Organellar Genomes Research Articles

Organellar genome comparisons of Sargassum polycystum and S. plagiophyllum (Fucales, Phaeophyceae) with other Sargassum species

BackgroundSargassum polycystum C. Agardh and Sargassum plagiophyllum C. Agardh are inhabitants of tropical coastal areas, their populations are negatively influenced by global warming and marine environment changes. The mitochondrial and chloroplast genomes of these species have not been sequenced.ResultsThe mitochondrial genomes of S. polycystum and S. plagiophyllum were 34,825 bp and 34,862 bp, respectively, and their corresponding chloroplast genomes were 124,493 bp and 124,536 bp, respectively. The mitochondrial and chloroplast genomes of these species share conserved synteny, sequence regions and gene number when compared with the organellar genomes of other Sargassum species. Based on sequence analysis of 35 protein-coding genes, we deduced that S. polycystum and S. plagiophyllum were closely related with S. ilicifolium; these species diverged approximately 0.3 million years ago (Ma; 0.1–0.53 Ma) during the Pleistocene period (0.01–2.59 Ma). Rates of synonymous and non-synonymous substitutions in the mitochondrial genome of the Sargassum genus were 3 times higher than those in the chloroplast genome. In the mitochondrial genome, rpl5, rpl31 and rps11 had the highest synonymous substitution rates. In the chloroplast genome, psaE, rpl14 and rpl27 had the highest synonymous substitution rates.ConclusionsPhylogenetic analysis confirms the close relationship between the two sequenced species and S. ilicifolium. Both synonymous and non-synonymous substitution rates show significant divergence between the group of mitochondrial genomes versus the group of chloroplast genomes. The deciphering of complete mitochondrial and chloroplast genomes is significant as it advances our understanding of the evolutionary and phylogenetic relationships between species of brown seaweeds.

Engineering the plastid and mitochondrial genomes of flowering plants.

Engineering the plastid genome based on homologous recombination is well developed in a few model species. Homologous recombination is also the rule in mitochondria, but transformation of the mitochondrial genome has not been realized in the absence of selective markers. The application of transcription activator-like (TAL) effector-based tools brought about a dramatic change because they can be deployed from nuclear genes and targeted to plastids or mitochondria by an N-terminal targeting sequence. Recognition of the target site in the organellar genomes is ensured by the modular assembly of TALE repeats. In this paper, I review the applications of TAL effector nucleases and TAL effector cytidine deaminases for gene deletion, base editing and mutagenesis in plastids and mitochondria. I also review emerging technologies such as post-transcriptional RNA modification to regulate gene expression, Agrobacterium- and nanoparticle-mediated organellar genome transformation, and self-replicating organellar vectors as production platforms.

Sorting of mitochondrial and plastid heteroplasmy in Arabidopsis is extremely rapid and depends on MSH1 activity

The fate of new mitochondrial and plastid mutations depends on their ability to persist and spread among the numerous organellar genome copies within a cell (heteroplasmy). The extent to which heteroplasmies are transmitted across generations or eliminated through genetic bottlenecks is not well understood in plants, in part because their low mutation rates make these variants so infrequent. Disruption of MutS Homolog 1 (MSH1), a gene involved in plant organellar DNA repair, results in numerous de novo point mutations, which we used to quantitatively track the inheritance of single nucleotide variants in mitochondrial and plastid genomes in Arabidopsis. We found that heteroplasmic sorting (the fixation or loss of a variant) was rapid for both organelles, greatly exceeding rates observed in animals. In msh1 mutants, plastid variants sorted faster than those in mitochondria and were typically fixed or lost within a single generation. Effective transmission bottleneck sizes (N) for plastids and mitochondria were N ∼ 1 and 4, respectively. Restoring MSH1 function further increased the rate of heteroplasmic sorting in mitochondria (N ∼ 1.3), potentially because of its hypothesized role in promoting gene conversion as a mechanism of DNA repair, which is expected to homogenize genome copies within a cell. Heteroplasmic sorting also favored GC base pairs. Therefore, recombinational repair and gene conversion in plant organellar genomes can potentially accelerate the elimination of heteroplasmies and bias the outcome of this sorting process.

