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.     

Extensive structural variations between mitochondrial genomes of CMS and normal peppers (Capsicum annuum L.) revealed by complete nucleotide sequencing

Background

Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp.

Results

We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (ψatp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and ψatp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes.

Conclusions

Although large portion of sequence context was shared by mitochondrial genomes of CMS and male-fertile pepper lines, extensive genome rearrangements were detected. CMS candidate genes located on the edges of highly-rearranged CMS-specific DNA regions and near to repeat sequences. These characteristics were detected among CMS-associated genes in other species, implying a common mechanism might be involved in the evolution of CMS-associated genes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-561) contains supplementary material, which is available to authorized users.     

A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing

A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.     

Automated sequencing of complete mitochondrial genomes from laser-capture microdissected samples

Mitochondrial DNA mutations have been related to both aging and a variety of diseases such as cancer. Due to the relatively small size of the genome (16 kb) and with the use of automated DNA sequencing, the entire genome can be sequenced from clinical specimens in days. We present a reliable approach to complete mitochondrial genome sequencing from laser-capture microdissected human clinical cancer specimens that overcome the inherent limitations of relatively small tissue samples and partial DNA degradation, which are unavoidable when laser-capture microdissection is used to attain pure populations of cells from heterogeneous tissues obtained from surgical procedures. The acquisition of sufficient template combined with a standard set of 18 pairs of PCR primers allows for the efficient amplification of the genome. Subsequent single-stranded amplification is performed using 36 sequencing primers, and samples are run on an ABI PRISM 3100 Genetic Analyzer. The use of this procedure should allow even investigators with little experience sequencing from clinical specimens success in complete mitochondrial genome sequencing.     

The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants

Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and rps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann     

Baboon phylogeny as inferred from complete mitochondrial genomes

Baboons (genus Papio) are an interesting phylogeographical primate model for the evolution of savanna species during the Pleistocene. Earlier studies, based on partial mitochondrial sequence information, revealed seven major haplogroups indicating multiple para‐ and polyphylies among the six baboon species. The most basal splits among baboon lineages remained unresolved and the credibility intervals for divergence time estimates were rather large. Assuming that genetic variation within the two studied mitochondrial loci so far was insufficient to infer the apparently rapid early radiation of baboons we used complete mitochondrial sequence information of ten specimens, representing all major baboon lineages, to reconstruct a baboon phylogeny and to re‐estimate divergence times. Our data confirmed the earlier tree topology including the para‐ and polyphyletic relationships of most baboon species; divergence time estimates are slightly younger and credibility intervals narrowed substantially, thus making the estimates more precise. However, the most basal relationships could not be resolved and it remains open whether (1) the most southern population of baboons diverged first or (2) a major split occurred between southern and northern clades. Our study shows that complete mitochondrial genome sequences are more effective to reconstruct robust phylogenies and to narrow down estimated divergence time intervals than only short portions of the mitochondrial genome, although there are also limitations in resolving phylogenetic relationships. Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.     

Afrotherian phylogeny as inferred from complete mitochondrial genomes

Afrotheria is a huge assemblage of various mammals encompassing six orders that were once classified as distantly related groups. This superordinal relationship may have resulted from the break-up of Gondowanaland followed by the isolation of the African continent between 105 and 40 million years ago. Although the monophyly of Afrotheria is well supported by recent molecular studies, the interrelationships within afrotherian mammals remain unclarified. In this study, we determined the sequence of the complete mitochondrial genomes of hyrax, golden mole, and elephant shrew. These sequences were compared with those of other eutherians to analyze the phylogenetic relationships among afrotherians and, in particular, those among paenungulates. Our mitochondrial genome analysis supports the monophyly of Tethytheria.     

The complete nucleotide sequence of the mitochondrial genome of Tetraodon nigroviridis.

