April 21, 2020  |  

The genomes of polyextremophilic Cyanidiales contain 1% horizontally transferred genes with diverse adaptive functions.

The role and extent of horizontal gene transfer (HGT) in eukaryotes are hotly disputed topics that impact our understanding of the origin of metabolic processes and the role of organelles in cellular evolution. We addressed this issue by analyzing 10 novel Cyanidiales genomes and determined that 1% of their gene inventory is HGT-derived. Numerous HGT candidates share a close phylogenetic relationship with prokaryotes that live in similar habitats as the Cyanidiales and encode functions related to polyextremophily. HGT candidates differ from native genes in GC-content, number of splice sites, and gene expression. HGT candidates are more prone to loss, which may explain the absence of a eukaryotic pan-genome. Therefore, the lack of a pan-genome and cumulative effects fail to provide substantive arguments against our hypothesis of recurring HGT followed by differential loss in eukaryotes. The maintenance of 1% HGTs, even under selection for genome reduction, underlines the importance of non-endosymbiosis related foreign gene acquisition. © 2019, Rossoni et al.


April 21, 2020  |  

A siphonous macroalgal genome suggests convergent functions of homeobox genes in algae and land plants.

Genome evolution and development of unicellular, multinucleate macroalgae (siphonous algae) are poorly known, although various multicellular organisms have been studied extensively. To understand macroalgal developmental evolution, we assembled the ~26?Mb genome of a siphonous green alga, Caulerpa lentillifera, with high contiguity, containing 9,311 protein-coding genes. Molecular phylogeny using 107 nuclear genes indicates that the diversification of the class Ulvophyceae, including C. lentillifera, occurred before the split of the Chlorophyceae and Trebouxiophyceae. Compared with other green algae, the TALE superclass of homeobox genes, which expanded in land plants, shows a series of lineage-specific duplications in this siphonous macroalga. Plant hormone signalling components were also expanded in a lineage-specific manner. Expanded transport regulators, which show spatially different expression, suggest that the structural patterning strategy of a multinucleate cell depends on diversification of nuclear pore proteins. These results not only imply functional convergence of duplicated genes among green plants, but also provide insight into evolutionary roots of green plants. Based on the present results, we propose cellular and molecular mechanisms involved in the structural differentiation in the siphonous alga. © The Author(s) 2019. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.


September 22, 2019  |  

Anisogamy evolved with a reduced sex-determining region in volvocine green algae

Male and female gametes differing in size—anisogamy—emerged independently from isogamous ancestors in various eukaryotic lineages, although genetic bases of this emergence are still unknown. Volvocine green algae are a model lineage for investigating the transition from isogamy to anisogamy. Here we focus on two closely related volvocine genera that bracket this transition—isogamous Yamagishiella and anisogamous Eudorina. We generated de novo nuclear genome assemblies of both sexes of Yamagishiella and Eudorina to identify the dimorphic sex-determining chromosomal region or mating-type locus (MT) from each. In contrast to the large (>1?Mb) and complex MT of oogamous Volvox, Yamagishiella and Eudorina MT are smaller (7–268?kb) and simpler with only two sex-limited genes—the minus/male-limited MID and the plus/female-limited FUS1. No prominently dimorphic gametologs were identified in either species. Thus, the first step to anisogamy in volvocine algae presumably occurred without an increase in MT size and complexity.


September 22, 2019  |  

The genome of Ectocarpus subulatus highlights unique mechanisms for stress tolerance in brown algae

Brown algae are multicellular photosynthetic organisms belonging to the stramenopile lineage. They are successful colonizers of marine rocky shores world-wide. The genus Ectocarpus, and especially strain Ec32, has been established as a genetic and genomic model for brown algae. A related species, Ectocarpus subulatus Kuetzing, is characterized by its high tolerance of abiotic stress. Here we present the genome and metabolic network of a haploid male strain of E. subulatus, establishing it as a comparative model to study the genomic bases of stress tolerance in Ectocarpus. Our analyses indicate that E. subulatus has separated from Ectocarpus sp. Ec32 via allopatric speciation. Since this event, its genome has been shaped by the activity of viruses and large retrotransposons, which in the case of chlorophyll-binding proteins, may be related to the expansion of this gene family. We have identified a number of further genes that we suspect to contribute to stress tolerance in E. subulatus, including an expanded family of heat shock proteins, the reduction of genes involved in the production of halogenated defense compounds, and the presence of fewer cell wall polysaccharide-modifying enzymes. However, 96% of genes that differed between the two examined Ectocarpus species, as well as 90% of genes under positive selection, were found to be lineage-specific and encode proteins of unknown function. This underlines the uniqueness of brown algae with respect to their stress tolerance mechanisms as well as the significance of establishing E. subulatus as a comparative model for future functional studies.