Deep sequencing of the epitype specimen of Synarthrophyton patena (Hooker f. & Harvey) R.A.Townsend (Hapalidiales, Rhodophyta) confirms the correct application of this name

Synarthrophyton patena (Hooker f. & Harvey) R.A.Townsend is a discoid marine coralline red alga distributed in the southwestern Pacific Ocean (type locality: southeast North Island, New Zealand). It is the generitype of Synarthrophyton R.A.Townsend, a genus of taxonomic debate. High-throughput sequencing was performed on the herein designated epitype specimen of S. patena to characterize its genetic markers and organellar genome structure. The complete plastid genome of S. patena is 181,685 bp in length and contains 232 genes. A partial mitogenome was assembled amounting to 25,779 bp and encodes 46 genes. Both genomes show a high level of gene synteny to the organellar genomes of S. chejuense. DNA markers rbcL, psbA, and cox1 are identical or very similar to sequences deposited in GenBank and analyzed here under the same name from Australia (including Tasmania) and New Zealand. A combined phylogenetic analysis of S. patena using rbcL and psbA gene sequences fully resolved it in a clade with other S. patena, Synarthrophyton spp., and unidentified Hapalidiales. These data confirm the accurate application of the binomial S. patena, stabilize the use of the generitype, and contribute to future congeneric evolutionary and taxonomic studies in the Hapalidiaceae.

Organellar transcripts dominate the cellular mRNA pool across plants of varying ploidy levels

Mitochondrial and plastid functions depend on coordinated expression of proteins encoded by genomic compartments that have radical differences in copy number of organellar and nuclear genomes. In polyploids, doubling of the nuclear genome may add challenges to maintaining balanced expression of proteins involved in cytonuclear interactions. Here, we use ribo-depleted RNA sequencing (RNA-seq) to analyze transcript abundance for nuclear and organellar genomes in leaf tissue from four different polyploid angiosperms and their close diploid relatives. We find that even though plastid genomes contain <1% of the number of genes in the nuclear genome, they generate the majority (69.9 to 82.3%) of messenger RNA (mRNA) transcripts in the cell. Mitochondrial genes are responsible for a much smaller percentage (1.3 to 3.7%) of the leaf mRNA pool but still produce much higher transcript abundances per gene compared to nuclear genome. Nuclear genes encoding proteins that functionally interact with mitochondrial or plastid gene products exhibit mRNA expression levels that are consistently more than 10-fold lower than their organellar counterparts, indicating an extreme cytonuclear imbalance at the RNA level despite the predominance of equimolar interactions at the protein level. Nevertheless, interacting nuclear and organellar genes show strongly correlated transcript abundances across functional categories, suggesting that the observed mRNA stoichiometric imbalance does not preclude coordination of cytonuclear expression. Finally, we show that nuclear genome doubling does not alter the cytonuclear expression ratios observed in diploid relatives in consistent or systematic ways, indicating that successful polyploid plants are able to compensate for cytonuclear perturbations associated with nuclear genome doubling.

The evolution of extremely diverged plastomes in Selaginellaceae (lycophyte) is driven by repeat patterns and the underlying DNA maintenance machinery.

Two factors are proposed to account for the unusual features of organellar genomes: the disruptions of organelle-targeted DNA replication, repair, and recombination (DNA-RRR) systems in the nuclear genome and repetitive elements in organellar genomes. Little is known about how these factors affect organellar genome evolution. The deep-branching vascular plant family Selaginellaceae is known to have a deficient DNA-RRR system and convergently evolved organellar genomes. However, we found that the plastid genome (plastome) of Selaginella sinensis has extremely accelerated substitution rates, a low GC content, pervasive repeat elements, a dynamic network structure, and it lacks direct or inverted repeats. Unexpectedly, its organelle DNA-RRR system is short of a plastid-targeted Recombinase A1 (RecA1) and a mitochondrion-targeted RecA3, in line with other explored Selaginella species. The plastome contains a large collection of short- and medium-sized repeats. Given the absence of RecA1 surveillance, we propose that these repeats trigger illegitimate recombination, accelerated mutation rates, and structural instability. The correlations between repeat quantity and architectural complexity in the Selaginella plastomes support these conclusions. We, therefore, hypothesize that the interplay of the deficient DNA-RRR system and the high repeat content has led to the extraordinary divergence of the S. sinensis plastome. Our study not only sheds new light on the mechanism of plastome divergence by emphasizing the power of cytonuclear integration, but it also reconciles the longstanding contradiction on the effects of DNA-RRR system disruption on genome structure evolution.