The fresh water pufferfish Tetraodon nigroviridis is a model organism for studying evolution of genome and gene functions, but its mitochondrial genome (mtDNA) sequence is still not available. We determined the complete nucleotide sequence of its mtDNA using shotgun sequencing. The T. nigroviridis mtDNA was 16,462 bp, and contained 13 protein coding genes, 22 tRNAs, 2 rRNAs and a major non-coding region. The gene order was identical to the common type of vertebrate mtDNA, whereas the G + C content in the sense strand was 46.9%, much higher than most other fish species. One hundred and three SNPs were detected in the control region of the mtDNA of 35 individuals, a majority of SNPs were detected in the 5' end of the control region. A phylogenetic study including 21 fish species was performed on concatenated amino acid sequences of 12 protein coding genes, and revealed that the T. nigroviridis was clustered with Fugu rubripes into a group. The complete mtDNA sequence and SNPs in its control region will be useful in studying fish evolution, in differentiating different Tetraodon species and in analyzing genetic diversity within T. nigroviridis.     

Unusual features in the photosynthetic machinery of Halorhodospira halochloris DSM 1059 revealed by complete genome sequencing

Halorhodospira halochloris is an anaerobic, halophilic, purple photosynthetic bacterium belonging to γ-Proteobacteria. H. halochloris is also characteristic as a thermophilic phototrophic isolate producing bacteriochlorophyll (BChl) b. Here, we report the complete genome sequence of H. halochloris DSM 1059. The genetic arrangement for this bacterium’s photosynthetic apparatus is of particular interest; its genome contains two sets of puf operons encoding the reaction center and core light-harvesting 1 (LH1) complexes having almost identical nucleotide sequences (e.g., 98.8–99.9% of nucleotide identities between two sets of pufLM genes, but 100% of deduced amino acid sequence identities). This duplication of photosynthetic genes may provide a glimpse at natural selection in action. The β-polypeptides of the LH1 complex in purple bacteria usually contain two histidine residues to bind BChl a; however, those of H. halochloris were revealed to have four histidine residues, indicating unusual pigment organization in the LH1 complex of this species. Like in other BChl b-producing phototrophs, the genome of H. halochloris lacks the divinyl reductase genes bciA and bciB. The phylogeny of chlorophyllide a oxidoreductase, which catalyzes committed steps in the synthesis of BChl a and BChl b, indicates that evolution toward BChl b production is convergent. Geranylgeranyl reductase (BchP) of H. halochloris has an insertion region in its primary structure, which could be important for its unusual sequential reduction reactions.

     

Structural dynamics of the mitochondrial ADP/ATP carrier revealed by molecular dynamics simulation studies

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier has been recently crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). In the crystal structure, the six-transmembrane helix bundle that defines the nucleotide translocation pathway is closed on the matrix side due to sharp kinks in the odd-numbered helices. The closed conformation is further sealed by the loops protruding into the matrix that interact through an intricate network of charge-pairs. To gain insight into its structural dynamics we performed molecular dynamics (MD) simulation studies of the ADP/ATP carrier with and without its cocrystallized inhibitor. The two trajectories sampled a conformational space around two different configurations characterized by distinct salt-bridge networks with a significant shift from inter- to intrarepeat bonding on the matrix side in the absence of CATR. Analysis of the geometrical parameters defining the transmembrane helices showed that even-numbered helices can undergo a face rotation, whereas odd-numbered helices can undergo a change in the wobble angle with a conserved proline acting as molecular hinge. Our results provide new information on the dynamical properties of the ADP/ATP carrier and for the first time yield a detailed picture of a stable carrier conformation in absence of the inhibitor.     

Temporal dynamics in the evolution of the sunflower genome as revealed by sequencing and annotation of three large genomic regions

Improved knowledge of genome composition, especially of its repetitive component, generates important informations in both theoretical and applied research. In this study, we provide the first insight into the local organization of the sunflower genome by sequencing and annotating 349,380 bp from 3 BAC clones, each including one single-copy gene. These analyses resulted in the identification of 11 putative gene sequences, 18 full-length LTR retrotransposons, 6 incomplete LTR retrotransposons, 2 non-autonomous LTR-retroelements (LINEs), 2 putative DNA transposons fragments and one putative helitron. Among LTR-retrotransposons, non-autonomous elements (the so-called LARDs), which do not carry any protein-encoding sequence, were discovered for the first time in the sunflower. The insertion time of intact retroelements was measured, based on sister LTRs divergence. All isolated elements were inserted relatively recently, especially those belonging to the Gypsy superfamily. Retrotransposon families related to those identified in the BAC clones are present also in other species of Helianthus, both annual and perennial, and even in other Asteraceae. In one of the three BAC clones, we found five copies of a lipid transfer protein (LTP) encoding gene within less than 100,000 bp, four of which are potentially functional. Two of these are interrupted by LTR retrotransposons, in the intron and in the coding sequence, respectively. The divergence between sister LTRs of the retrotransposons inserted within the genes indicates that LTP gene duplication started earlier than 1.749 MYRS ago. On the whole, the results reported in this study confirm that the sunflower is an excellent system to study transposons dynamics and evolution.     

