Nucleotide diversity of the Chlamydomonas reinhardtii plastid genome: addressing the mutational-hazard hypothesis

Background  

The mutational-hazard hypothesis argues that the noncoding-DNA content of a genome is a consequence of the mutation rate (μ) and the effective number of genes per locus in the population (N _g ). The hypothesis predicts that genomes with a high N _g μ will be more compact than those with a small N _g μ. Approximations of N _g μ can be gained by measuring the nucleotide diversity at silent sites (π_silent). We addressed the mutation-hazard hypothesis apropos plastid-genome evolution by measuring π_silent of the Chlamydomonas reinhardtii plastid DNA (ptDNA), the most noncoding-DNA-dense plastid genome observed to date. The data presented here in conjunction with previously published values of π_silent for the C. reinhardtii mitochondrial and nuclear genomes, which are respectively compact and bloated, allow for a complete analysis of nucleotide diversity and genome compactness in all three genetic compartments of this model organism.     

Nucleotide Diversity of the Colorless Green Alga Polytomella parva (Chlorophyceae,Chlorophyta): High for the Mitochondrial Telomeres,Surprisingly Low Everywhere Else*

ABSTRACT. Silent‐site nucleotide diversity data (π_silent) can provide insights into the forces driving genome evolution. Here we present π_silent statistics for the mitochondrial and nuclear DNAs of Polytomella parva, a nonphotosynthetic green alga with a highly reduced, linear fragmented mitochondrial genome. We show that this species harbors very little genetic diversity, with the exception of the mitochondrial telomeres, which have an excess of polymorphic sites. These data are compared with previously published π_silent values from the mitochondrial and nuclear genomes of the model species Chlamydomonas reinhardtii and Volvox carteri, which are close relatives of P. parva, and are used to understand the modes and tempos of genome evolution within green algae.     

Expression and nucleotide diversity of the poplar <Emphasis Type="Italic">COBL</Emphasis> gene

The COBRA gene encodes a putative glycosyl-phosphatidylinositol-anchored protein that regulates cellulose deposition and oriented cell expansion in the plant cell wall. This study reports the identification of PtCOBL4, a first COBRA gene from the tree Populus tomentosa. The full-length cDNA of PtCOBL4 was isolated from a xylem cDNA library. The deduced protein sequence shares 72.7% identity with Arabidopsis AtCOBL4 protein involved in secondary cell wall deposition. Analysis of differential tissue expression by real-time polymerase chain reaction (PCR) indicated that PtCOBL4 is expressed predominantly in the mature xylem zone. By using the sequenced whole genome and DNA microarray data of Populus, we demonstrated that COBRA is a multigene family of 11 members, each of which exhibit different tissue-specific expression patterns. To evaluate the functional consequences of nucleotide polymorphisms in the PtCOBL4 locus, the patterns of variation in a 2,002-bp region of the gene were surveyed in 40 unrelated individuals representative of almost the entire natural range of P. tomentosa. Sixty-one single-nucleotide polymorphisms (SNPs) were identified at a frequency of one SNP per 32.8 bp of sequence, giving an estimated nucleotide diversity of π _T = 0.00800 and θ _w = 0.00716. Within coding regions, nonsynonymous diversity (π _nonsyn = 0.00285) was markedly lower than synonymous diversity (π _syn = 0.02128); the π _nonsyn/π _syn ratio was 0.13, significantly less than 1, indicating that the synonymous sites were subject to strong purifying selection. These results provide the necessary foundation for improving the quantity and quality of cellulose via genetic engineering or by candidate-gene-based association genetics in P. tomentosa.     

Compartmentalisation of [FeFe]‐hydrogenase maturation in Chlamydomonas reinhardtii

Molecular hydrogen (H₂) can be produced in green microalgae by [FeFe]‐hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub‐cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub‐cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase‐deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H₂ production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H₂ production comparable to the wild type, as did the transformants expressing full‐length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm‐targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H₂‐producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.     

The <Emphasis Type="Italic">Dunaliella salina</Emphasis> organelle genomes: large sequences,inflated with intronic and intergenic DNA

Background  

Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri.     