Rampant nuclear-mitochondrial-plastid phylogenomic discordance in globally distributed calcifying microalgae.

Summary Incongruent phylogenies have been widely observed between nuclear and plastid or mitochondrial genomes in terrestrial plants and animals. However, few studies have examined these patterns in microalgae or the discordance between the two organelles.Here we investigated the nuclear–mitochondrial–plastid phylogenomic incongruence in Emiliania–Gephyrocapsa, a group of cosmopolitan calcifying phytoplankton with enormous populations and recent speciations. We assembled mitochondrial and plastid genomes of 27 strains from across global oceans and temperature regimes, and analyzed the phylogenomic histories of the three compartments using concatenation and coalescence methods.Six major clades with varying morphology and distribution are well recognized in the nuclear phylogeny, but such relationships are absent in the mitochondrial and plastid phylogenies, which also differ substantially from each other. The rampant phylogenomic discordance is due to a combination of organellar capture (introgression), organellar genome recombination, and incomplete lineage sorting of ancient polymorphic organellar genomes. Hybridization can lead to replacements of whole organellar genomes without introgression of nuclear genes and the two organelles are not inherited as a single cytoplasmic unit.This study illustrates the convoluted evolution and inheritance of organellar genomes in isogamous haplodiplontic microalgae and provides a window into the phylogenomic complexity of marine unicellular eukaryotes.

Plastid and mitochondrial phylogenomics reveal correlated substitution rate variation in Koenigia (Polygonoideae, Polygonaceae) and a reduced plastome for Koenigia delicatula including loss of all ndh genes

Koenigia, a genus proposed by Linnaeus, has a contentious taxonomic history. In particular, relationships among species and the circumscription of the genus relative to Aconogonon remain uncertain. To explore phylogenetic relationships of Koenigia with other members of tribe Persicarieae and to establish the timing of major evolutionary diversification events, genome skimming of organellar sequences was used to assemble plastomes and mitochondrial genes from 15 individuals representing 13 species. Most Persicarieae plastomes exhibit a conserved structure and content relative to other flowering plants. However, Koenigia delicatula has lost functional copies of all ndh genes and the intron from atpF. In addition, the rpl32 gene was relocated in the K. delicatula plastome, which likely occurred via overlapping inversions or differential expansion and contraction of the inverted repeat. The highly supported but conflicting relationships between plastome and mitochondrial trees and among gene trees complicates the circumscription of Koenigia, which could be caused by rapid diversification within a short period. Moreover, the plastome and mitochondrial trees revealed correlated variation in substitution rates among Persicarieae species, suggesting a shared underlying mechanism promoting evolutionary rate variation in both organellar genomes. The divergence of dwarf K. delicatula from other Koenigia species may be associated with the well-known Eocene Thermal Maximum 2 or Early Eocene Climatic Optimum event, while diversification of the core-Koenigia clade associates with the Mid-Miocene Climatic Optimum and the uplift of Qinghai-Tibetan Plateau and adjacent areas.

Dynamic plastid and mitochondrial genomes in Chaetopeltidales (Chlorophyceae) and characterization of a new chlorophyte taxon.

