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

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

Complete mitochondrial genomes of prasinophyte algae Pyramimonas parkeae and Cymbomonas tetramitiformis

Mitochondria are archetypal eukaryotic organelles that were acquired by endosymbiosis of an ancient species of alpha‐proteobacteria by the last eukaryotic common ancestor. The genetic information contained within the mitochondrial genome has been an important source of information for resolving relationships among eukaryotic taxa. In this study, we utilized mitochondrial and chloroplast genomes to explore relationships among prasinophytes. Prasinophytes are represented by diverse early‐diverging green algae whose physical structures and genomes have the potential to elucidate the traits of the last common ancestor of the Viridiplantae (or Chloroplastida). We constructed de novo mitochondrial genomes for two prasinophyte algal species, Pyramimonas parkeae and Cymbomonas tetramitiformis, representing the prasinophyte clade. Comparisons of genome structure and gene order between these species and to those of other prasinophytes revealed that the mitochondrial genomes of P. parkeae and C. tetramitiformis are more similar to each other than to other prasinophytes, consistent with other molecular inferences of the close relationship between these two species. Phylogenetic analyses using the inferred amino acid sequences of mitochondrial and chloroplast protein‐coding genes resolved a clade consisting of P. parkeae and C. tetramitiformis; and this group (representing the prasinophyte clade I) branched with the clade II, consistent with previous studies based on the use of nuclear gene markers.     

Intra‐individual polymorphism in chloroplasts from NGS data: where does it come from and how to handle it?

Next‐generation sequencing allows access to a large quantity of genomic data. In plants, several studies used whole chloroplast genome sequences for inferring phylogeography or phylogeny. Even though the chloroplast is a haploid organelle, NGS plastome data identified a nonnegligible number of intra‐individual polymorphic SNPs. Such observations could have several causes such as sequencing errors, the presence of heteroplasmy or transfer of chloroplast sequences in the nuclear and mitochondrial genomes. The occurrence of allelic diversity has practical important impacts on the identification of diversity, the analysis of the chloroplast data and beyond that, significant evolutionary questions. In this study, we show that the observed intra‐individual polymorphism of chloroplast sequence data is probably the result of plastid DNA transferred into the mitochondrial and/or the nuclear genomes. We further assess nine different bioinformatics pipelines’ error rates for SNP and genotypes calling using SNPs identified in Sanger sequencing. Specific pipelines are adequate to deal with this issue, optimizing both specificity and sensitivity. Our results will allow a proper use of whole chloroplast NGS sequence and will allow a better handling of NGS chloroplast sequence diversity.     

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.     

Development of genomic resources for Nothofagus species using next‐generation sequencing data

Using next‐generation sequencing, we developed the first whole‐genome resources for two hybridizing Nothofagus species of the Patagonian forests that crucially lack genomic data, despite their ecological and industrial value. A de novo assembly strategy combining base quality control and optimization of the putative chloroplast gene map yielded ~32 000 contigs from 43% of the reads produced. With 12.5% of assembled reads, we covered ~96% of the chloroplast genome and ~70% of the mitochondrial gene content, providing functional and structural annotations for 112 and 52 genes, respectively. Functional annotation was possible on 15% of the contigs, with ~1750 potentially novel nuclear genes identified for Nothofagus species. We estimated that the new resources (13.41 Mb in total) included ~4000 gene regions representing ~6.5% of the expected genic partition of the genome, the remaining contigs potentially being nongenic DNA. A high‐quality single nucleotide polymorphisms resource was developed by comparing various filtering methods, and preliminary results indicate a strong conservation of cpDNA genomes in contrast to numerous exclusive nuclear polymorphisms in both species. Finally, we characterized 2274 potential simple sequence repeat (SSR) loci, designed primers for 769 of them and validated nine of 29 loci in 42 individuals per species. Nothofagus obliqua had more alleles (4.89) on average than N. nervosa (2.89), 8 SSRs were efficient to discriminate species, and three were successfully transferred in three other Nothofagus species. These resources will greatly help for future inferences of demographic, adaptive and hybridizing events in Nothofagus species, and for conserving and managing natural populations.     

