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.     

Complete Chloroplast Genome Sequence of <Emphasis Type="Italic">Glycine max</Emphasis> and Comparative Analyses with other Legume Genomes

Lack of complete chloroplast genome sequences is still one of the major limitations to extending chloroplast genetic engineering technology to useful crops. Therefore, we sequenced the soybean chloroplast genome and compared it to the other completely sequenced legumes, Lotus and Medicago. The chloroplast genome of Glycine is 152,218 basepairs (bp) in length, including a pair of inverted repeats of 25,574 bp of identical sequence separated by a small single copy region of 17,895 bp and a large single copy region of 83,175 bp. The genome contains 111 unique genes, and 19 of these are duplicated in the inverted repeat (IR). Comparisons of Glycine, Lotus and Medicago confirm the organization of legume chloroplast genomes based on previous studies. Gene content of the three legumes is nearly identical. The rpl22 gene is missing from all three legumes, and Medicago is missing rps16 and one copy of the IR. Gene order in Glycine, Lotus, and Medicago differs from the usual gene order for angiosperm chloroplast genomes by the presence of a single, large inversion of 51 kilobases (kb). Detailed analyses of repeated sequences indicate that many of the Glycine repeats that are located in the intergenic spacer regions and introns occur in the same location in the other legumes and in Arabidopsis, suggesting that they may play some functional role. The presence of small repeats of psbA and rbcL in legumes that have lost one copy of the IR indicate that this loss has only occurred once during the evolutionary history of legumes.     

Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae)

The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.     

Localization and nucleotide sequences of the tRNA GCC Gly,tRNA GUC Asp and tRNA GCA Cys genes from wheat chloroplast

The location on the wheat chloroplast DNA map and the nucleotide sequences of the genes coding for tRNA _GCC ^Gly (trnG-GCC), tRNA _GUC ^Asp (trnD-GUC) and tRNA _GCA ^Cys (trnC-GCA) have been determined. These three genes are located in the large single copy region of the chloroplast genome, about half-way between one of the inverted repeats and the gene for the α subunit of ATP synthase. They are located on two Bam H1 fragments, called B6 and B18 by Bowmanet al. (1), which are separated by about 450 bp and which were cloned in our laboratory to allow sequencing. ThetrnD-GUC andtrnC-GCA sequences show 98.6 and 89% homology, respectively, with the corresponding spinach chloroplast tRNA genes sequences (2), which are the only other higher plant chloroplasttrnD-GUC andtrnC-GCA sequenced so far, while no othertrnG-GCC sequence has been published. ThetrnG-GCC sequence shows only 58% homology with the corresponding gene sequence inEuglena chloroplasts (3).     

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。     

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

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

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.     

Mapping of the ribosomal RNA genes on spinach chloroplast DNA.

Spinach chloroplast ribosomal RNAs have been hybridized to restriction endonuclease fragments of spinach chloroplast DNA. All three RNA species (23S, 16S and 5S) hybridized to a single large fragment when the DNA was digested with either Sall or Pstl. Hybridization of 23S RNA to fragments produced by Smal yielded two radioactive bands which corresponded to the bi-molar 2.5 X 10(6) and 1.15 X 10(6) Mr fragments. 16S RNA also hybridized to two, bi-molar Smal fragments (3.4 X 10(6) and 2.5 X 10(6) Mr) and 5S RNA hybridized to the 1.15 X 10(6) Mr bi-molar Smal fragment. The 23S RNA and 16S RNA cistrons were each also shown to contain a single EcoRI site. From the data it was possible to conclude that the ribosomal RNA genes are located on the inverted repeat region of the spinach chloroplast DNA restriction map [1,2], that the sequence of the cistrons is 16S - 23S - 5S and that the size of the spacer between the 16S and 23S RNA cistrons is approximately 0.90 X 10(6) Mr.     