September 22, 2019  |  

Mapping and characterizing N6-methyladenine in eukaryotic genomes using single-molecule real-time sequencing.

N6-Methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m6dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m6dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m6dA events in eukaryotic genomes.© 2018 Zhu et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies.

Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+ Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5′-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ~50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family. Copyright © 2018 the Author(s). Published by PNAS.


September 22, 2019  |  

The Chara genome: Secondary complexity and implications for plant terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote. Copyright © 2018 Elsevier Inc. All rights reserved.


September 22, 2019  |  

Analysis of the draft genome of the red seaweed Gracilariopsis chorda provides insights into genome size evolution in Rhodophyta.

Red algae (Rhodophyta) underwent two phases of large-scale genome reduction during their early evolution. The red seaweeds did not attain genome sizes or gene inventories typical of other multicellular eukaryotes. We generated a high-quality 92.1 Mb draft genome assembly from the red seaweed Gracilariopsis chorda, including methylation and small (s)RNA data. We analyzed these and other Archaeplastida genomes to address three questions: 1) What is the role of repeats and transposable elements (TEs) in explaining Rhodophyta genome size variation, 2) what is the history of genome duplication and gene family expansion/reduction in these taxa, and 3) is there evidence for TE suppression in red algae? We find that the number of predicted genes in red algae is relatively small (4,803-13,125 genes), particularly when compared with land plants, with no evidence of polyploidization. Genome size variation is primarily explained by TE expansion with the red seaweeds having the largest genomes. Long terminal repeat elements and DNA repeats are the major contributors to genome size growth. About 8.3% of the G. chorda genome undergoes cytosine methylation among gene bodies, promoters, and TEs, and 71.5% of TEs contain methylated-DNA with 57% of these regions associated with sRNAs. These latter results suggest a role for TE-associated sRNAs in RNA-dependent DNA methylation to facilitate silencing. We postulate that the evolution of genome size in red algae is the result of the combined action of TE spread and the concomitant emergence of its epigenetic suppression, together with other important factors such as changes in population size.


September 22, 2019  |  

Genome analyses of the microalga Picochlorum provide insights into the evolution of thermotolerance in the green lineage.

While the molecular events involved in cell responses to heat stress have been extensively studied, our understanding of the genetic basis of basal thermotolerance, and particularly its evolution within the green lineage, remains limited. Here, we present the 13.3-Mb haploid genome and transcriptomes of a halotolerant and thermotolerant unicellular green alga, Picochlorum costavermella (Trebouxiophyceae) to investigate the evolution of the genomic basis of thermotolerance. Differential gene expression at high and standard temperatures revealed that more of the gene families containing up-regulated genes at high temperature were recently evolved, and less originated at the ancestor of green plants. Inversely, there was an excess of ancient gene families containing transcriptionally repressed genes. Interestingly, there is a striking overlap between the thermotolerance and halotolerance transcriptional rewiring, as more than one-third of the gene families up-regulated at 35?°C were also up-regulated under variable salt concentrations in Picochlorum SE3. Moreover, phylogenetic analysis of the 9,304 protein coding genes revealed 26 genes of horizontally transferred origin in P. costavermella, of which five were differentially expressed at higher temperature. Altogether, these results provide new insights about how the genomic basis of adaptation to halo- and thermotolerance evolved in the green lineage.


September 22, 2019  |  

Exploring the limits and causes of plastid genome expansion in volvocine green algae.

Plastid genomes are not normally celebrated for being large. But researchers are steadily uncovering algal lineages with big and, in rare cases, enormous plastid DNAs (ptDNAs), such as volvocine green algae. Plastome sequencing of five different volvocine species has revealed some of the largest, most repeat-dense plastomes on record, including that of Volvox carteri (~525?kb). Volvocine algae have also been used as models for testing leading hypotheses on organelle genome evolution (e.g., the mutational hazard hypothesis), and it has been suggested that ptDNA inflation within this group might be a consequence of low mutation rates and/or the transition from a unicellular to multicellular existence. Here, we further our understanding of plastome size variation in the volvocine line by examining the ptDNA sequences of the colonial species Yamagishiella unicocca and Eudorina sp. NIES-3984 and the multicellular Volvox africanus, which are phylogenetically situated between species with known ptDNA sizes. Although V. africanus is closely related and similar in multicellular organization to V. carteri, its ptDNA was much less inflated than that of V. carteri. Synonymous- and noncoding-site nucleotide substitution rate analyses of these two Volvox ptDNAs suggest that there are drastically different plastid mutation rates operating in the coding versus intergenic regions, supporting the idea that error-prone DNA repair in repeat-rich intergenic spacers is contributing to genome expansion. Our results reinforce the idea that the volvocine line harbors extremes in plastome size but ultimately shed doubt on some of the previously proposed hypotheses for ptDNA inflation within the lineage.