Structural alterations from multiple displacement amplification of a human genome revealed by mate-pair sequencing

Comprehensive identification of the acquired mutations that cause common cancers will require genomic analyses of large sets of tumor samples. Typically, the tissue material available from tumor specimens is limited, which creates a demand for accurate template amplification. We therefore evaluated whether phi29-mediated whole genome amplification introduces false positive structural mutations by massive mate-pair sequencing of a normal human genome before and after such amplification. Multiple displacement amplification led to a decrease in clone coverage and an increase by two orders of magnitude in the prevalence of inversions, but did not increase the prevalence of translocations. While multiple strand displacement amplification may find uses in translocation analyses, it is likely that alternative amplification strategies need to be developed to meet the demands of cancer genomics.     

Entire nucleotide sequences of Gossypium raimondii and G. arboreum mitochondrial genomes revealed A‐genome species as cytoplasmic donor of the allotetraploid species

• Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A–G and K, and an allotetraploid genomic group, AD. Gossypium raimondii (D₅) and G. arboreum (A₂) are the putative contributors to the progenitor of G. hirsutum (AD₁), the economically important fibre‐producing cotton species.
• Mitochondrial DNA from week‐old etiolated seedlings was extracted from isolated organelles using discontinuous sucrose density gradient method. Mitochondrial genomes were sequenced, assembled, annotated and analysed in orderly.
• Gossypium raimondii (D₅) and G. arboreum (A₂) mitochondrial genomes were provided in this study. The mitochondrial genomes of two diploid species harboured circular genome of 643,914 bp (D₅) and 687,482 bp (A₂), respectively. They differ in size and number of repeat sequences, both contain illuminating triplicate sequences with 7317 and 10,246 bp, respectively, demonstrating dynamic difference and rearranged genome organisations. Comparing the D₅ and A₂ mitogenomes with mitogenomes of tetraploid Gossypium species (AD₁, G. hirsutum; AD₂, G. barbadense), a shared 11 kbp fragment loss was detected in allotetraploid species, three regions shared by G. arboreum (A₂), G. hirsutum (AD₁) and G. barbadense (AD₂), while eight regions were specific to G. raimondii (D₅). The presence/absence variations and gene‐based phylogeny supported that A‐genome is a cytoplasmic donor to the progenitor of allotetraploid species G. hirsutum and G. barbadense.
• The results present structure variations and phylogeny of Gossypium mitochondrial genome evolution.

     

Overlapping open reading frames revealed by complete nucleotide sequencing of turnip yellow mosaic virus genomic RNA.

The complete nucleotide sequence of turnip yellow mosaic virus (TYMV) genomic RNA has been determined on a set of overlapping cDNA clones using a sequential sequencing strategy. The RNA is 6318 nucleotides long, excluding the cap structure. The genome organization deduced from the sequence confirms previous results of in vitro translation. A novel open reading frame (ORF) putatively encoding a Pro-rich and very basic 69K (K = kilodalton) protein is detected at the 5' end of the genome. It is initiated at the first AUG codon on the RNA and overlaps the major ORF that encodes the non structural 206K (previously referred to as 195K) protein of TYMV; its function is unknown. Several amino acid consensus sequences already described among plant and animal viruses are also found in the TYMV-encoded polypeptides. A comparison with other viruses whose RNA sequence is known leads to the conclusion that TYMV belongs to the "Sindbis-like" supergroup of viruses and could be related to Semliki forest virus.     

