Contrasting modes and tempos of genome evolution in land plant organelles

Despite inhabiting the same cell lineage for roughly a billion years and being dependent on the same nucleus for most of their gene products and genetic control, the two organelle genomes of land plants exhibit remarkably different tempos and patterns of evolutionary change. With a few notable exceptions, chloroplast genomes are highly conserved in size and gene arrangement, whereas mitochondrial genomes vary enormously in size and organization. Conversely, nucleotide substitution rates are on average several times higher in chloroplast DNA than in mitochondrial DNA. Mechanistic and selective forces underlying these differences are only poorly understood.     

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle‐scale barcodes. Next‐generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high‐quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long‐range PCR and sequenced using next‐generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early‐diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome‐scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.     

藻类植物叶绿体基因组研究进展

藻类植物的cpDNA结构复杂,普遍缺失反向重复序列IR,且存在IR的藻类植物种类的cpDNA也有IR变短退化迹象.藻类植物的cpDNA包含的基因一般比高等植物要多,编码能力更强.藻类植物cpDNA全序列的测定方法主要是Fosmid文库构建,配合使用Long-PCR技术.该文对国内外有关藻类植物叶绿体基因组结构、叶绿体编码基因、叶绿体基因组在藻类系统发育中的应用以及藻类植物叶绿体基因组的提取和序列测定方法等进行综述,为藻类植物的系统发育和叶绿体起源以及功能基因组学的研究提供理论依据.     

Chloroplast Genome Evolution in the Euglenaceae

Over the last few years multiple studies have been published outlining chloroplast genomes that represent many of the photosynthetic euglenid genera. However, these genomes were scattered throughout the euglenophyceaean phylogenetic tree, and focused on comparisons with Euglena gracilis. Here, we present a study exclusively on taxa within the Euglenaceae. Six new chloroplast genomes were characterized, those of Cryptoglena skujai, E. gracilis var. bacillaris, Euglena viridis, Euglenaria anabaena, Monomorphina parapyrum, and Trachelomonas volvocina, and added to six previously published chloroplast genomes to determine if trends existed within the family. With this study: at least one genome has now been characterized for each genus, the genomes of different strains from two taxa were characterized to explore intraspecific variability, and a second taxon has been characterized for the genus Monomorphina to examine intrageneric variability. Overall results showed a large amount of variability among the genomes, though a few trends could be identified both within Euglenaceae and within Euglenophyta. In addition, the intraspecific analysis indicated that the similarity of a genome sequence between strains was taxon dependent, and the intrageneric analysis indicated that the majority of the evolutionary changes within the Euglenaceae occurred intergenerically.     

Linkage disequilibrium and phylogenetic congruence between chloroplast and mitochondrial haplotypes in Silene vulgaris

Both the chloroplast and mitochondrial genomes are used extensively in studies of plant population genetics and systematics. In the majority of angiosperms, the chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) are each primarily transmitted maternally, but rare biparental transmission is possible. The extent to which the cpDNA and mtDNA are in linkage disequilibrium is argued to be dependent on the fidelity of co-transmission and the population structure. This study reports complete linkage disequilibrium between cpDNA and mtDNA haplotypes in 86 individuals from 17 populations of Silene vulgaris, a gynodioecious plant species. Phylogenetic analysis of cpDNA and mtDNA haplotypes within 14 individuals supports a hypothesis that the evolutionary histories of the chloroplasts and mitochondria are congruent within S. vulgaris, as might be expected if this association persists for long periods. This provides the first documentation of the evolutionary consequences of long-term associations between chloroplast and mitochondrial genomes within a species. Factors that contribute to the phylogenetic and linkage associations, as well as the potential for intergenomic hitchhiking resulting from selection on genes in one organellar genome are discussed.     

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein‐coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein‐coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya).     

被子植物叶绿体基因组的结构变异研究进展

叶绿体是绿色植物特有的细胞器,其基因组信息被广泛应用于植物系统发育和比较基因组学研究。目前,越来越多的物种有了叶绿体全基因组序列,人们对叶绿体基因组的结构及其变异规律有了更深入的了解。该文对近年来国内外有关被子植物叶绿体基因组插入/缺失、短片段倒位与重复、基因组结构重排以及基因丢失等结构变异式样的研究进展进行综述,并分析了叶绿体基因组结构研究中仍存在的问题以及该领域未来的发展趋势。     

Microsatellite analysis in organelle genomes of Chlorophyta

Simple Sequence Repeats (SSRs) or microsatellites constitute a significant portion of genomes however; their significance in organellar genomes has not been completely understood. The availability of organelle genome sequences allows us to understand the organization of SSRs in their genic and intergenic regions. In the present work, SSRs were identified and categorized in 14 mitochondrial and 22 chloroplast genomes of algal species belonging to Chlorophyta. Based on the study, it was observed that number of SSRs in non-coding region were more as compared to coding region and frequency of mononucleotides repeats were highest followed by dinucleotides in both mitochondrial and chloroplast genomes. It was also observed that maximum number of SSRs was found in genes encoding for beta subunit of RNA polymerase in chloroplast genomes and NADH dehydrogenase in mitochondrial genomes. This is the first and original report on whole genomes sequence analysis of organellar genomes of green algae.     

