Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.     

Characterization of chloroplast DNA microsatellites from Saccharum spp and related species

Microsatellites, or simple sequence repeats (SSRs), and their flanking regions in chloroplast genomes (plastomes) of some species of the family Poaceae were analyzed in silico to look for DNA sequence variations. Comparison of the complete chloroplast DNA sequences (cpDNAs) of sugarcane (Saccharum hybrid cv. SP-80-3280 and S. officinarum cv. NCo310) and related species, Agrostis stolonifera, Brachypodium distachyon, Hordeum vulgare subsp vulgare, Lolium perenne, Oryza nivara, O. sativa subsp indica, O. sativa subsp japonica, Sorghum bicolor, Triticum aestivum, Zea mays, and Z. mays cv. B73, allowed us to examine the organization of chloroplast SSRs (cpSSRs) in genic and intergenic regions. We identified 204 cpSSRs in the sugarcane cpDNA; 22.5% were in genic regions. The ndh, rps, trn, and rpl gene clusters of the chloroplasts had the most repeats. Mononucleotide repeats were the most abundant cpSSRs in these species; however, di-, tri-, tetra-, penta-, and hexanucleotide repeats were also identified. Many base substitutions and deletions/insertions were identified in the cpSSR loci and their flanking regions. Multiple alignments of all cpSSR sequences of Poaceae species made identification of nucleotide variability possible; repeat motifs are not uniformly distributed across the Poaceae plastomes, but are mostly confined to intergenic regions. Phylogeny was determined by maximum parsimony and neighbor-joining inference methods. The cpSSRs of these species were found to be polymorphic. It appears that individual cpSSRs in the Poaceae are stable, at least over short periods of evolutionary time. We conclude that the plastome database can be exploited for phylogenetic analysis and biotechnological development.     

Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication

The spacer between the 16S and 23S rRNA genes of the chloroplast DNA has been implicated as an origin of replication in several species of plants. In the evening primrose, Oenothera, this site was found to vary greatly in size, with plastid genomes (plastomes) being readily distinguished. To determine whether plastome "strength" in transmission could be correlated with variation at oriB, the 16S rRNA-trnI spacer was sequenced from five plastomes. The size variation was found to be due to differential amplification (and deletion) of combinations of sequences belonging to seven families of direct repeats. From these comparisons, one short series of direct repeats and one region capable of forming a hairpin structure were identified as candidates for the factor that could be responsible for the differences between strong and weak plastome types. Ample sequence variation allowed phylogenetic inferences to be made about the relationships among the plastomes. Phylogenetic trees also could be constructed for most of the families of direct repeats. The amplifications and deletions of repeats that account for the size variation at oriB are proposed to have occurred through extensive replication slippage at this site.      

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Complete sequencing and comparative analyses of the pepper (Capsicum annuum L.) plastome revealed high frequency of tandem repeats and large insertion/deletions on pepper plastome

Plants in the family Solanaceae are used as model systems in comparative and evolutionary genomics. The complete chloroplast genomes of seven solanaceous species have been sequenced, including tobacco, potato and tomato, but not peppers. We analyzed the complete chloroplast genome sequence of the hot pepper, Capsicum annuum. The pepper chloroplast genome was 156,781 bp in length, including a pair of inverted repeats (IR) of 25,783 bp. The content and the order of 133 genes in the pepper chloroplast genome were identical to those of other solanaceous plastomes. To characterize pepper plastome sequence, we performed comparative analysis using complete plastome sequences of pepper and seven solanaceous plastomes. Frequency and contents of large indels and tandem repeat sequences and distribution pattern of genome-wide sequence variations were investigated. In addition, a phylogenetic analysis using concatenated alignments of coding sequences was performed to determine evolutionary position of pepper in Solanaceae. Our results revealed two distinct features of pepper plastome compared to other solanaceous plastomes. Firstly, large indels, including insertions on accD and rpl20 gene sequences, were predominantly detected in the pepper plastome compared to other solanaceous plastomes. Secondly, tandem repeat sequences were particularly frequent in the pepper plastome. Taken together, our study represents unique features of evolution of pepper plastome among solanaceous plastomes.     