Strict paternal transmission of mitochondrial DNA of Chlamydomonas species is explained by selection against maternal nucleoids

Summary.  The non-Mendelian inheritance of organelle DNA is common in most plants and animals. Here we examined inheritance mechanisms involved in the transfer of mitochondrial DNA. We successively backcrossed (to F₅) two interfertile strains of the unicellular isogamous haploid algae Chlamydomonas reinhardtii and Chlamydomonas smithii to match nuclear backgrounds and examine transmission patterns of mitochondrial DNA by PCR analysis of cob gene sequences. Mitochondrial DNA was strictly transmitted paternally. To investigate the behavior of parental mitochondrial DNA, we used F₅ progeny to form zygotes and isolated single zygotes. The results showed selective disappearance of maternal mitochondrial nucleoids occurred between 3 and 6 h after zygote formation. Received July 11, 2002; accepted September 28, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan.     

Genetic connectivity among swarming sites in the wide ranging and recently declining little brown bat (Myotis lucifugus)

Characterizing movement dynamics and spatial aspects of gene flow within a species permits inference on population structuring. As patterns of structuring are products of historical and current demographics and gene flow, assessment of structure through time can yield an understanding of evolutionary dynamics acting on populations that are necessary to inform management. Recent dramatic population declines in hibernating bats in eastern North America from white‐nose syndrome have prompted the need for information on movement dynamics for multiple bat species. We characterized population genetic structure of the little brown bat, Myotis lucifugus, at swarming sites in southeastern Canada using 9 nuclear microsatellites and a 292‐bp region of the mitochondrial genome. Analyses of F_ST, Φ_ST, and Bayesian clustering (STRUCTURE) found weak levels of genetic structure among swarming sites for the nuclear and mitochondrial genome (Global F_ST = 0.001, P < 0.05, Global Φ_ST = 0.045, P < 0.01, STRUCTURE K = 1) suggesting high contemporary gene flow. Hierarchical AMOVA also suggests little structuring at a regional (provincial) level. Metrics of nuclear genetic structure were not found to differ between males and females suggesting weak asymmetries in gene flow between the sexes. However, a greater degree of mitochondrial structuring does support male‐biased dispersal long term. Demographic analyses were consistent with past population growth and suggest a population expansion occurred from approximately 1250 to 12,500 BP, following Pleistocene deglaciation in the region. Our study suggests high gene flow and thus a high degree of connectivity among bats that visit swarming sites whereby mainland areas of the region may be best considered as one large gene pool for management and conservation.     

Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation

Summary The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 × 10^–7) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 × 10^–7). No colonies were seen on control plates (frequency < 0.96 × 10^–9). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.     

Analysis of genetic structure of the red shrimp <Emphasis Type="Italic">Aristeus antennatus</Emphasis> from the Western Mediterranean employing two mitochondrial regions

In this paper we addressed the phylogeographical genetic structure of the economically important red shrimp, Aristeus antennatus (Crustacea, Aristeidae) in the Western Mediterranean. Partial mitochondrial regions of the cytochrome c oxidase subunit I (514 bp) and ribosomal 16S subunit (547 bp) were sequenced in 137 individuals collected at three localities: Catalan Sea, Ligurian Sea and the southern Tyrrhenian Sea. Values of haplotypic diversity were h = 0.552–0.724, whereas those for nucleotide diversity were π = 0.0012–0.0026. Among-sample genetic diversity was not significant and no geographical patterns in the distribution of haplotypes were apparent. Results of the present study are consistent with a past population expansion that occurred <2,000 years ago. Despite the current fishing pressure, genetic variability appears to be sufficiently high to keep A. antennatus populations stable over time. Dispersal-related life history traits may account for the shallow genetic structure. Our results are not in contrast with the hypothesis of sustainability of Western Mediterranean red shrimp fisheries predicted on the basis of previously obtained biological results.     

Influence of Selenium on Hepatic Mitochondrial Antioxidant Capacity in Ducklings Intoxicated with Aflatoxin B<Subscript>1</Subscript>

The aim of the study was to investigate the effect of selenium on hepatic mitochondrial antioxidant capacity in ducklings administrated with aflatoxin B₁ (AFB₁). Ninety 7-day-old ducklings were randomly divided into three groups (groups I–III). Group I was used as a blank control. Group II was administered with AFB₁ (0.1 mg/kg body weight). Group III was administered with AFB₁ (0.1 mg/kg body weight) plus selenium (sodium selenite, 1 mg/kg body weight). All treatments were given once daily for 21 days. The results showed that the activities of mitochondrial superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR) in group II ducklings significantly decreased when compared with group I (P < 0.01). Furthermore, the content of hepatic mitochondrial malondialdehyde (MDA) significantly increased (P < 0.01). However, the activities of hepatic mitochondrial SOD, CAT, GSH-Px, and GR in group III ducklings significantly increased when compared with group II (P < 0.05). In addition, the content of hepatic mitochondrial MDA significantly decreased (P < 0.01). These results revealed that AFB₁ significantly induced hepatic mitochondrial antioxidant dysfunction. However, sodium selenite could significantly ameliorate the negative effect induced by AFB₁.     

