Molecular phylogeny of fucoxanthin-chlorophyll a/c proteins from Chaetoceros gracilis and Lhcq/Lhcf diversity.

Diatoms adapt to various aquatic light environments and play major roles in the global carbon cycle using their unique light-harvesting system, i.e. fucoxanthin chlorophyll a/c binding proteins (FCPs). Structural analyses of photosystem II (PSII)-FCPII and photosystem I (PSI)-FCPI complexes from the diatom Chaetoceros gracilis have revealed the localization and interactions of many FCPs; however, the entire set of FCPs has not been characterized. Here, we identify 46 FCPs in the newly assembled genome and transcriptome of C. gracilis. Phylogenetic analyses suggest that these FCPs can be classified into five subfamilies: Lhcr, Lhcf, Lhcx, Lhcz, and the novel Lhcq, in addition to a distinct type of Lhcr, CgLhcr9. The FCPs in Lhcr, including CgLhcr9 and some Lhcqs, have orthologous proteins in other diatoms, particularly those found in the PSI-FCPI structure. By contrast, the Lhcf subfamily, some of which were found in the PSII-FCPII complex, seems to be diversified in each diatom species, and the number of Lhcqs differs among species, indicating that their diversification may contribute to species-specific adaptations to light. Further phylogenetic analyses of FCPs/light-harvesting complex (LHC) proteins using genome data and assembled transcriptomes of other diatoms and microalgae in public databases suggest that our proposed classification of FCPs is common among various red-lineage algae derived from secondary endosymbiosis of red algae, including Haptophyta. These results provide insights into the loss and gain of FCP/LHC subfamilies during the evolutionary history of the red algal lineage.

A simple and practical workflow for genotyping of CRISPR-Cas9-based knockout phenotypes using multiplexed amplicon sequencing.

Founder animals carrying high proportions of somatic mutation induced by CRISPR-Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next-generation sequencing is the most suitable approach for large-scale management of multiple samples and accurate evaluation of the efficiency of Cas9-induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR-Cas9-based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single-indexed library on the Illumina MiSeq platform. Additionally, a user-friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype-phenotype correlations in CRISPR-Cas9-based loss-of-function screening of numerous target genes in various organisms.

MBGD update 2018: microbial genome database based on hierarchical orthology relations covering closely related and distantly related comparisons.

The Microbial Genome Database for Comparative Analysis (MBGD) is a database for comparative genomics based on comprehensive orthology analysis of bacteria, archaea and unicellular eukaryotes. MBGD now contains 6318 genomes. To utilize the database for both closely related and distantly related genomes, MBGD previously provided two types of ortholog tables: the standard ortholog table containing one representative genome from each genus covering the entire taxonomic range and the taxon specific ortholog tables for each taxon. However, this approach has a drawback in that the standard ortholog table contains only genes that are conserved in the representative genomes. To address this problem, we developed a stepwise procedure to construct ortholog tables hierarchically in a bottom-up manner. By using this approach, the new standard ortholog table now covers the entire gene repertoire stored in MBGD. In addition, we have enhanced several functionalities, including rapid and flexible keyword searching, profile-based sequence searching for orthology assignment to a user query sequence, and displaying a phylogenetic tree of each taxon based on the concatenated core gene sequences. For integrative database searching, the core data in MBGD are represented in Resource Description Framework (RDF) and a SPARQL interface is provided to search them. MBGD is available at http://mbgd.genome.ad.jp/.

MBGD update 2015: microbial genome database for flexible ortholog analysis utilizing a diverse set of genomic data.

The microbial genome database for comparative analysis (MBGD) (available at http://mbgd.genome.ad.jp/) is a comprehensive ortholog database for flexible comparative analysis of microbial genomes, where the users are allowed to create an ortholog table among any specified set of organisms. Because of the rapid increase in microbial genome data owing to the next-generation sequencing technology, it becomes increasingly challenging to maintain high-quality orthology relationships while allowing the users to incorporate the latest genomic data available into an analysis. Because many of the recently accumulating genomic data are draft genome sequences for which some complete genome sequences of the same or closely related species are available, MBGD now stores draft genome data and allows the users to incorporate them into a user-specific ortholog database using the MyMBGD functionality. In this function, draft genome data are incorporated into an existing ortholog table created only from the complete genome data in an incremental manner to prevent low-quality draft data from affecting clustering results. In addition, to provide high-quality orthology relationships, the standard ortholog table containing all the representative genomes, which is first created by the rapid classification program DomClust, is now refined using DomRefine, a recently developed program for improving domain-level clustering using multiple sequence alignment information.