Molecular Analysis of the Hot Spot Region Related to Length Mutations in Wheat Chloroplast Dnas. I. Nucleotide Divergence of Genes and Intergenic Spacer Regions Located in the Hot Spot Region

The nucleotide divergence of chloroplast DNAs around the hot spot region related to length mutation in Triticum (wheat) and Aegilops was analyzed. DNA sequences (ca. 4.5 kbp) of three chloroplast genome types of wheat complex were compared with one another and with the corresponding region of other grasses. The sequences region contained rbcL and psaI, two open reading frames, and a pseudogene, rpl23' (pseudogene for ribosomal protein L23) disrupted by AT-rich intergic spacer regions. The evolution of these genes in the closely related wheat complex is characterized by nonbiased nucleotide substitutions in terms of being synonymous/nonsynonymous, having A-T pressure transitions over transversions, and frequent changes at the third codon position, in contrast with the gene evolution among more distant plant groups where biased nucleotide substitutions have frequently occurred. The sequences of these genes had diverged almost in proportion to taxonomic distance. The sequence of the pseudogene rpl23' changed approximately two times faster than that of the coding region. Sequence comparison between the pseudogene and its protein-coding counterpart revealed different degrees of nucleotide homology in wheat, rice and maize, suggesting that the transposition timing of the pseudogene differed and/or that different rates of gene conversion operated on the pseudogene in the cpDNA of the three plant groups in Gramineae. The intergenic spacer regions diverged approximately ten times faster than the genes. The divergence of wheat from barley, and that from rice are estimated based on the nucleotide similarity to be 1.5, 10 and 40 million years, respectively.     

Do nonasterid holoparasitic flowering plants have plastid genomes?

Past work involving the plastid genome (plastome) of holoparasitic plants has been confined to Scrophulariaceae (or Orobanchaceae) which have truncated plastomes owing to loss of photosynthetic and other genes. Nonasterid holoparasites from Balanophoraceae (Corynaea), Hydnoraceae (Hydnora) and Cytinaceae (Cytinus) were tested for the presence of plastid genes and a plastome. Using PCR, plastid 16S rDNA was successfully amplified and sequenced from the above three holoparasites. The sequence of Cytinus showed 121 single base substitutions relative to Nicotiana (8% of the molecule) whereas higher sequence divergence was observed in Hydnora and Corynaea (287 and 513 changes, respectively). Secondary structural models for these 16S rRNAs show that most changes are compensatory, thus suggesting they are functional. Probes constructed for 16S rDNA and for four plastid-encoded ribosomal protein genes (rps2, rps4, rps7 and rpl16) were used in Southern blots of digested genomic DNA from the three holoparasites. Positive hybridizations were obtained using each of the five probes only for Cytinus. For SmaI digests, all plastid gene probes hybridized to a common fragment ca. 20 kb in length in this species. Taken together, these data provide preliminary evidence suggestive of the retention of highly diverged and truncated plastid genome in Cytinus. The greater sequence divergence for 16S rDNA and the negative hybridization results for Hydnora and Corynaea suggests two possibilities: the loss of typically conserved elements of their plastomes or the complete absence of a plastome.     

Loss of the rpl32 gene from the chloroplast genome and subsequent acquisition of a preexisting transit peptide within the nuclear gene in Populus

Gene transfer events from organelle genomes (mitochondria and chloroplasts in plants) to the nuclear genome are important processes in the evolution of the eukaryotic cell. It is highly likely that the gene transfer event is still an ongoing process in higher plant mitochondria and chloroplasts. The number and order of genes encoded in the chloroplast genome of higher plants are highly conserved. Recently, several exceptional cases of gene loss from the chloroplast genome have been discovered as the number of complete chloroplast genome sequences has increased. The Populus chloroplast genome has lost the rpl32 gene, while the corresponding the chloroplast rpl32 (cp rpl32) gene has been identified in the nuclear genome. Nuclear genes transferred from the chloroplast genome need to gain a sequence that encodes a transit peptide. Here, we revealed that the nuclear cp rpl32 gene has acquired the exon sequence, which is highly homologous to a transit peptide derived from the chloroplast Cu-Zn superoxide dismutase (cp sod-1) gene. The cp rpl32 gene has acquired the sequence that encodes not only for the transit peptide, but also for the conserved N-terminal portion of the mature SOD protein from the cp sod-1 gene, suggesting the occurrence of DNA sequence duplication. Unlike cp SOD-1, cp RPL32 did not show biased localization in the chloroplasts. This difference may be caused by mutations accumulated in the sequence of the SOD domain on the cp rpl32 gene. We provide new insight into the fate of the inherent sequence derived from a transit peptide.     

Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences.