Low number of mitochondrial pseudogenes in the chicken (<Emphasis Type="Italic">Gallus gallus</Emphasis>) nuclear genome: implications for molecular inference of population history and phylogenetics

Background  

Mitochondrial DNA has been detected in the nuclear genome of eukaryotes as pseudogenes, or Numts. Human and plant genomes harbor a large number of Numts, some of which have high similarity to mitochondrial fragments and thus may have been inadvertently included in population genetic and phylogenetic studies using mitochondrial DNA. Birds have smaller genomes relative to mammals, and the genome-wide frequency and distribution of Numts is still unknown. The release of a preliminary version of the chicken (Gallus gallus) genome by the Genome Sequencing Center at Washington University, St. Louis provided an opportunity to search this first avian genome for the frequency and characteristics of Numts relative to those in human and plants.     

Characterization of singlet oxygen-accumulating mutants isolated in a screen for altered oxidative stress response in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background  

When photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. In Chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene GPXH/GPX5 during high light stress, but little is known about the cellular mechanisms involved in this response. To investigate components affecting singlet oxygen signaling in C. reinhardtii, a mutant screen was performed.     

An <Emphasis Type="Italic">in silico</Emphasis> analysis of the mitochondrial protein import apparatus of plants

Background  

An in silico analysis of the mitochondrial protein import apparatus from a variety of species; including Chlamydomonas reinhardtii, Chlorella variabilis, Ectocarpus siliculosus, Cyanidioschyzon merolae, Physcomitrella patens, Selaginella moellendorffii, Picea glauca, Oryza sativa and Arabidopsis thaliana was undertaken to determine if components differed within and between plant and non-plant species.     

Mitochondrially-targeted expression of a cytoplasmic male sterility-associated <Emphasis Type="Italic">orf220</Emphasis> gene causes male sterility in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Background  

The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility.     

The complete mitochondrial genome of the oriental fruit moth <Emphasis Type="Italic">Grapholita molesta</Emphasis> (Busck) (Lepidoptera: Tortricidae)

The oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) currently is one of the economically most destructive pest species of stone and pome fruits worldwide. Here we sequenced the complete mitochondrial genome of this pest. This genome is 15,776 bp long, with an A + T content of 81.24%, containing 37 typical animal mitochondrial genes and an A + T-rich region. All gene are arranged as hypothesized ancestral gene order of insects except for trnM, which was shuffled from 3′ downstream of trnQ to 5′ upstream of trnI. cox1 gene uses unusual CGA start codon, as that in all other sequenced lepidopteran mitochondrial genome. The secondary structures for the two rRNA genes were predicted. All helices typically present in insect mitochondrial rRNA genes are generated. A microsatellite sequence was inserted into the region of H2347 in rrnL in G. molesta and two other sequenced tortricid mitochondrial genomes, indicating that the insertion event in this helix might occurred anciently in family Tortricidae. All of the 22 typical animal tRNA genes have a typical cloverleaf structure except for trnS2, in which the D-stem pairings in the DHU arm are absent. An intergenic sequence is present between trnQ and nad2 as well as in other sequenced lepidopteran mitochondrial genomes, which was presumed to be a remnant of trnM gene and its boundary sequences after the duplication of trnM to the upstream of trnI in Lepidoptera. The A + T-rich region is 836 bp, containing six repeat sequences of “TTATTATTATTATTAAATA(G)TTT.”     

