tRNAs, a single‐copy ribosomal RNA cistron, a tmRNA, and the RNase P RNA.     

Identification of functional lox sites in the plastid genome

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.     

Covarion structure in plastid genome evolution: a new statistical test

Covarion models of molecular evolution allow the rate of evolution of a site to vary through time. There are few simple and effective tests for covarion evolution, and consequently, little is known about the presence of covarion processes in molecular evolution. We describe two new tests for covarion evolution and demonstrate with simulations that they perform well under a wide range of conditions. A survey of covarion evolution in sequenced plastid genomes found evidence of covarion drift in at least 26 out of 57 genes. Covarion evolution is most evident in first and second codon positions of the plastid genes, and there is no evidence of covarion evolution in third codon positions. Therefore, the significant covarion tests are likely due to changes in the selective constraints of amino acids. The frequency of covarion evolution within the plastid genome suggests that covarion processes of evolution were important in generating the observed patterns of sequence variation among plastid genomes.     

Plastid‐targeted forms of restriction endonucleases enhance the plastid genome rearrangement rate and trigger the reorganization of its genomic architecture

Plant cells have acquired chloroplasts (plastids) with a unique genome (ptDNA), which developed during the evolution of endosymbiosis. The gene content and genome structure of ptDNAs in land plants are considerably stable, although those of algal ptDNAs are highly varied. Plant cells seem, therefore, to be intolerant of any structural or organizational changes in the ptDNA. Genome rearrangement functions as a driver of genomic evolutionary divergence. Here, we aimed to create various types of rearrangements in the ptDNA of Arabidopsis genomes using plastid‐targeted forms of restriction endonucleases (pREs). Arabidopsis plants expressing each of the three specific pREs, i.e., pTaqI, pHinP1I, and pMseI, were generated; they showed the leaf variegation phenotypes associated with impaired chloroplast development. We confirmed that these pREs caused double‐stranded breaks (DSB) at their recognition sites in ptDNAs. Genome‐wide analysis of ptDNAs revealed that the transgenic lines exhibited a large number of rearrangements such as inversions and deletions/duplications, which were dominantly repaired by microhomology‐mediated recombination and microhomology‐mediated end‐joining, and less by non‐homologous end‐joining. Notably, pHinP1I, which recognized a small number of sites in ptDNA, induced drastic structural changes, including regional copy number variations throughout ptDNAs. In contrast, the transient expression of either pTaqI or pMseI, whose recognition site numbers were relatively larger, resulted in small‐scale changes at the whole genome level. These results indicated that DSB frequencies and their distribution are major determinants in shaping ptDNAs.     

石松类和蕨类植物质体基因组结构演化研究进展

近年来, 随着测序技术的发展, 石松类和蕨类植物的核基因组、质体基因组以及线粒体基因组研究发展迅速, 质体基因组研究工作更是呈爆发式增长。截至2019年3月1日, GenBank公布的石松类和蕨类植物的175个质体基因组中, 约3/4为最近两年新增。研究内容从早期对个别质体基因组结构和序列特征的简单报道, 逐渐发展到综合性的比较基因组学和系统发育基因组学研究。目前已发表的质体基因组覆盖了石松类和蕨类植物的所有目和大部分科, 这两大类群的质体基因组结构变异和系统发育的基本框架已逐渐清晰。这些研究为我们理解维管植物的早期演化提供了重要参考。本文对石松类和蕨类植物的质体基因组结构特征进行了系统梳理, 发现其结构变异主要包括大片段倒位、IR区边界变动、基因或内含子丢失等, 其中一些结构变异可作为较高分类阶元的共衍征。RNA编辑和长片段非编码序列插入普遍存在于石松类和蕨类植物的质体基因组中, 但其起源、演化机制和功能等仍不清楚。我们对质体基因组的应用、系统发育研究中质体和核基因组的优劣性, 以及系统发育基因组学的前景进行了评述。     

A divergent plastid genome inConopholis americana,an achlorophyllous parasitic plant