CPGView: A package for visualizing detailed chloroplast genome structures

Chloroplast genomes have been widely used in studying plant phylogeny and evolution. Several chloroplast genome visualization tools have been developed to display the distribution of genes on the genome. However, these tools do not draw features, such as exons, introns, repetitive elements, and variable sites, disallowing in-depth examination of the genome structures. Here, we developed and validated a software package called Chloroplast Genome Viewers (CPGView). CPGView can draw three maps showing (i) the distributions of genes, variable sites, and repetitive sequences, including microsatellites, tandem and dispersed repeats; (ii) the structure of the cis-splicing genes after adjusting the exon-intron boundary positions using a coordinate scaling algorithm, and (iii) the structure of the trans-splicing gene rps12. To test the accuracy of CPGView, we sequenced, assembled, and annotated 31 chloroplast genomes from 31 genera of 22 families. CPGView drew maps correctly for all the 31 chloroplast genomes. Lastly, we used CPGView to examine 5998 publicly released chloroplast genomes from 2513 genera of 553 families. CPGView succeeded in plotting maps for 5882 but failed to plot maps for 116 chloroplast genomes. Further examination showed that the annotations of these 116 genomes had various errors needing manual correction. The test on newly generated data and publicly available data demonstrated the ability of CPGView to identify errors in the annotations of chloroplast genomes. CPGView will become a widely used tool to study the detailed structure of chloroplast genomes. The web version of CPGView can be accessed from http://www.1kmpg.cn/cpgview .     

Origin and phylogeny of chloroplasts revealed by a simple correlation analysis of complete genomes

The complete sequenced genomes of chloroplast have provided much information on the origin and evolution of this organelle. In this paper we attempt to use these sequences to test a novel approach for phylogenetic analysis of complete genomes based on correlation analysis of compositional vectors. All protein sequences from 21 complete chloroplast genomes are analyzed in comparison with selected archaea, eubacteria, and eukaryotes. The distance-based analysis shows that the chloroplast genomes are most closely related to cyanobacteria, consistent with the endosymbiotic origin of chloroplasts. The chloroplast genomes are separated to two major clades corresponding to chlorophytes (green plants) s.l. and rhodophytes (red algae) s.l. The interrelationships among the chloroplasts are largely in agreement with the current understanding on chloroplast evolution. For instance, the analysis places the chloroplasts of two chromophytes (Guillardia and Odontella) within the rhodophyte lineage, supporting secondary endosymbiosis as the source of these chloroplasts. The relationships among the green algae and land plants in our tree also agree with results from traditional phylogenetic analyses. Thus, this study establishes the value of our simple correlation analysis in elucidating the evolutionary relationships among genomes. It is hoped that this approach will provide insights on comparative genome analysis.     

Gene duplication,transfer, and evolution in the chloroplast genome

In addition to the nuclear genome, organisms have organelle genomes. Most of the DNA present in eukaryotic organisms is located in the cell nucleus. Chloroplasts have independent genomes which are inherited from the mother. Duplicated genes are common in the genomes of all organisms. It is believed that gene duplication is the most important step for the origin of genetic variation, leading to the creation of new genes and new gene functions. Despite the fact that extensive gene duplications are rare among the chloroplast genome, gene duplication in the chloroplast genome is an essential source of new genetic functions and a mechanism of neo-evolution. The events of gene transfer between the chloroplast genome and nuclear genome via duplication and subsequent recombination are important processes in evolution. The duplicated gene or genome in the nucleus has been the subject of several recent reviews. In this review, we will briefly summarize gene duplication and evolution in the chloroplast genome. Also, we will provide an overview of gene transfer events between chloroplast and nuclear genomes.     

Mating Type Linked Mutations Which Disrupt the Uniparental Transmission of Chloroplast Genes in Chlamydomonas

In Chlamydomonas reinhardtii, chloroplast genomes are normally transmitted by the mating type plus (mt+) parent and mitochondrial genomes by the mating type minus (mt-) parent. In this paper we describe three new nuclear mutations, designated mat-3-1 to -3, which are tightly linked to the mt+ allele and permit high transmission of chloroplast genomes from the mt- parent, but have no effect on transmission of mitochondrial genomes. We also show that mat-1, reported by others to be a nuclear mutation linked to mt- which promotes transmission of chloroplast genomes by the mt- parent, is probably a vegetative diploid since it contains both mt+ and mt- alleles. Vegetative diploids behave as if they are mt- with respect to mating, but possess a level of chloroplast gene transmission intermediate between that of haploid mt- and mt+ stocks.     