The complete mitochondrial genome analysis of the tiger (<Emphasis Type="Italic">Panthera tigris</Emphasis>)

The complete mitochondrial genomes of five tiger samples from three subspecies (P. t. sumatrae, P. t. altica, and P. t. tigris) were successfully obtained by using 26 specifically designed Panthera-specific primer sets. The genome organization and gene arrangement of the five tiger samples were similar to each other; however polymorphic tandem repeat sequences were observed in the control region (CR). This led to a difference in the genome lengths obtained from these five samples with an average size of 16,994 bp for the five tiger mitochondrial genomes. The nucleotide base composition was on average as follows: A, 31.8%; T, 27.0%; C, 26.6%; G, 14.6% and exhibited compositional asymmetry. Most of tiger mitochondrial genome characteristics are similar to those of other common vertebrate species; however, some distinctive features were observed in the CR. First, the repetitive sequence 2 (RS 2) contained two repeat units of 80 bp and the first 15 bp of what would be the third repeat motif. The repetitive sequence 3 (RS 3) contained 47–50 repeat motifs of a shorter 8 bp (ACGTAYAC)_n. Second, length heteroplasmy polycystosine (poly-C) stretches was observed at the end of the HV I locus in all tiger samples.     

Lineage-specific variations of congruent evolution among DNA sequences from three genomes,and relaxed selective constraints on <Emphasis Type="Italic">rbc</Emphasis>L in <Emphasis Type="Italic">Cryptomonas</Emphasis> (Cryptophyceae)

Background  

Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbc L genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbc L genes, and to compare the rbc L gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.     

Genetic variation at mtDNA and microsatellite loci in Chinese longsnout catfish (<Emphasis Type="Italic">Leiocassis longirostris</Emphasis>)

Genetic variability and population structure of the Chinese longsnout catfish Leiocassis longirostris Günther in the Yangtze River was examined with mitochondrial control region sequences and nuclear microsatellite markers. A 705-bp segment of the mitochondrial DNA control region was sequenced from 132 samples, which identified a total of 61 haplotypes. The Chinese longsnout catfish in the Yangtze River was characterized with high haplotype diversity (h = 0.9770 ± 0.0041) but low nucleotide diversity (π = 0.0081 ± 0.0043). Median-joining network analysis revealed a star-shaped pattern and mismatch distribution analysis found a smooth unimodal distribution, which suggested that this species in the Yangtze River underwent a population expansion following bottlenecks and/or they originated from a small size of founding population. It was estimated that the possible time of population expansion was 139,000–435,000 years before present, a time period in the middle Pleistocene. The analysis of molecular variance and phylogenetic reconstructions did not detect significant geographic structure between different river sections. This pattern of genetic variation was further evidenced with nuclear microsatellite markers. The genetic differentiation between above and below the Gezhouba Dam and Three Gorges Dam is very small at mitochondrial and nuclear levels, which suggested that these recently developed dams might have not significantly resulted in population genetic fragmentation in the Chinese longsnout catfish. However, the potential exacerbation of genetic structuring by the dams should not be overlooked in the future.     

Genetic differentiation between populations of swimming crab <Emphasis Type="Italic">Portunus trituberculatus</Emphasis> along the coastal waters of the East China Sea

Portunus trituberculatus is a commercially important species widely spread in the East China Sea. Intraspecific variation of the mitochondrial DNA cytochrome oxidase subunit I (mtDNA COI) gene was investigated in 213 individuals from six localities (Changjiang Estuary, Shengsi Islands, Zhoushan Islands, Dongtou Islands, Dinghai Bay, and Quanzhou Bay) ranging from north (31°21′N) to south (24°55′N) coastal waters of the East China Sea. Overall, a total of 27 mtDNA haplotypes and 21 variable sites were detected in the 787 bp segment of COI gene. Analysis of mtDNA COI sequence data revealed that crabs from the six localities were characterized by moderately high haplotypic diversity (h = 0.787 ± 0.026), while sequence divergence values between haplotypes were relatively low (π = 0.00241 ± 0.00098). Each population was characterized by a single most frequent haplotype, shared among all six localities, and a small number of rare ones, typically present in only one or two individuals and representative of a specific population. However, neither the neighbor-joining tree nor the minimum spanning network (MSN) based on the haplotype data exhibited geographical patterns of the six populations. Mismatch distribution analysis of P. trituberculatus individuals sampled from the six localities suggested that sudden population expansion might have occurred in CJ and SS population that might be consistent with over-exploitation of the swimming crab. Analysis of molecular variance (AMOVA) and F _ST statistics showed that significant genetic differentiation existed among the SS, ZS, DT, DH, and QZ populations, suggesting that gene flow might be reduced, even between the geographically close sites, despite the high potential of dispersal. The possible causes of the observed genetic heterogeneity among the P. trituberculatus populations and the potential applications of the mtDNA COI marker in the artificial breeding and fisheries management are discussed. Handling editor: C. Sturmbauer