Epigenetic modification maintains intrinsic limb-cell identity in Xenopus limb bud regeneration.

Many amphibians can regenerate limbs, even in adulthood. If a limb is amputated, the stump generates a blastema that makes a complete, new limb in a process similar to developmental morphogenesis. The blastema is thought to inherit its limb-patterning properties from cells in the stump, and it retains the information despite changes in morphology, gene expression, and differentiation states required by limb regeneration. We hypothesized that these cellular properties are maintained as epigenetic memory through histone modifications. To test this hypothesis, we analyzed genome-wide histone modifications in Xenopus limb bud regeneration. The trimethylation of histone H3 at lysine 4 (H3K4me3) is closely related to an open chromatin structure that allows transcription factors access to genes, whereas the trimethylation of histone H3 at lysine 27 (H3K27me3) is related to a closed chromatin state that blocks the access of transcription factors. We compared these two modification profiles by high-throughput sequencing of samples prepared from the intact limb bud and the regenerative blastema by chromatin immunoprecipitation. For many developmental genes, histone modifications at the transcription start site were the same in the limb bud and the blastema, were stable during regeneration, and corresponded well to limb properties. These results support our hypothesis that histone modifications function as a heritable cellular memory to maintain limb cell properties, despite dynamic changes in gene expression during limb bud regeneration in Xenopus.

RNA-seq Transcriptional Profiling of an Arbuscular Mycorrhiza Provides Insights into Regulated and Coordinated Gene Expression in Lotus japonicus and Rhizophagus irregularis.

Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a HiSeq 2000 next-generation sequencer with a Cufflinks assembly and de novo transcriptome assembly. There were 3,641 genes differentially expressed during arbuscular mycorrhizal development in L. japonicus, approximately 80% of which were up-regulated. The up-regulated genes included secreted proteins, transporters, proteins involved in lipid and amino acid metabolism, ribosomes and histones. We also detected many genes that were differentially expressed in small-secreted peptides and transcription factors, which may be involved in signal transduction or transcription regulation during symbiosis. Co-regulated genes between arbuscular mycorrhizal and root nodule symbiosis were not particularly abundant, but transcripts encoding for membrane traffic-related proteins, transporters and iron transport-related proteins were found to be highly co-up-regulated. In transcripts of arbuscular mycorrhizal fungi, expansion of cytochrome P450 was observed, which may contribute to various metabolic pathways required to accommodate roots and soil. The comprehensive gene expression data of both plants and arbuscular mycorrhizal fungi provide a powerful platform for investigating the functional and molecular mechanisms underlying arbuscular mycorrhizal symbiosis.

MBGD update 2013: the microbial genome database for exploring the diversity of microbial world.

The microbial genome database for comparative analysis (MBGD, available at http://mbgd.genome.ad.jp/) is a platform for microbial genome comparison based on orthology analysis. As its unique feature, MBGD allows users to conduct orthology analysis among any specified set of organisms; this flexibility allows MBGD to adapt to a variety of microbial genomic study. Reflecting the huge diversity of microbial world, the number of microbial genome projects now becomes several thousands. To efficiently explore the diversity of the entire microbial genomic data, MBGD now provides summary pages for pre-calculated ortholog tables among various taxonomic groups. For some closely related taxa, MBGD also provides the conserved synteny information (core genome alignment) pre-calculated using the CoreAligner program. In addition, efficient incremental updating procedure can create extended ortholog table by adding additional genomes to the default ortholog table generated from the representative set of genomes. Combining with the functionalities of the dynamic orthology calculation of any specified set of organisms, MBGD is an efficient and flexible tool for exploring the microbial genome diversity.

Molecular impact of juvenile hormone agonists on neonatal Daphnia magna.

Daphnia magna has been used extensively to evaluate organism- and population-level responses to pollutants in acute toxicity and reproductive toxicity tests. We have previously reported that exposure to juvenile hormone (JH) agonists results in a reduction of reproductive function and production of male offspring in a cyclic parthenogenesis, D. magna. Recent advances in molecular techniques have provided tools to understand better the responses to pollutants in aquatic organisms, including D. magna. DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to JH agonists: methoprene (125, 250 and 500 ppb), fenoxycarb (0.5, 1 and 2 ppb) and epofenonane (50, 100 and 200 ppb). Exposure to these JH analogs resulted in chemical-specific patterns of gene expression. The heat map analyses based on hierarchical clustering revealed a similar pattern between treatments with a high dose of methoprene and with epofenonane. In contrast, treatment with low to middle doses of methoprene resulted in similar profiles to fenoxycarb treatments. Hemoglobin and JH epoxide hydrolase genes were clustered as JH-responsive genes. These data suggest that fenoxycarb has high activity as a JH agonist, methoprene shows high toxicity and epofenonane works through a different mechanism compared with other JH analogs, agreeing with data of previously reported toxicity tests. In conclusion, D. magna DNA microarray is useful for the classification of JH analogs and identification of JH-responsive genes.