Chaetopeltidales is a poorly characterized order in the Chlorophyceae, with only two plastid and no mitochondrial genomes published. Here we describe a new taxon in Chaetopeltidales, Gormaniella terricola gen. et sp. nov. and characterize both of its organellar genomes. Gormaniella terricola was inadvertently isolated from a surface-sterilized hornwort thallus. Light microscopy was used to characterize its vegetative morphology. Organellar genomes were assembled, annotated, and analyzed using a variety of software packages. The mitochondrial genome (66,927 bp) represents the first complete mitochondrial genome published for Chaetopeltidales. The chloroplast genome, measuring 428,981 bp, is one of the largest plastid genomes published to date and shares this large size and an incredible number of short, dispersed repeats with the other sequenced chloroplast genomes in Chaetopeltidales. Despite these shared features, the chloroplast genomes of Chaetopeltidales appear to be highly rearranged when compared to one another, with numerous inversions, translocations, and duplications, suggesting a particularly dynamic chloroplast genome. Both the chloroplast and mitochondrial genomes of G. terricola contain a number of mobile group I and group II introns, which appear to have invaded separately. Three of the introns within the mitochondrial genome encode homing endonucleases that are phylogenetically nested within those found in fungi, rather than algae, suggesting a possible case of horizontal gene transfer. These results help to shed light on a poorly understood group of algae and their unusual organellar genomes, raising additional questions about the unique patterns of genome evolution within Chaetopeltidales.

Highlighted articles for June 2022

Highlighted articles for June 2022

E3 ligase BRUTUS Is a Negative Regulator for the Cellular Energy Level and the Expression of Energy Metabolism-Related Genes Encoded by Two Organellar Genomes in Leaf Tissues.

E3 ligase BRUTUS (BTS), a putative iron sensor, is expressed in both root and shoot tissues in seedlings of Arabidopsis thaliana. The role of BTS in root tissues has been well established. However, its role in shoot tissues has been scarcely studied. Comparative transcriptome analysis with shoot and root tissues revealed that BTS is involved in regulating energy metabolism by modulating expression of mitochondrial and chloroplast genes in shoot tissues. Moreover, in shoot tissues of bts-1 plants, levels of ADP and ATP and the ratio of ADP/ATP were greatly increased with a concomitant decrease in levels of soluble sugar and starch. The decreased starch level in bts-1 shoot tissues was restored to the level of shoot tissues of wild-type plants upon vanadate treatment. Through this study, we expand the role of BTS to regulation of energy metabolism in the shoot in addition to its role of iron deficiency response in roots.

Molecular analyses reveal a new species of Palmariaceae from Subantarctic Chile: Devaleraea yagan sp. nov. (Palmariales, Rhodophyta)

ABSTRACT The red algal family Palmariaceae is distributed in the northern and southern hemispheres and currently includes four genera and 25 species. Recent studies based on transcriptome and organellar genome data strongly support a monophyletic family; however, this condition was not confirmed among its constituent genera. Additionally, no reliable morphological diagnostic features have been identified for delimiting Devaleraea and Palmaria. During expeditions to Subantarctic Chile along isolated channels and fjords to explore macroalgae diversity, we collected samples of specimens from the intertidal and assessed them using morphology and molecular markers (cox1, rbcL, nuclear-encoded ITS rDNA). Here we describe the new species Devaleraea yagan based on multilocus phylogeny and confirm the low genetic divergences in palmariaceaean species using principal coordinate analysis (PCoA). This new species is diagnosed by having its diminutive thalli attached to the substratum by a reduced holdfast and by the presence of rhizoidal cells in the medulla. Further analyses including genomic data of members of Devaleraea, Halosaccion and Neohalosacciocolax lineages are expected to support the taxonomic restructuring of the family.

The first genome for the Cape Primrose Streptocarpus rexii (Gesneriaceae), a model plant for studying meristem-driven shoot diversity.