Biogenesis of photosynthetic complexes in the chloroplast of Chlamydomonas reinhardtii requires ARSA1, a homolog of prokaryotic arsenite transporter and eukaryotic TRC40 for guided entry of tail‐anchored proteins

as1, for antenna size mutant 1, was obtained by insertion mutagenesis of the unicellular green alga Chlamydomonas reinhardtii. This strain has a low chlorophyll content, 8% with respect to the wild type, and displays a general reduction in thylakoid polypeptides. The mutant was found to carry an insertion into a homologous gene, prokaryotic arsenite transporter (ARSA), whose yeast and mammal counterparts were found to be involved in the targeting of tail‐anchored (TA) proteins to cytosol‐exposed membranes, essential for several cellular functions. Here we present the characterization in a photosynthetic organism of an insertion mutant in an ARSA‐homolog gene. The ARSA1 protein was found to be localized in the cytosol, and yet its absence in as1 leads to a small chloroplast and a strongly decreased chlorophyll content per cell. ARSA1 appears to be required for optimal biogenesis of photosynthetic complexes because of its involvement in the accumulation of TOC34, an essential component of the outer chloroplast membrane translocon (TOC) complex, which, in turn, catalyzes the import of nucleus‐encoded precursor polypeptides into the chloroplast. Remarkably, the effect of the mutation appears to be restricted to biogenesis of chlorophyll‐binding polypeptides and is not compensated by the other ARSA homolog encoded by the C. reinhardtii genome, implying a non‐redundant function.     

Differential transmission of the Cucumis organellar genomes

 Although plants generally show maternal transmission of the organellar genomes, previous research has demonstrated that the mitochondrial (mt) genome of cucumber is paternally transmitted. In this study, we identified RFLPs in the organellar genomes of melon, squash, and watermelon to establish organellar DNA transmission. Serial dilutions of DNA demonstrated that our hybridizations revealed the presence of a polymorphic cytoplasm when it represented at least 1% of the DNA sample. At this level of sensitivity, the chloroplast genomes of melon, squash, and watermelon were maternally transmitted. The mitochondrial genomes of squash and watermelon were maternally transmitted; however, melon, like cucumber, showed paternal transmission of the mitochondrial genome. Because most angiosperms and the related genera Cucurbita and Citrullus show maternal transmission of the mtDNA, paternal transmission in Cucumis is likely the derived state. The Cucumis mitochondrial genomes are several-fold larger than those of other cucurbits. Based on 55 probe-enzyme combinations, mtDNA size differences could not be explained by duplication of the entire genome or partial duplication of regions hybridizing with the mitochondrial probes. Because the chloroplast, mitochondrial, and nuclear genomes of Cucumis are differentially transmitted, this genus is an excellent system to study the role of intergenomic transfer in the evolution of extremely large mitochondrial genomes. Received: 20 November 1997 / Accepted: 30 December 1997     

Phylogenetic position of the coral symbiont Ostreobium (Ulvophyceae) inferred from chloroplast genome data

The green algal genus Ostreobium is an important symbiont of corals, playing roles in reef decalcification and providing photosynthates to the coral during bleaching events. A chloroplast genome of a cultured strain of Ostreobium was available, but low taxon sampling and Ostreobium's early‐branching nature left doubt about its phylogenetic position. Here, we generate and describe chloroplast genomes from four Ostreobium strains as well as Avrainvillea mazei and Neomeris sp., strategically sampled early‐branching lineages in the Bryopsidales and Dasycladales respectively. At 80,584 bp, the chloroplast genome of Ostreobium sp. HV05042 is the most compact yet found in the Ulvophyceae. The Avrainvillea chloroplast genome is ~94 kbp and contains introns in infA and cysT that have nearly complete sequence identity except for an open reading frame (ORF) in infA that is not present in cysT. In line with other bryopsidalean species, it also contains regions with possibly bacteria‐derived ORFs. The Neomeris data did not assemble into a canonical circular chloroplast genome but a large number of contigs containing fragments of chloroplast genes and showing evidence of long introns and intergenic regions, and the Neomeris chloroplast genome size was estimated to exceed 1.87 Mb. Chloroplast phylogenomics and 18S nrDNA data showed strong support for the Ostreobium lineage being sister to the remaining Bryopsidales. There were differences in branch support when outgroups were varied, but the overall support for the placement of Ostreobium was strong. These results permitted us to validate two suborders and introduce a third, the Ostreobineae.     