Characterization of the far-red light absorbing light-harvesting chlorophyll a/b binding complex, a derivative of the distinctive Lhca gene family in green algae.

Prasiola crispa, an aerial green alga, exhibits remarkable adaptability to the extreme conditions of Antarctica by forming layered colonies capable of utilizing far-red light for photosynthesis. Despite a recent report on the structure of P. crispa's unique light-harvesting chlorophyll (Chl)-binding protein complex (Pc-frLHC), which facilitates far-red light absorption and uphill excitation energy transfer to photosystem II, the specific genes encoding the subunits of Pc-frLHC have not yet been identified. Here, we report a draft genome sequence of P. crispa strain 4113, originally isolated from soil samples on Ongul Island, Antarctica. We obtained a 92 Mbp sequence distributed in 1,045 scaffolds comprising 10,244 genes, reflecting 87.1% of the core eukaryotic gene set. Notably, 26 genes associated with the light-harvesting Chl a/b binding complex (LHC) were identified, including four Pc-frLHC genes, with similarity to a noncanonical Lhca gene with four transmembrane helices, such as Ot_Lhca6 in Ostreococcus tauri and Cr_LHCA2 in Chlamydomonas reinhardtii. A comparative analysis revealed that Pc-frLHC shares homology with certain Lhca genes found in Coccomyxa and Trebouxia species. This similarity indicates that Pc-frLHC has evolved from an ancestral Lhca gene with four transmembrane helices and branched out within the Trebouxiaceae family. Furthermore, RNA-seq analysis conducted during the initiation of Pc-frLHC gene induction under red light illumination indicated that Pc-frLHC genes were induced independently from other genes associated with photosystems or LHCs. Instead, the genes of transcription factors, helicases, chaperones, heat shock proteins, and components of blue light receptors were identified to coexpress with Pc-frLHC. Those kinds of information could provide insights into the expression mechanisms of Pc-frLHC and its evolutional development.

RNA-Seq Analysis Reveals Genes Related to Photoreception, Nutrient Uptake, and Toxicity in a Noxious Red-Tide Raphidophyte Chattonella antiqua.

Aquaculture industries are under threat from noxious red tides, but harm can be mitigated by precautions such as early harvesting and restricting fish feeding to just before the outbreak of a red tide. Therefore, accurate techniques for forecasting red-tide outbreaks are strongly needed. Omics analyses have the potential to expand our understanding of the eco-physiology of these organisms at the molecular level, and to facilitate identification of molecular markers for forecasting their population dynamics and occurrence of damages to fisheries. Red tides of marine raphidophytes, especially Chattonella species, often extensively harm aquaculture industries in regions with a temperate climate around the world. A red tide of Chattonella tends to develop just after an input of nutrients along the coast. Chattonella displays diurnal vertical migration regulated by a weak blue light, so it photosynthesizes in the surface layer during the daytime and takes up nutrients in the bottom layer during the nighttime. Superoxide produced by Chattonella cells is a strong candidate for the cause of its toxicity to bacteria and fishes. Here we conducted mRNA-seq of Chattonella antiqua to identify genes with functions closely related to the dynamics of the noxious red tide, such as photosynthesis, photoreception, nutrient uptake, and superoxide production. The genes related to photosynthetic pigment biosynthesis and nutrient uptake had high similarity with those of model organisms of plants and algae and other red-tide microalgae. We identified orthologous genes of photoreceptors such as aureochrome (newly five genes), the cryptochrome/photolyase (CRY/PHR) family (6-4PHR, plant CRY or cyclobutane pyrimidine dimer [CPD] Class III, CPD Class II, and CRY-DASH), and phytochrome (four genes), which regulate various physiological processes such as flagellar motion and cell cycle in model organisms. Six orthologous genes of NADPH oxidase, which produces superoxide on the cell membrane, were found and divided into two types: one with 5-6 transmembrane domains and another with 11 transmembrane domains. The present study should open the way for analyzing the eco-physiological features of marine raphidophytes at the molecular level.