Cape Primroses (Streptocarpus, Gesneriaceae) are an ideal study system for investigating the genetics underlying species diversity in angiosperms. Streptocarpus rexii has served as a model species for plant developmental research for over five decades due to its unusual extended meristem activity present in the leaves. In this study, we sequenced and assembled the complete nuclear, chloroplast, and mitochondrial genomes of S. rexii using Oxford Nanopore Technologies long read sequencing. Two flow cells of PromethION sequencing resulted in 32 billion reads and were sufficient to generate a draft assembly including the chloroplast, mitochondrial and nuclear genomes, spanning 776 Mbp. The final nuclear genome assembly contained 5,855 contigs, spanning 766 Mbp of the 929‐Mbp haploid genome with an N50 of 3.7 Mbp and an L50 of 57 contigs. Over 70% of the draft genome was identified as repeats. A genome repeat library of Gesneriaceae was generated and used for genome annotation, with a total of 45,045 genes annotated in the S. rexii genome. Ks plots of the paranomes suggested a recent whole genome duplication event, shared between S. rexii and Primulina huaijiensis. A new chloroplast and mitochondrial genome assembly method, based on contig coverage and identification, was developed, and successfully used to assemble both organellar genomes of S. rexii . This method was developed into a pipeline and proved widely applicable. The nuclear genome of S. rexii and other datasets generated and reported here will be invaluable resources for further research to aid in the identification of genes involved in morphological variation underpinning plant diversification.

Cytonuclear coevolution in a holoparasitic plant with highly disparate organellar genomes.

Contrasting substitution rates in the organellar genomes of Lophophytum agree with the DNA repair, replication, and recombination gene content. Plastid and nuclear genes whose products form multisubunit complexesco-evolve. The organellar genomes of the holoparasitic plant Lophophytum (Balanophoraceae) show disparate evolution. In the plastid, the genome has been severely reduced and presents a > 85% AT content, while in the mitochondria most protein-coding genes have been replaced by homologs acquired by horizontal gene transfer (HGT) from their hosts (Fabaceae). Both genomes carry genes whose products form multisubunit complexes with those of nuclear genes, creating a possible hotspot of cytonuclear coevolution. In this study, we assessed the evolutionary rates of plastid, mitochondrial and nuclear genes, and their impact on cytonuclear evolution of genes involved in multisubunit complexes related to lipid biosynthesis and proteolysis in the plastid and those in charge of the oxidative phosphorylation in the mitochondria. Genes from the plastid and the mitochondria (both native and foreign) of Lophophytum showed extremely high and ordinary substitution rates, respectively. These results agree with the biased loss of plastid-targeted proteins involved in angiosperm organellar repair, replication, and recombination machinery. Consistent with the high rate of evolution of plastid genes, nuclear-encoded subunits of plastid complexes showed disproportionate increases in non-synonymous substitution rates, while those of the mitochondrial complexes did not show different rates than the control (i.e. non-organellar nuclear genes). Moreover, the increases in the nuclear-encoded subunits of plastid complexes were positively correlated with the level of physical interaction they possess with the plastid-encoded ones. Overall, these results suggest that a structurally-mediated compensatory factor may be driving plastid-nuclear coevolution in Lophophytum, and that mito-nuclear coevolution was not altered by HGT.

A new lineage of non-photosynthetic green algae with extreme organellar genomes

BackgroundThe plastid genomes of the green algal order Chlamydomonadales tend to expand their non-coding regions, but this phenomenon is poorly understood. Here we shed new light on organellar genome evolution in Chlamydomonadales by studying a previously unknown non-photosynthetic lineage. We established cultures of two new Polytoma-like flagellates, defined their basic characteristics and phylogenetic position, and obtained complete organellar genome sequences and a transcriptome assembly for one of them.ResultsWe discovered a novel deeply diverged chlamydomonadalean lineage that has no close photosynthetic relatives and represents an independent case of photosynthesis loss. To accommodate these organisms, we establish the new genus Leontynka, with two species (L. pallida and L. elongata) distinguishable through both their morphological and molecular characteristics. Notable features of the colourless plastid of L. pallida deduced from the plastid genome (plastome) sequence and transcriptome assembly include the retention of ATP synthase, thylakoid-associated proteins, the carotenoid biosynthesis pathway, and a plastoquinone-based electron transport chain, the latter two modules having an obvious functional link to the eyespot present in Leontynka. Most strikingly, the ~362 kbp plastome of L. pallida is by far the largest among the non-photosynthetic eukaryotes investigated to date due to an extreme proliferation of sequence repeats. These repeats are also present in coding sequences, with one repeat type found in the exons of 11 out of 34 protein-coding genes, with up to 36 copies per gene, thus affecting the encoded proteins. The mitochondrial genome of L. pallida is likewise exceptionally large, with its >104 kbp surpassed only by the mitogenome of Haematococcus lacustris among all members of Chlamydomonadales hitherto studied. It is also bloated with repeats, though entirely different from those in the L. pallida plastome, which contrasts with the situation in H. lacustris where both the organellar genomes have accumulated related repeats. Furthermore, the L. pallida mitogenome exhibits an extremely high GC content in both coding and non-coding regions and, strikingly, a high number of predicted G-quadruplexes.ConclusionsWith its unprecedented combination of plastid and mitochondrial genome characteristics, Leontynka pushes the frontiers of organellar genome diversity and is an interesting model for studying organellar genome evolution.