The families within the class Mollicutes are distinguished by their morphologies, nutritional requirements, and abilities to metabolize certain compounds. Biosystematic classification of the plant-pathogenic mycoplasmalike organisms (MLOs) has been difficult because these organisms have not been cultured in vitro, and hence their nutritional requirements have not been determined nor have physiological characterizations been possible. To investigate the evolutionary relationship of the MLOs to other members of the class Mollicutes, a segment of a ribosomal protein operon was cloned and sequenced from an aster yellows-type MLO which is pathogenic for members of the genus Oenothera and from Acholeplasma laidlawii. The deduced amino acid sequence data from the rpl22 and rps3 genes indicate that the MLOs are more closely related to A. laidlawii than to animal mycoplasmas, confirming previous results from 16S rRNA sequence comparisons. This conclusion is also supported by the finding that the UGA codon is not read as a tryptophan codon in the MLO and A. laidlawii, in contrast to its usage in Mycoplasma capricolum.     

The nucleotide sequence of five ribosomal protein genes from the cyanelles ofCyanophora paradoxa: Implications concerning the phylogenetic relationship between cyanelles and chloroplasts

Summary The nucleotide sequences of the ribosomal protein genesrps18, rps19, rpl2, rpl33, and partial sequence ofrpl22 from cyanelles, the photosynthetic organelles of the protistCyanophora paradoxa, have been determined. These genes form two clusters oriented in opposite and divergent directions. One cluster contains therpl33 andrps18 genes; the other contains therpl2, rps19, andrpl22 genes, in that order. Phylogenetic trees were constructed from both the DNA sequences and the deduced protein sequences of cyanelles,Euglena gracilis and land plant chloroplasts, andEscherichia coli, using parsimony or maximum likelihood methods. In addition, a phylogenetic tree was built from a distance matrix comparing the number of nucleotide substitutions per site. The phylogeny inferred from all these methods suggests that cyanelles fall within the chloroplast line of evolution and that the evolutionary distances between cyanelles and land plant chloroplasts are shorter than betweenE. gracilis chloroplasts and land plant chloroplasts.     

Mitochondrial DNA sequences of primates: Tempo and mode of evolution

Summary We cloned and sequenced a segment of mitochondrial DNA from human, chimpanzee, gorilla, orangutan, and gibbon. This segment is 896 bp in length, contains the genes for three transfer RNAs and parts of two proteins, and is homologous in all 5 primates. The 5 sequences differ from one another by base substitutions at 283 positions and by a deletion of one base pair. The sequence differences range from 9 to 19% among species, in agreement with estimates from cleavage map comparisons, thus confirming that the rate of mtDNA evolution in primates is 5 to 10 times higher than in nuclear DNA. The most striking new finding to emerge from these comparisons is that transitions greatly outnumber transversions. Ninety-two percent of the differences among the most closely related species (human, chimpanzee, and gorilla) are transitions. For pairs of species with longer divergence times, the observed percentage of transitions falls until, in the case of comparisons between primates and non-primates, it reaches a value of 45. The time dependence is probably due to obliteration of the record of transitions by multiple substitutions at the same nucleotide site. This finding illustrates the importance of choosing closely related species for analysis of the evolutionary process. The remarkable bias toward transitions in mtDNA evolution necessitates the revision of equations that correct for multiple substitutions at the same site. With revised equations, we calculated the incidence of silent and replacement substitutions in the two protein-coding genes. The silent substitution rate is 4 to 6 times higher than the replacement rate, indicating strong functional constraints at replacement sites. Moreover, the silent rate for these two genes is about 10% per million years, a value 10 times higher than the silent rate for the nuclear genes studied so far. In addition, the mean substitution rate in the three mitochondrial tRNA genes is at least 100 times higher than in nuclear tRNA genes. Finally, genealogical analysis of the sequence differences supports the view that the human lineage branched off only slightly before the gorilla and chimpanzee lineages diverged and strengthens the hypothesis that humans are more related to gorillas and chimpanzees than is the orangutan.Abbreviations mtDNA mitochondrial DNA - bp base pair - URF unidentified reading frame     

Human Mitochondrial DNA Variation and Evolution: Analysis of Nucleotide Sequences from Seven Individuals