Complete Genome Sequence of Aurantimicrobium minutum Type Strain KNCT, a Planktonic Ultramicrobacterium Isolated from River Water.

Aurantimicrobium minutum type strain KNC(T) is a planktonic ultramicrobacterium isolated from river water in western Japan. Strain KNC(T) has an extremely small, streamlined genome of 1,622,386 bp comprising 1,575 protein-coding sequences. The genome annotation suggests that strain KNC(T) has an actinorhodopsin-based photometabolism.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

Construction of an ortholog database using the semantic web technology for integrative analysis of genomic data.

Recently, various types of biological data, including genomic sequences, have been rapidly accumulating. To discover biological knowledge from such growing heterogeneous data, a flexible framework for data integration is necessary. Ortholog information is a central resource for interlinking corresponding genes among different organisms, and the Semantic Web provides a key technology for the flexible integration of heterogeneous data. We have constructed an ortholog database using the Semantic Web technology, aiming at the integration of numerous genomic data and various types of biological information. To formalize the structure of the ortholog information in the Semantic Web, we have constructed the Ortholog Ontology (OrthO). While the OrthO is a compact ontology for general use, it is designed to be extended to the description of database-specific concepts. On the basis of OrthO, we described the ortholog information from our Microbial Genome Database for Comparative Analysis (MBGD) in the form of Resource Description Framework (RDF) and made it available through the SPARQL endpoint, which accepts arbitrary queries specified by users. In this framework based on the OrthO, the biological data of different organisms can be integrated using the ortholog information as a hub. Besides, the ortholog information from different data sources can be compared with each other using the OrthO as a shared ontology. Here we show some examples demonstrating that the ortholog information described in RDF can be used to link various biological data such as taxonomy information and Gene Ontology. Thus, the ortholog database using the Semantic Web technology can contribute to biological knowledge discovery through integrative data analysis.

Diofenolan induces male offspring production through binding to the juvenile hormone receptor in Daphnia magna.

Juvenile hormone (JH) and JH agonists have been reported to induce male offspring production in various daphnid species including Daphnia magna. We recently established a short-term in vivo screening assay to detect chemicals having male offspring induction activity in adult D. magna. Diofenolan has been developed as a JH agonist for insect pest control, but its male offspring induction activity in daphnids has not been investigated yet. In this study, we found that the insect growth regulator (IGR) diofenolan exhibited a potent male offspring induction activity at low ng/L to µg/L concentrations, as demonstrated by the short-term in vivo screening assay and the recently developed TG211 ANNEX 7 test protocol. A two-hybrid assay performed using the D. magna JH receptor confirmed that diofenolan had a strong JH activity. Global whole body transcriptome analysis of D. magna exposed to 10 ng/L diofenolan showed an up-regulation of JH-responsive genes and modulation of several genes involved in the ecdysone receptor signaling pathway. These results clearly demonstrate that diofenolan has strong JH activity and male offspring induction activity, and that a combination of modified standardized regulatory testing protocols and rapid in vitro and in vivo screening assays are able to identify potential endocrine disruptors in D. magna. The observation that diofenolan modulates multiple endocrine signaling pathways in D. magna suggests that further investigation of potential interference with growth, development and reproduction is warranted.

Analysis of expressed sequence tags of the water flea Daphnia magna.

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.

Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: implication for land plant evolution.

The mosses and flowering plants diverged >400 million years ago. The mosses have haploid-dominant life cycles, whereas the flowering plants are diploid-dominant. The common ancestors of land plants have been inferred to be haploid-dominant, suggesting that genes used in the diploid body of flowering plants were recruited from the genes used in the haploid body of the ancestors during the evolution of land plants. To assess this evolutionary hypothesis, we constructed an EST library of the moss Physcomitrella patens, and compared the moss transcriptome to the genome of Arabidopsis thaliana. We constructed full-length enriched cDNA libraries from auxin-treated, cytokinin-treated, and untreated gametophytes of P. patens, and sequenced both ends of >40,000 clones. These data, together with the mRNA sequences in the public databases, were assembled into 15,883 putative transcripts. Sequence comparisons of A. thaliana and P. patens showed that at least 66% of the A. thaliana genes had homologues in P. patens. Comparison of the P. patens putative transcripts with all known proteins, revealed 9,907 putative transcripts with high levels of similarity to vascular plant genes, and 850 putative transcripts with high levels of similarity to other organisms. The haploid transcriptome of P. patens appears to be quite similar to the A. thaliana genome, supporting the evolutionary hypothesis. Our study also revealed that a number of genes are moss specific and were lost in the flowering plant lineage.