Identification of the Mitochondrial Genome in the Chrysophyte Alga Ochromonas danica

ABSTRACT. Analysis of total DNA isolated from the Chrysophyte alga Ochromonas danica revealed, in addition to nuclear DNA, two genomes present as numerous copies per cell. The larger genome (?120 kilobase pairs or kbp) is the plastid DNA, which is identified by its hybridization to plasmids containing sequences for the photosynthesis genes rbcL, psbA, and psbC. The smaller genome (40 kbp) is the mitochondrial genome as identified by its hybridization with plasmids containing gene sequences of plant cytochrome oxidase subunits I and II. Both the 120- and 40-kbp genomes contain genes for the small and large subunits of rDNA. The mitochondrial genome is linear with terminal inverted repeats of about 1.6 kbp. Two other morphologically similar species were examined, Ochromonas minuta and Poteriochromonas malhamensis. All three species have linear mitochondrial DNA of 40 kbp. Comparisons of endonuclease restriction-fragment patterns of the mitochondrial and chloroplast DNAs as well as those of their nuclear rDNA repeats failed to reveal any fragment shared by any two of the species. Likewise, no common fragment size was detected by hybridization with plasmids containing heterologous DNA or with total mitochondrial DNA of O. danica; these observations support the taxonomic assignment of these three organisms to different species. The Ochromonas mitochondrial genomes are the first identified in the chlorophyll a/c group of algae. Combining these results with electron microscopic observations of putative mitochondrial genomes reported for other chromophytes and published molecular studies of other algal groups suggests that all classes of eukaryote algae may have mitochondrial genomes < 100 kbp in size, more like other protistans than land plants.     

Population genomic analyses from low‐coverage RAD‐Seq data: a case study on the non‐model cucurbit bottle gourd

Restriction site‐associated DNA sequencing (RAD‐Seq), a next‐generation sequencing‐based genome ‘complexity reduction’ protocol, has been useful in population genomics in species with a reference genome. However, the application of this protocol to natural populations of genomically underinvestigated species, particularly under low‐to‐medium sequencing depth, has not been well justified. In this study, a Bayesian method was developed for calling genotypes from an F₂ population of bottle gourd [Lagenaria siceraria (Mol.) Standl.] to construct a high‐density genetic map. Low‐depth genome shotgun sequencing allowed the assembly of scaffolds/contigs comprising approximately 50% of the estimated genome, of which 922 were anchored for identifying syntenic regions between species. RAD‐Seq genotyping of a natural population comprising 80 accessions identified 3226 single nuclear polymorphisms (SNPs), based on which two sub‐gene pools were suggested for association with fruit shape. The two sub‐gene pools were moderately differentiated, as reflected by the Hudson's F_ST value of 0.14, and they represent regions on LG7 with strikingly elevated F_ST values. Seven‐fold reduction in heterozygosity and two times increase in LD (r²) were observed in the same region for the round‐fruited sub‐gene pool. Outlier test suggested the locus LX3405 on LG7 to be a candidate site under selection. Comparative genomic analysis revealed that the cucumber genome region syntenic to the high F_ST island on LG7 harbors an ortholog of the tomato fruit shape gene OVATE. Our results point to a bright future of applying RAD‐Seq to population genomic studies for non‐model species even under low‐to‐medium sequencing efforts. The genomic resources provide valuable information for cucurbit genome research.     

Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

Prospects on the evolutionary mitogenomics of plants: A case study on the olive family (Oleaceae)

The mitogenome is rarely used to reconstruct the evolutionary history of plants, contrary to nuclear and plastid markers. Here, we evaluate the usefulness of mitochondrial DNA for molecular evolutionary studies in Oleaceae, in which cases of cytoplasmic male sterility (CMS) and of potentially contrasted organelle inheritance are known. We compare the diversity and the evolution of mitochondrial and chloroplast genomes by focusing on the olive complex and related genera. Using high‐throughput techniques, we reconstructed complete mitogenomes (ca. 0.7 Mb) and plastomes (ca. 156 kb) for six olive accessions and one Chionanthus. A highly variable organization of mitogenomes was observed at the species level. In olive, two specific chimeric genes were identified in the mitogenome of lineage E3 and may be involved in CMS. Plastid‐derived regions (mtpt) were observed in all reconstructed mitogenomes. Through phylogenetic reconstruction, we demonstrate that multiple integrations of mtpt regions have occurred in Oleaceae, but mtpt regions shared by all members of the olive complex derive from a common ancestor. We then assembled 52 conserved mitochondrial gene regions and complete plastomes of ten additional accessions belonging to tribes Oleeae, Fontanesieae and Forsythieae. Phylogenetic congruence between topologies based on mitochondrial regions and plastomes suggests a strong disequilibrium linkage between both organellar genomes. Finally, while phylogenetic reconstruction based on plastomes fails to resolve the evolutionary history of maternal olive lineages in the Mediterranean area, their phylogenetic relationships were successfully resolved with complete mitogenomes. Overall, our study demonstrates the great potential of using mitochondrial DNA in plant phylogeographic and metagenomic studies.     