We have analyzed nucleotide sequence variation in an approximately 900-base pair region of the human mitochondrial DNA molecule encompassing the heavy strand origin of replication and the D-loop. Our analysis has focused on nucleotide sequences available from seven humans. Average nucleotide diversity among the sequences is 1.7%, several-fold higher than estimates from restriction endonuclease site variation in mtDNA from these individuals and previously reported for other humans. This disparity is consistent with the rapidly evolving nature of this noncoding region. However, several instances of convergent or parallel gain and loss of restriction sites due to multiple substitutions were observed. In addition, other results suggest that restriction site (as well as pairwise sequence) comparisons may underestimate the total number of substitutions that have occurred since the divergence of two mtDNA sequences from a common ancestral sequence, even at low levels of divergence. This emphasizes the importance of recognizing the large standard errors associated with estimates of sequence variability, particularly when constructing phylogenies among closely related sequences. Analysis of the observed number and direction of substitutions revealed several significant biases, most notably a strand dependence of substitution type and a 32-fold bias favoring transitions over transversions. The results also revealed a significantly nonrandom distribution of nucleotide substitutions and sequence length variation. Significantly more multiple substitutions were observed than expected for these closely related sequences under the assumption of uniform rates of substitution. The bias for transitions has resulted in predominantly convergent or parallel changes among the observed multiple substitutions. There is no convincing evidence that recombination has contributed to the mtDNA sequence diversity we have observed.     

Molecular systematic studies on the tribe Polygonateae (s. l.) in China based on RFLPs data of PCR-amplified chloroplast DNA fragments

Phylogenetic relationships in the tribe Polygonateae s. l. of Liliaceae were investigated with an RFLP analysis of two PCR-amplified chloroplast genome DNA fragments. One fragment is the transfer RNA gene for lysine( trnK gene) including the maturase-encoding gene matK, and the other is the rpl16 gene which codes for a chloroplast ribosomal protein in large subunit. The trnK gene is ca. 2600 bp in length in all the taxa, but the rpl16 gene ranges from ca. 1140 bp to ca. 1320 bp in length among different genera and in Polygonatum. The results suggested that the tribe Polygonateae s. str. consisting of Polygonatum, Disporopsis, Smilacina and Maianthemum is closely related to Convallaria in Convallarieae, and supported the earlier results that Streptopus and Disporum should be removed out from the tribe Polygonateae s. l.. The results also showed that Polygonatum formed a clad with Disporopsis, while Smilacina lumped with Maianthemum in the tribe Polygonateae s. str.. In addition, the present study supports the view that S. ginfoshanicumshould be transferred from Smilacina to Polygonatum.     

The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations

The frequency and specificity of mutations produced in vitro by eucaryotic DNA polymerase-beta have been determined in a forward mutation assay using a 250-base target sequence in M13mp2 DNA. Homogeneous DNA polymerase-beta, isolated from four different sources, produces mutations at a frequency of 4-6%/single round of gap-filling DNA synthesis. DNA sequence analyses of 460 independent mutants resulting from this error-prone DNA synthesis demonstrate a wide variety of mutational events. Frameshift and base substitutions are made at approximately equal frequency and together comprise about 90% of all mutations. Two mutational "hot spots" for frameshift and base substitution mutations were observed. The characteristics of the mutations at these sites suggest that certain base substitution errors result from dislocation of template bases rather than from direct mispair formation by DNA polymerase-beta. When considering the entire target sequence, single-base frameshift mutations occur primarily in runs of identical bases, usually pyrimidines. The loss of a single base occurs 20-80 times more frequently than single-base additions and much more frequently than the loss of two or more bases. Base substitutions occur at many sites throughout the target, representing a wide spectrum of mispair formations. Averaged over a large number of phenotypically detectable sites, the base substitution error frequency is greater than one mistake for every 5000 bases polymerized. Large deletion mutations are also observed, at a frequency more than 10-fold over background, indicating that purified DNA polymerases alone are capable of producing such deletions. These data are discussed in relation to the physical and kinetic properties of the purified enzymes and with respect to the proposed role for this DNA polymerase in vivo.     

Molecular analysis of the complete set of length mutations found in the plastomes of Triticum-Aegilops species.

Precise location and nature of each of 14 length mutations detected among chloroplast DNAs of Triticum-Aegilops species by RFLP analysis were determined at the nucleotide sequence level. Each mutation was compared with at least three non-mutated wild-type plastomes as standards. These 14 length mutations were classified into 4 duplications and 10 deletions. One duplication occurred in the small single-copy region close to the border of the inverted repeat, and the remaining 13 length mutations took place in the large single-copy region. All length mutations occurred in the intergenic regions, suggesting that these length mutations do not affect plastid gene expression. Saltatory replication was the cause of all duplications, whereas intramolecular recombination mediated by short direct repeats played a substantial role in the deletions. Recurrent occurrences of certain deletion events were found in some AT-rich regions, which constituted hot spots for deletion. Out of four hypervariable regions detected among the grass plastomes, two (downstream of rbcL and a tRNA gene accumulated region) were still active after differentiation of Triticum and Aegilops complex.     