Analysis of three leafminers' complete mitochondrial genomes

Liriomyza trifolii (Burgess), Liriomyza huidobrensis (Blanchard), and Liriomyza bryoniae (Kaltenbach), are three closely related and economically important leafminer pests in the world. This study examined the complete mitochondrial genomes of L. trifolii, L. huidobrensis and L. bryoniae, which were 16141 bp, 16236 bp and 16183 bp in length, respectively. All of them displayed 37 typical animal mitochondrial genes and an A + T-rich region. The genomes were highly compact with only 60–68 bp of non-coding intergenic spacer. However, considerable differences in the A + T-rich region were detected among the three species. Results of this study also showed the two ribosomal RNA genes of the three species had very limited variable sites and thus should not provide much information in the study of population genetics of these species. Data generated from three leafminers' complete mitochondrial genomes should provide valuable information in studying phylogeny of Diptera, and developing genetic markers for species identification in leafminers.     

Ecophysiology of Thioploca ingrica as revealed by the complete genome sequence supplemented with proteomic evidence

Large sulfur-oxidizing bacteria, which accumulate a high concentration of nitrate, are important constituents of aquatic sediment ecosystems. No representative of this group has been isolated in pure culture, and only fragmented draft genome sequences are available for these microorganisms. In this study, we successfully reconstituted the genome of Thioploca ingrica from metagenomic sequences, thereby generating the first complete genome sequence from this group. The Thioploca samples for the metagenomic analysis were obtained from a freshwater lake in Japan. A PCR-free paired-end library was constructed from the DNA extracted from the samples and was sequenced on the Illumina MiSeq platform. By closing gaps within and between the scaffolds, we obtained a circular chromosome and a plasmid-like element. The reconstituted chromosome was 4.8 Mbp in length with a 41.2% GC content. A sulfur oxidation pathway identical to that suggested for the closest relatives of Thioploca was deduced from the reconstituted genome. A full set of genes required for respiratory nitrate reduction to dinitrogen gas was also identified. We further performed a proteomic analysis of the Thioploca sample and detected many enzymes/proteins involved in sulfur oxidation, nitrate respiration and inorganic carbon fixation as major components of the protein extracts from the sample, suggesting that these metabolic activities are strongly associated with the physiology of T. ingrica in lake sediment.     

Progress in the complete mitochondrial genomes of the Acari

蜱螨亚纲包括蜱类和螨类,是节肢动物中物种多样性最高的类群之一.本文综述了当前已测序的28种蜱螨线粒体基因组的研究成果.概括起来,蜱螨线粒体基因组具有以下特点:(1)大小变异显著,其中柑橘全爪螨Panonychus citri线粒体基因组在目前已测节肢动物中最小(13077 bp);(2)一般碱基组成偏向A和T,但6种蜱螨具有相反的GC-偏斜(正值);(3)基因组的碱基组成及A+T富集区的位置、长度和拷贝数等变异显著,其中4种叶螨的A+T含量最高,其A+T富集区在目前已测节肢动物中最短(44 -57 bp);(4)基因高度重排,特别是真螨总目的种类,但重排与高分类阶元无相关性;(5)真螨总目部分螨类的tRNA基因极度缩短,不能形成经典的三叶草二级结构.作者建议要进一步测定更多蜱螨的线粒体基因组,验证蜱螨非典型tRNA基因的生物学功能性,分析蜱螨线粒体基因组的分子进化机制,开展蜱螨线粒体转录组研究等.     

蜱螨线粒体基因组研究进展

蜱螨亚纲包括蜱类和螨类, 是节肢动物中物种多样性最高的类群之一。本文综述了当前已测序的28种蜱螨线粒体基因组的研究成果。概括起来, 蜱螨线粒体基因组具有以下特点： （1）大小变异显著, 其中柑橘全爪螨Panonychus citri线粒体基因组在目前已测节肢动物中最小（13 077 bp）; （2）一般碱基组成偏向A和T, 但6种蜱螨具有相反的GC-偏斜（正值）; （3）基因组的碱基组成及A+T富集区的位置、 长度和拷贝数等变异显著, 其中4种叶螨的A+T含量最高, 其A+T富集区在目前已测节肢动物中最短（44~57 bp）; （4）基因高度重排, 特别是真螨总目的种类, 但重排与高分类阶元无相关性; （5）真螨总目部分螨类的tRNA基因极度缩短, 不能形成经典的三叶草二级结构。作者建议要进一步测定更多蜱螨的线粒体基因组, 验证蜱螨非典型tRNA基因的生物学功能性, 分析蜱螨线粒体基因组的分子进化机制, 开展蜱螨线粒体转录组研究等。