Independent Size Expansions and Intron Proliferation in Red Algal Plastid and Mitochondrial Genomes.

Proliferation of selfish genetic elements has led to significant genome size expansion in plastid and mitochondrial genomes of various eukaryotic lineages. Within the red algae, such expansion events are only known in the plastid genomes of the Proteorhodophytina, a highly diverse group of mesophilic microalgae. By contrast, they have never been described in the much understudied red algal mitochondrial genomes. Therefore, it remains unclear how widespread such organellar genome expansion events are in this eukaryotic phylum. Here, we describe new mitochondrial and plastid genomes from 25 red algal species, thereby substantially expanding the amount of organellar sequence data available, especially for Proteorhodophytina, and show that genome expansions are common in this group. We confirm that large plastid genomes are limited to the classes Rhodellophyceae and Porphyridiophyceae, which, in part, are caused by lineage-specific expansion events. Independently expanded mitochondrial genomes—up to three times larger than typical red algal mitogenomes—occur across Proteorhodophytina classes and a large shift toward high GC content occurred in the Stylonematophyceae. Although intron proliferation is the main cause of plastid and mitochondrial genome expansion in red algae, we do not observe recent intron transfer between different organelles. Phylogenomic analyses of mitochondrial and plastid genes from our expanded taxon sampling yielded well-resolved phylogenies of red algae with strong support for the monophyly of Proteorhodophytina. Our work shows that organellar genomes followed different evolutionary dynamics across red algal lineages.

Pfprex from Plasmodium falciparum can bypass oxidative stress-induced DNA lesions.

Apicomplexans such as the malaria parasite Plasmodium falciparum possess a unique organelle known as the apicoplast that has its own circular genome. The apicoplast genome is AT rich and is subjected to oxidative stress from the byproducts of the normal biochemical pathways that operate in the apicoplast. It is expected that oxidative stress will lead to the appearance of DNA lesions such as 2-hydroxydeoxyadenine, thymine glycol, and 8-oxodeoxyguanine in the apicoplast genome. The apicoplast genome is replicated by the DNA polymerase activity present in the Pfprex enzyme. We have named the polymerase module of Pfprex as PfpPol and the enzyme belongs to the A family of DNA polymerases. Similar to other members of this family, PfpPol also exhibits high fidelity of DNA synthesis. We show that this enzyme is also capable of carrying out translesion DNA synthesis past common DNA lesions that arise due to oxidative stress. The residues N505 and Y509 from the fingers sub-domain, which are unique to PfpPol, play an important role in the ability of PfpPol to bypass the three lesions. The observed lesion-bypass ability of the Pfprex enzyme will minimize the adverse effects of oxidative stress on the apicoplast genome of the malaria parasite. These findings also have implications regarding the evolution of the machinery responsible for replication of organellar genomes.