Intergenic DNA sequences flanking the pseudo alpha globin genes of human and chimpanzee.

We have determined the sequence of 2400 base pairs upstream from the human pseudo alpha globin (psi alpha) gene, and for comparison, 1100 base pairs of DNA within and upstream from the chimpanzee psi alpha gene. The region upstream from the promoter of the psi alpha gene shows no significant homology to the intergenic regions of the adult alpha 2 and alpha 1 globin genes. The chimpanzee gene has a coding defect in common with the human psi alpha gene, showing that the product of this gene, if any, was inactivated before the divergence of human and chimpanzee. However the chimpanzee gene contains a normal ATG initiation codon in contrast to the human gene which has GTG as the initiation codon. The psi alpha genes of both human and chimpanzee are flanked by the same Alu family member. The structure and position of this repeat have not been altered since the divergence of human and chimpanzee, and it is at least as well conserved as its immediate flanking sequence. Comparing human and chimpanzee, the 300 bp Alu repeat has accumulated only two base substitutions and one length mutation; the adjacent 300 bp flanking region has accumulated five base substitutions and twelve length mutations.     

Identification of functional open reading frames in chloroplast genomes

We have used a rapid computer dot-matrix comparison method to identify all DNA regions which have been evolutionarily conserved between the completely sequenced chloroplast genomes of tobacco and a liverwort. Analysis of these regions reveals 74 homologous open reading frames (ORFs) which have been conserved as to length and amino acid sequence; these ORFs also have an excess of nucleotide substitutions at silent sites of codons. Since the nonfunctional parts of these genomes have become saturated with mutations and show no sequence similarity whatsoever, the homologous ORFs are almost certainly functional. A further four pairs of ORFs show homology limited to only a short part of their putative gene products. Amino acid sequence identities range between 50 and 99%; some chloroplast proteins are seen to be among the most slowly evolving of all known proteins. A search of the nucleotide and amino acid sequence databanks has revealed several previously unidentified genes in chloroplast sequences from other species, but no new homologies to prokaryotic genes.     

Persistence of Repeated Sequences That Evolve by Replication Slippage

The evolution of short repeated sequences by replication slippage under the assumption of selective neutrality is modeled using a linear birth and death process. The equilibrium distribution, the distribution of the life expectancy of a repeated sequence when the process starts from two repeats, the age distribution of repeats, the probability of obtaining two genes with i and j copies which diverged t generations ago and the conditional variance of copy number given the repeat number is more than one are computed. The distributions of life expectancy and age are shown to have long tails. Also the statistic which estimates the conditional variance is shown to have a large coefficient of variation. Using these theoretical results, we develop an approximate test of our model and analyze persistent repeated sequences found in the primate beta-globin gene region and Oenothera chloroplast DNA which are polymorphic within species. We found one sequence in Oenothera chloroplast DNA which does not fit to our neutral model.     

叶绿体DNA片段的RFLP分析在黄精族系统学研究中的应用

对广义百合科黄精族6属23种及铃兰族1属1种的叶绿体基因组trnK和rpl16两个基因片段进 行了PCR-RFLP分析,结果表明：trnK基因的PCR产物在各类群间几乎不存在长度变异,均约2600bp,而rpl16基因则在各属之间及黄精属内表现出长度变异,变异范围在1140~1320bp之间;限制性酶切位点的同源性分析显示,黄精属、竹根七属、鹿药属和舞鹤草属构成的狭义黄精族与铃兰族中的铃兰属有较近的亲缘关系,并支持将扭柄花属和万寿竹属从广义百合科黄精族中分出的观点;在狭义黄精族内,黄精属与竹根七属聚成一支,鹿药属与舞鹤草属聚成另一支,为探讨族内属间的系统演化关系提供了分子生物学方面的证据。另外,本研究结果支持将金佛山黄精从鹿药属转隶至黄精属的观点。     

Maize Mitochondrial Plasmid S-1 Sequences Share Homology with Chloroplast Gene psbA

The linear, 6397-base pair (bp), mitochondrial S-1 DNA molecule from maize contains a 420-bp segment that is homologous with the chloroplast gene (psbA) that codes for the quinone binding protein of photosystem II. This is the first report of a chloroplast sequence in a naturally occurring viral-like or plasmid DNA. The complete sequence of the S-1 chloroplast segment has been compared with homologous regions of six different chloroplast genes. The S-1 segment has diverged from the other genes both by length mutation and base substitution. Several of the length mutations are exact adjacent tandem duplications of 4 and 5 bp similar to "footprints" left after excision of transposable elements in maize nuclear DNA.