September 22, 2019  |  

Insights into the evolution of multicellularity from the sea lettuce genome.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva’s rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance “green tides.” Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage. Copyright © 2018 Elsevier Ltd. All rights reserved.


September 22, 2019  |  

Haematococcus lacustris: the makings of a giant-sized chloroplast genome.

Recent work on the chlamydomonadalean green alga Haematococcus lacustris uncovered the largest plastid genome on record: a whopping 1.35 Mb with >90 % non-coding DNA. A 500-word description of this genome was published in the journal Genome Announcements. But such a short report for such a large genome leaves many unanswered questions. For instance, the H. lacustris plastome was found to encode only 12 tRNAs, less than half that of a typical plastome, it appears to have a non-standard genetic code, and is one of only a few known plastid DNAs (ptDNAs), out of thousands of available sequences, not biased in adenine and thymine. Here, I take a closer look at the H. lacustris plastome, comparing its size, content and architecture to other large organelle DNAs, including those from close relatives in the Chlamydomonadales. I show that the H. lacustris plastid coding repertoire is not as unusual as initially thought, representing a standard set of rRNAs, tRNAs and protein-coding genes, where the canonical stop codon UGA appears to sometimes signify tryptophan. The intergenic spacers are dense with repeats, and it is within these regions where potential answers to the source of such extreme genomic expansion lie. By comparing ptDNA sequences of two closely related strains of H. lacustris, I argue that the mutation rate of the non-coding DNA is high and contributing to plastome inflation. Finally, by exploring publicly available RNA-sequencing data, I find that most of the intergenic ptDNA is transcriptionally active.


September 22, 2019  |  

Genomic characterization reveals significant divergence within Chlorella sorokiniana (Chlorellales, Trebouxiophyceae)

Selection of highly productive algal strains is crucial for establishing economically viable biomass and biopro- duct cultivation systems. Characterization of algal genomes, including understanding strain-specific differences in genome content and architecture is a critical step in this process. Using genomic analyses, we demonstrate significant differences between three strains of Chlorella sorokiniana (strain 1228, UTEX 1230, and DOE1412). We found that unique, strain-specific genes comprise a substantial proportion of each genome, and genomic regions with> 80% local nucleotide identity constitute <15% of each genome among the strains, indicating substantial strain specific evolution. Furthermore, cataloging of meiosis and other sex-related genes in C. sor- okiniana strains suggests strategic breeding could be utilized to improve biomass and bioproduct yields if a sexual cycle can be characterized. Finally, preliminary investigation of epigenetic machinery suggests the pre- sence of potentially unique transcriptional regulation in each strain. Our data demonstrate that these three C. sorokiniana strains represent significantly different genomic content. Based on these findings, we propose in- dividualized assessment of each strain for potential performance in cultivation systems.


September 22, 2019  |  

Genomic analysis of Picochlorum species reveals how microalgae may adapt to variable environments.

Understanding how microalgae adapt to rapidly changing environments is not only important to science but can help clarify the potential impact of climate change on the biology of primary producers. We sequenced and analyzed the nuclear genome of multiple Picochlorum isolates (Chlorophyta) to elucidate strategies of environmental adaptation. It was previously found that coordinated gene regulation is involved in adaptation to salinity stress, and here we show that gene gain and loss also play key roles in adaptation. We determined the extent of horizontal gene transfer (HGT) from prokaryotes and their role in the origin of novel functions in the Picochlorum clade. HGT is an ongoing and dynamic process in this algal clade with adaptation being driven by transfer, divergence, and loss. One HGT candidate that is differentially expressed under salinity stress is indolepyruvate decarboxylase that is involved in the production of a plant auxin that mediates bacteria-diatom symbiotic interactions. Large differences in levels of heterozygosity were found in diploid haplotypes among Picochlorum isolates. Biallelic divergence was pronounced in P. oklahomensis (salt plains environment) when compared with its closely related sister taxon Picochlorum SENEW3 (brackish water environment), suggesting a role of diverged alleles in response to environmental stress. Our results elucidate how microbial eukaryotes with limited gene inventories expand habitat range from mesophilic to halophilic through allelic diversity, and with minor but important contributions made by HGT. We also explore how the nature and quality of genome data may impact inference of nuclear ploidy.


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