Maturyoshka: A maturase inside a maturase, and other peculiarities of the novel chloroplast genomes of marine euglenophytes

Organellar genomes often carry group II introns, which occasionally encode proteins called maturases that are important for splicing. The number of introns varies substantially among various organellar genomes, and bursts of introns have been observed in multiple eukaryotic lineages, including euglenophytes, with more than 100 introns in their plastid genomes. To examine the evolutionary diversity and history of maturases, an essential gene family among euglenophytes, we searched for their homologs in newly sequenced and published plastid genomes representing all major euglenophyte lineages. We found that maturase content in plastid genomes has a patchy distribution, with a maximum of eight of them present in Eutreptiella eupharyngea. The most basal lineages of euglenophytes, Eutreptiales, share the highest number of maturases, but the lowest number of introns. We also identified a peculiar convoluted structure of a gene located in an intron, in a gene within an intron, within yet another gene, present in some Eutreptiales. Further investigation of functional domains of identified maturases show that most of them lost at least one of the functional domains, which implies that the patchy maturase distribution is due to frequent inactivation and eventual loss over time. Finally, we identified the diversified evolutionary origin of analysed maturases, which were acquired along with the green algal plastid or horizontally transferred. These findings indicate that euglenophytes' plastid maturases have experienced a surprisingly dynamic history due to gains from diversified donors, their retention, and loss.

5WBF: a low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of Plasmodium falciparum clinical isolates

BackgroundWhole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programmes. A major drawback of this approach is the large amount of human DNA compared to parasite DNA extracted from unprocessed whole blood. As red blood cells (RBCs) have a diameter of about 7–8 µm and exhibit some deformability, it was hypothesized that cheap and commercially available 5 µm filters might retain leukocytes but much less of Plasmodium falciparum-infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for 5 µm Whole Blood Filtration), may provide highly enriched parasite material suitable for P. falciparum WGS.MethodsWhole blood was collected from five patients experiencing a P. falciparum malaria episode (ring-stage parasitaemia range: 0.04–5.5%) and from mock samples obtained by mixing synchronized, ring-stage cultured P. falciparum 3D7 parasites with uninfected human whole blood (final parasitaemia range: 0.02–1.1%). These whole blood samples (50 to 400 µL) were diluted in RPMI 1640 medium or PBS 1× buffer and filtered with a syringe connected to a 5 µm commercial filter. DNA was extracted from 5WBF-treated and unfiltered counterpart blood samples using a commercial kit. The 5WBF method was evaluated on the ratios of parasite:human DNA assessed by qPCR and by sequencing depth and percentages of coverage from WGS data (Illumina NextSeq 500). As a comparison, the popular selective whole-genome amplification (sWGA) method, which does not rely on blood filtration, was applied to the unfiltered counterpart blood samples.ResultsAfter applying 5WBF, qPCR indicated an average of twofold loss in the amount of parasite template DNA (Pf ARN18S gene) and from 4096- to 65,536-fold loss of human template DNA (human β actin gene). WGS analyses revealed that > 95% of the parasite nuclear and organellar genomes were all covered at ≥ 10× depth for all samples tested. In sWGA counterparts, the organellar genomes were poorly covered and from 47.7 to 82.1% of the nuclear genome was covered at ≥ 10× depth depending on parasitaemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5460 genes; mean coverage: 91×; median coverage: 93×; 5th percentile: 70×; 95th percentile: 103×), allowing the identification of gene copy number variations such as for gch1. This later analysis was not possible for sWGA-treated samples, as a much more heterogeneous distribution of reads across gene sequences was observed (mean coverage: 80×; median coverage: 51×; 5th percentile: 7×; 95th percentile: 245×).ConclusionsThe novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized 5 µm filters which cost from 1.0 to 1.7€ per unit depending on suppliers. 5WBF permits extensive genome-wide analysis of P. falciparum ring-stage isolates from minute amounts of whole blood even with parasitaemias as low as 0.02%.

Spatial Features and Functional Implications of Plant 3D Genome Organization

The advent of high-throughput sequencing-based methods for chromatin conformation, accessibility, and immunoprecipitation assays has been a turning point in 3D genomics. Altogether, these new tools have been pushing upward the interpretation of pioneer cytogenetic evidence for a higher order in chromatin packing. Here, we review the latest development in our understanding of plant spatial genome structures and different levels of organization and discuss their functional implications. Then, we spotlight the complexity of organellar (i.e., mitochondria and plastids) genomes and discuss their 3D packing into nucleoids. Finally, we propose unaddressed research axes to investigate functional links between chromatin-like dynamics and transcriptional regulation within organellar nucleoids.