We have used heterologous probes to investigate the degree of sequence conservation in the plastid genome ofConopholis americana, a totally achlorophyllous angiosperm which exists as a root parasite on red oaks. AlthoughConopholis is completely nonphotosynthetic, it retains a plastid genome in which certain regions, including that which contains the ribosomal RNA genes, are highly conserved. Other regions, including those containing the genes for numerous photosynthesis proteins, are either absent or highly divergent. We also find that the 16S and 23S ribosomal genes of theConopholis plastid are transcribed and processed, implying a potentially functional genetic apparatus. These results are in agreement with findings reported recently for a related root parasite,Epifagus virginiana (de Pamphilis and Palmer, 1990). Furthermore, the plastid genome is maintained in high copy number in fruit tissue, whereas mature seeds have an approximately 10-fold lower copy number.     

High-quality draft genome sequence of nematocidal Bacillus thuringiensis Sbt003

Bacillus thuringiensis represents one of the six species of "Bacillus cereus group" in the genus Bacillus within the family Bacillaceae. Strain Sbt003 was isolated from soil and identified as B. thuringiensis. It harbors at least seven plasmids and produces three shapes of parasporal crystals including oval, bipyramidal and rice. SDS-PAGE analysis of spore-crystal suspension of this strain reveals six major protein bands, which implies the presence of multiple parasporal crystal genes. Bioassay of this strain reveals that it shows specific activity against nematodes and human cancer cells. In this study, we report the whole genomic shotgun sequences of Sbt003. The high-quality draft of the genome is 6,175,670 bp long (including chromosome and plasmids) with 6,372 protein-coding and 80 RNA genes.     

Comparative plastid genomics of Pinus species: Insights into sequence variations and phylogenetic relationships

Pinus L. is the largest genus of conifers and provides a classical model for studying species divergence and phylogenetic evolution by gymnosperms. However, our poor understanding of sequence divergence in the whole plastid genomes of Pinus species severely hinders studies of their evolution and phylogeny. Thus, we analyzed the sequences of 97 Pinus plastid genomes, including four newly sequenced genomes and 93 previously published plastomes, to explore the evolution and phylogenetic relationships in the genus Pinus. The complete chloroplast genomes of Pinus species ranged in size from 109 640 bp (P. cembra L.) to 121 976 bp (P. glabra Walter), and these genomes comprised circular DNA molecules in a similar manner to those of most gymnosperms. We identified 9108 repeats where most of the repeats comprised the dispersed type with 3983 (44%), followed by tandem repeats with 2999 (33%), and then palindromic repeats with 2126 (23%). Sixteen divergence hotspot regions were identified in Pinus plastid genomes, which could be useful molecular markers for future population genetics studies. Phylogenetic analysis showed that Pinus species could be divided into two diverged clades comprising the subgenera Strobus (single needle section) and Pinus (double needles section). Molecular dating suggested that the genus Pinus originated approximately 130.38 Mya during the late Cretaceous. The two subgenera subsequently split 85.86 Mya, which was largely consistent with the other molecular results based on partial DNA markers. These findings provide important insights into the sequence variations and phylogenetic evolution of Pinus plastid genomes.     

Hypothesis: Gene‐rich plastid genomes in red algae may be an outcome of nuclear genome reduction