Combined hybridization capture and shotgun sequencing for ancient DNA analysis of extinct wild and domestic dromedary camel

The performance of hybridization capture combined with next‐generation sequencing (NGS) has seen limited investigation with samples from hot and arid regions until now. We applied hybridization capture and shotgun sequencing to recover DNA sequences from bone specimens of ancient‐domestic dromedary (Camelus dromedarius) and its extinct ancestor, the wild dromedary from Jordan, Syria, Turkey and the Arabian Peninsula, respectively. Our results show that hybridization capture increased the percentage of mitochondrial DNA (mtDNA) recovery by an average 187‐fold and in some cases yielded virtually complete mitochondrial (mt) genomes at multifold coverage in a single capture experiment. Furthermore, we tested the effect of hybridization temperature and time by using a touchdown approach on a limited number of samples. We observed no significant difference in the number of unique dromedary mtDNA reads retrieved with the standard capture compared to the touchdown method. In total, we obtained 14 partial mitochondrial genomes from ancient‐domestic dromedaries with 17–95% length coverage and 1.27–47.1‐fold read depths for the covered regions. Using whole‐genome shotgun sequencing, we successfully recovered endogenous dromedary nuclear DNA (nuDNA) from domestic and wild dromedary specimens with 1–1.06‐fold read depths for covered regions. Our results highlight that despite recent methodological advances, obtaining ancient DNA (aDNA) from specimens recovered from hot, arid environments is still problematic. Hybridization protocols require specific optimization, and samples at the limit of DNA preservation need multiple replications of DNA extraction and hybridization capture as has been shown previously for Middle Pleistocene specimens.     

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome‐assisted research in barley (Hordeum vulgare) including a whole‐genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole‐genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA‐coding exome reduces barley genomic complexity more than 50‐fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in‐solution hybridization‐based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full‐length cDNAs and de novo assembled RNA‐Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA‐coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping‐by‐sequencing and genetic diversity analyzes.     

Genome size‐dependent pcna gene copy number in dinoflagellates and molecular evidence of retroposition as a major evolutionary mechanism

Proliferating cell nuclear antigen (PCNA) plays critical roles in eukaryotic DNA replication and replication‐associated processes. It is typically encoded by one or two gene copies (pcna) in eukaryotic genomes. Recently reported higher copy numbers of pcna in some dinoflagellates raised a question of how this gene has uniquely evolved in this phylum. Through real‐time PCR quantification, we found a wide range of pcna copy number (2–287 copies) in 11 dinoflagellate species (n = 38), and a strong positive correlation between pcna copy number and genome size (log₁₀–log₁₀ transformed). Intraspecific pcna diverged up to 21% and are dominated by nonsynonymous substitutions, indicating strong purifying selection pressure on and hence functional necessity of this gene. By surveying pcna copy numbers in eukaryotes, we observed a genome size threshold at 4 pg DNA, above which more than two pcna copies are found. To examine whether retrotransposition is a mechanism of pcna duplication, we measured the copy number of retroposed pcna, taking advantage of the 22‐nt dinoflagellate‐specific spliced leader (DinoSL) capping the 5′ end of dinoflagellate nuclear‐encoded mRNAs, which would exist in the upstream region of a retroposed gene copy. We found that retroposed pcna copy number increased with total pcna copy number and genome size. These results indicate co‐evolution of dinoflagellate pcna copy number with genome size, and retroposition as a major mechanism of pcna duplication in dinoflagellates. Furthermore, we posit that the demand of faithful replication and maintenance of the large dinoflagellate genomes might have favored the preservation of the retroposed pcna as functional genes.