Translation in chloroplasts

The discovery that chloroplasts have semi-autonomous genetic systems has led to many insights into the biogenesis of these organelles and their evolution from free-living photosynthetic bacteria. Recent developments of our understanding of the molecular mechanisms of translation in chloroplasts suggest selective pressures that have maintained the 100-200 genes of the ancestral endosymbiont in chloroplast genomes. The ability to introduce modified genes into chloroplast genomes by homologous recombination and the recent development of an in vitro chloroplast translation system have been exploited for analyses of the cis-acting requirements for chloroplast translation. Trans-acting translational factors have been identified by genetic and biochemical approaches. Several studies have suggested that chloroplast mRNAs are translated in association with membranes.     

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.     

Fifteen novel universal primer pairs for sequencing whole chloroplast genomes and a primer pair for nuclear ribosomal DNAs

Chloroplast genome information helps improve the phylogenetic resolution and can act as organelle-scale barcodes in recently radiated plant groups. Previously we reported that nine universal primer pairs could amplify angiosperm whole chloroplast genomes by long-range polymerase chain reaction and using next-generation sequencing. Although these primers show high universality and efficiency for sequencing whole chloroplast genomes in angiosperms, they did not fully resolve the following two issues surrounding sequencing angiosperm chloroplast genomes: (i) approximately 30% of angiosperms cannot be amplified successfully; and (ii) only fresh leaves can be applied. In this study, we designed another set of 15 universal primer pairs for amplifying angiosperm whole chloroplast genomes to complement the original nine primer pairs. Furthermore, we designed a primer pair for nuclear ribosomal DNAs (nrDNAs). To validate the functionality of the primers, we tested 44 species with silica gel-dried leaves and 15 species with fresh leaves that have been shown to not be amplified with the original nine primer pairs. The result showed that, in 65.9% and 88.6% of the 44 species with silica gel-dried leaves, the whole chloroplast genome and nrDNAs could be amplified, respectively. In addition, all 15 fresh leaf samples could have the whole chloroplast genome successfully amplified. The nrDNAs comprise partial sequences of 18S and 26S, along with the complete sequence of 5.8S and the internal transcribed spacers ITS1 and ITS2. The mean size of nrDNA was 5800 bp. This study shows that the 15 universal primer set is an indispensable tool for amplifying whole chloroplast genomes in angiosperms, and these are an important supplement to the nine reported primer pairs.     

A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea

Extensive DNA sequencing of the chloroplast genome of the red alga Porphyra purpurea has resulted in the detection of more than 125 genes. Fifty-eight (approximately 46%) of these genes are not found on the chloroplast genomes of land plants. These include genes encoding 17 photosynthetic proteins, three tRNAs, and nine ribosomal proteins. In addition, nine genes encoding proteins related to biosynthetic functions, six genes encoding proteins involved in gene expression, and at least five genes encoding miscellaneous proteins are among those not known to be located on land plant chloroplast genomes. The increased coding capacity of the P. purpurea chloroplast genome, along with other characteristics such as the absence of introns and the conservation of ancestral operons, demonstrate the primitive nature of the P. purpurea chloroplast genome. In addition, evidence for a monophyletic origin of chloroplasts is suggested by the identification of two groups of genes that are clustered in chloroplast genomes but not in cyanobacteria.     

Nuclear-encoded chloroplast RNA polymerase sigma factor SIG2 activates chloroplast-encoded phycobilisome genes in a red alga, Cyanidioschyzon merolae

The phycobilisome (PBS) is a photosynthetic light-harvesting complex in red algae, whose structural genes are separately encoded by both the nuclear and chloroplast genomes. While the expression of PBS genes in both genomes is responsive to environmental changes to modulate light-harvesting efficiency, little is known about how gene expression of the two genomes is coordinated. In this study, we focused on the four nuclear-encoded chloroplast sigma factors to understand aspects of this coordination, and found that SIG2 directs the expression of chloroplast PBS genes in the red alga Cyanidioschyzon merolae.     

Enslaved bacteria as new hope for plant biotechnologists

The most distinguishing feature of the plant cell is a DNA-containing organelle that sets plants apart from all other organisms: the chloroplast. Compelling evidence supports an endosymbiotic origin for chloroplasts. According to this theory, chloroplasts are descendants of formerly free-living cyanobacterial ancestors which entered an endosymbiotic relationship with a pre-eukaryotic cell and were ultimately integrated into the metabolism of the host cell. Chloroplasts retain many prokaryotic features and their gene expression system still closely resembles that of their eubacterial ancestors. During the past decade, our knowledge about chloroplast biology has benefited immensely from a most remarkable methodological breakthrough: the development of transformation technologies for chloroplast genomes. Moreover, recent advances in the manipulation of higher plant chloroplast genomes have created unprecedented opportunities for the genetic engineering of plants and promise to overcome many of the problems associated with conventional transgenic technologies. This review describes the state of the art in genetic engineering of higher plant chloroplast genomes and highlights the tremendous potential of these technologies for the biotechnology of the future. Received: 27 January 2000 / Received revision: 15 March 2000 / Accepted: 24 March 2000