Red algae (Rhodophyta) putatively diverged from the eukaryote tree of life >1.2 billion years ago and are the source of plastids in the ecologically important diatoms, haptophytes, and dinoflagellates. In general, red algae contain the largest plastid gene inventory among all such organelles derived from primary, secondary, or additional rounds of endosymbiosis. In contrast, their nuclear gene inventory is reduced when compared to their putative sister lineage, the Viridiplantae, and other photosynthetic lineages. The latter is thought to have resulted from a phase of genome reduction that occurred in the stem lineage of Rhodophyta. A recent comparative analysis of a taxonomically broad collection of red algal and Viridiplantae plastid genomes demonstrates that the red algal ancestor encoded ~1.5× more plastid genes than Viridiplantae. This difference is primarily explained by more extensive endosymbiotic gene transfer (EGT) in the stem lineage of Viridiplantae, when compared to red algae. We postulate that limited EGT in Rhodophytes resulted from the countervailing force of ancient, and likely recurrent, nuclear genome reduction. In other words, the propensity for nuclear gene loss led to the retention of red algal plastid genes that would otherwise have undergone intracellular gene transfer to the nucleus. This hypothesis recognizes the primacy of nuclear genome evolution over that of plastids, which have no inherent control of their gene inventory and can change dramatically (e.g., secondarily non‐photosynthetic eukaryotes, dinoflagellates) in response to selection acting on the host lineage.     

Plastid genome sequencing,comparative genomics,and phylogenomics:Current status and prospects

More than 190 plastid genomes have been completely sequenced during the past two decades due to advances in DNA sequencing technologies.Based on this unprecedented abundance of data,extensive genomic changes have been revealed in the plastid genomes.Inversion is the most common mechanism that leads to gene order changes.Several inversion events have been recognized as informative phylogenetic markers,such as a 30-kb inversion found in all living vascular plants minus lycopsids and two short inversions putat...     

Plastid genome sequencing,comparative genomics,and phylogenomics: Current status and prospects

Abstract More than 190 plastid genomes have been completely sequenced during the past two decades due to advances in DNA sequencing technologies. Based on this unprecedented abundance of data, extensive genomic changes have been revealed in the plastid genomes. Inversion is the most common mechanism that leads to gene order changes. Several inversion events have been recognized as informative phylogenetic markers, such as a 30‐kb inversion found in all living vascular plants minus lycopsids and two short inversions putatively shared by all ferns. Gene loss is a common event throughout plastid genome evolution. Many genes were independently lost or transferred to the nuclear genome in multiple plant lineages. The trnR‐CCG gene was lost in some clades of lycophytes, ferns, and seed plants, and all the ndh genes were absent in parasitic plants, gnetophytes, Pinaceae, and the Taiwan moth orchid. Certain parasitic plants have, in particular, lost plastid genes related to photosynthesis because of the relaxation of functional constraint. The dramatic growth of plastid genome sequences has also promoted the use of whole plastid sequences and genomic features to solve phylogenetic problems. Chloroplast phylogenomics has provided additional evidence for deep‐level phylogenetic relationships as well as increased phylogenetic resolutions at low taxonomic levels. However, chloroplast phylogenomics is still in its infant stage and rigorous analysis methodology has yet to be developed.     

Draft genome sequence of Bacillus amyloliquefaciens HB-26

Bacillus amyloliquefaciens HB-26, a Gram-positive bacterium was isolated from soil in China. SDS-PAGE analysis showed this strain secreted six major protein bands of 65, 60, 55, 34, 25 and 20 kDa. A bioassay of this strain reveals that it shows specific activity against P. brassicae and nematode. Here we describe the features of this organism, together with the draft genome sequence and annotation. The 3,989,358 bp long genome (39 contigs) contains 4,001 protein-coding genes and 80 RNA genes.     

Sequences of 72 plastid genes support the early divergence of Cornales in the asterids

Recent phylogenetic analyses of molecular data have supported different hypotheses of relationships among Cornales,Ericales,and core asterids.Such hypotheses have implications for the evolution of important morphological and embryological features of asterids.In this study we generated plastid genome-scale data of Davidia (Cornales) and Franklinia (Ericales) and combined them with published sequence data of eudicots.Our maximum parsimony,maximum likelihood,and Bayesian analyses generated strongly supported and congruent phylogenetic relationships among the three major lineages of the asterids.Cornales diverges first in asterids; Ericales is sister to the core asterids.Adding two more taxa helps mitigate long branch attraction in parsimony analyses.Sampling 26-28 plastid protein-coding genes may provide satisfactory resolution and support for relationships of eudicots including basal lineages of asterids.