Chlamydomonas reinhardii gene for the 32 000 mol. wt. protein of photosystem II contains four large introns and is located entirely within the chloroplast inverted repeat

The chloroplast psbA gene from the green unicellular alga Chlamydomonas reinhardii has been localized, cloned and sequenced. This gene codes for the rapidly-labeled 32-kd protein of photosystem II, also identified as as herbicide-binding protein. Unlike psbA in higher plants which is found in the large single copy region of the chloroplast genome and is uninterrupted, psbA in C. reinhardii is located entirely within the inverted repeat, hence present in two identical copies per circular chloroplast genome, and contains four large introns. These introns range from 1.1 to 1.8 kb in size and fall into the category of Group I introns. Two of the introns contain open reading frames which are in-frame with the preceding exon sequences. We present the nucleotide sequence for the C. reinhardii psbA 5'-and 3' -flanking sequences, the coding region contained in five exons and the deduced amino acid sequence. The algal gene codes for a protein of 352 amino acid residues which is 95% homologous, excluding the last eight amino acid residues, with the higher plant protein.     

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa.

A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.     

Chloroplast transfer RNAs and tRNA genes of wheat

Fractionation (by two-dimensional polyacrylamide gel electrophoresis) of total tRNA from wheat chloroplasts yields about 33 RNA spots. Of these, 30 have been identified by aminoacylation as containing tRNAs specific for 17 amino acids. Hybridization of labeled individual tRNAs to cloned chloroplast DNA fragments has revealed the location of at least nine pairs of tRNA genes in the segments of the inverted repeat, at least twelve tRNA genes in the large single copy region and one tRNA gene in the small single copy region. A comparison of this wheat chloroplast tRNA gene map to that of maize and of other higher plants suggests that gene rearrangements have occurred during evolution, even within cereal chloroplast DNA. These rearrangements have taken place within the inverted repeat, within the large single copy region and between the inverted repeat and the large single copy region.     

An AU-rich element in the 3' untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation.

In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.     

Cloning and characterization of the chloroplast elongation factor EF-Tu cDNA of Oryza sativa L

From the rice leaf cDNA library, we have cloned a cDNA encoding rice chloroplast translational elongation factor EF-Tu (tufA). The rice tufA cDNA clone contains 1678 nucleotides and codes for a 467 amino acid protein including a putative chloroplast transit peptide of 59 amino acid residues. The predicted molecular mass of the mature protein is approximately 45 kDa. This cDNA clone contains the 61 nucleotides of the 5' untranslated region (UTR) and the 213 nucleotides of 3' UTR. Amino acid sequence identity of the rice tufA with the mature chloroplast EF-Tu proteins of tobacco, pea, arabidopsis, and soybean ranges from 83% to 86%. The deduced polypeptide of the rice tufA cDNA contains GTP binding domains in its N-terminal region and chloroplast EF-Tu signature regions in the C-terminal region. The rice tufA appears to exist as a single copy gene, although its homologues of maize and oat exist as multiple copy genes. The rice tufA gene is located in chromosome 1 and is more highly expressed in the leaf than in root tissue.     

Positioning of protein-coding genes on the soybean chloroplast genome

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF₁ component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF₀ component of ATP synthase (atpH), for the cytochrome f (cytF), for the ‘32 Kd’ thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.     

Cloning of ndhK from soybean chloroplasts using antibodies raised to mitochondrial complex I

A soybean shoot cDNA expression library was screened with polyclonal antibodies raised against red beet complex I and several clones were identified. One clone, consisting of a 1 kb insert, was fully sequenced. The sequence of 1025 bp was found to contain two extended open reading frames and the proteins encoded were identified as the ndhK and ndhJ products of the chloroplast genome. Nuclear, mitochondrial and chloroplast DNA was isolated and probed with a ndhK-specific probe. The chloroplast DNA contained a single copy of the cloned insert. With nuclear DNA, positively hybridising bands of 1.2, 2.7 and 3.2 kb were observed indicating that at least one gene homologous to ndhK of the chloroplast genome, is also present in the nucleus. The mitochondrial DNA did not hybridise with the ndhK probe. Western analysis of thylakoid proteins with the mitochondrial complex I antibodies revealed several bands. It is suggested that soybean contains two copies of the ndhK gene, one, on the plastid genome, coding for a subunit of a chloroplast NAD(P)H dehydrogenase, and the other, in the nucleus, coding for a subunit of mitochondrial complex I.     

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis

Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5’ portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.