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Abstract Background OryzaGenome ( http://viewer.shigen.info/oryzagenome21detail/index.xhtml ), a feature within Oryzabase ( https://shigen.nig.ac.jp/rice/oryzabase/ ), is a genomic database for wild Oryza species that provides comparative and evolutionary genomics approaches for the rice research community. Results Here we release OryzaGenome2.1, the first major update of OryzaGenome. The main feature in this version is the inclusion of newly sequenced genotypes and their meta-information, giving a total of 217 accessions of 19 wild Oryza species (O. rufipogon, O. barthii, O. longistaminata, O. meridionalis, O. glumaepatula, O. punctata, O. minuta, O. officinalis, O. rhizomatis, O. eichingeri, O. latifolia, O. alta, O. grandiglumis, O. australiensis, O. brachyantha, O. granulata, O. meyeriana, O. ridleyi, and O. longiglumis). These 19 wild species belong to 9 genome types (AA, BB, CC, BBCC, CCDD, EE, FF, GG, and HHJJ), representing wide genomic diversity in the genus. Using the genotype information, we analyzed the genome diversity of Oryza species. Other features of OryzaGenome facilitate the use of information on single nucleotide polymorphisms (SNPs) between O. sativa and its wild progenitor O. rufipogon in rice research, including breeding as well as basic science. For example, we provide Variant Call Format (VCF) files for genome-wide SNPs of 33 O. rufipogon accessions against the O. sativa reference genome, IRGSP1.0. In addition, we provide a new SNP Effect Table function, allowing users to identify SNPs or small insertion/deletion polymorphisms in the 33 O. rufipogon accessions and to search for the effect of these polymorphisms on protein function if they reside in the coding region (e.g., are missense or nonsense mutations). Furthermore, the SNP Viewer for 446 O. rufipogon accessions was updated by implementing new tracks for possible selective sweep regions and highly mutated regions that were potentially exposed to selective pressures during the process of domestication. Conclusion OryzaGenome2.1 focuses on comparative genomic analysis of diverse wild Oryza accessions collected around the world and on the development of resources to speed up the identification of critical trait-related genes, especially from O. rufipogon. It aims to promote the use of genotype information from wild accessions in rice breeding and potential future crop improvements. Diverse genotypes will be a key resource for evolutionary studies in Oryza, including polyploid biology.

Most phasing programmes for sequencing data work well for genomes with low heterozygosity but drop in performance in regions of high heterozygosity. Here, Kajitani et al. develop the assembler Platanus-allee and demonstrate its utility in de novo assemblies of various genomes and the human MHC region.

Abstract Background Planarians are non-parasitic Platyhelminthes (flatworms) famous for their regeneration ability and for having a well-organized brain. Dugesia japonica is a typical planarian species that is widely distributed in the East Asia. Extensive cellular and molecular experimental methods have been developed to identify the functions of thousands of genes in this species, making this planarian a good experimental model for regeneration biology and neurobiology. However, no genome-level information is available for D. japonica, and few gene regulatory networks have been identified thus far. Results To obtain whole-genome information on this species and to study its gene regulatory networks, we extracted genomic DNA from 200 planarians derived from a laboratory-bred asexual clonal strain, and sequenced 476 Gb of data by second-generation sequencing. Kmer frequency graphing and fosmid sequence analysis indicated a complex genome that would be difficult to assemble using second-generation sequencing short reads. To address this challenge, we developed a new assembly strategy and improved the de novo genome assembly, producing a 1.56 Gb genome sequence (DjGenome ver1.0, including 202,925 scaffolds and N50 length 27,741 bp) that covers 99.4% of all 19,543 genes in the assembled transcriptome, although the genome is fragmented as 80% of the genome consists of repeated sequences (genomic frequency ≥ 2). By genome comparison between two planarian genera, we identified conserved non-coding elements (CNEs), which are indicative of gene regulatory elements. Transgenic experiments using Xenopus laevis indicated that one of the CNEs in the Djndk gene may be a regulatory element, suggesting that the regulation of the ndk gene and the brain formation mechanism may be conserved between vertebrates and invertebrates. Conclusion This draft genome and CNE analysis will contribute to resolving gene regulatory networks in planarians. The genome database is available at: http://www.planarian.jp.

Abstract Background In the previous paper published in 2017, we described the structure of Hox gene cluster of the ascidian, Halocynthia roretzi, and discussed the scenario for the disintegration of Hox gene clusters during evolution of ascidians. The description about the Hox gene cluster structure still represents the latest information, hence it has been left unchanged. In contrast, some points in Discussion, the description on the phylogenetic relationships among tunicates and the theoretical scenario for the disintegration of Hox gene cluster during evolution of ascidians, should be changed because the phylogenetic relationships among tunicates have recently been updated. The above mentioned points were made in accordance with the phylogenetic tree for tunicates based on the mitochondrial DNA sequences, which was the latest at the time of publication. In 2018, however, Kocot et al. and Delsuc et al. proposed new phylogenetic trees for tunicates based on a large number of nuclear gene sequences. The trees obtained by the two groups are essentially the same and different from the previous one in the phylogenetic positions of Appendicularia and Thaliacea, which leads to a change in the order of the emergence of ascidians and the Hox gene cluster disintegration during evolution of ascidians or tunicates. Results We add here a note to update the previous description on the phylogenetic relationships among tunicates and the theoretical scenario, including one Figure, so as to coincide with the new phylogenetic relationships among tunicates based on the nuclear gene sequences. Conclusion The previous summarized conclusion remains unchanged: we suggest that the Hox gene cluster of the ancestral ascidian experienced extensive genome shuffling during the course of evolution to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene organization on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to all ascidians and later lineage-specific events.

Eukaryotes often silence transposable elements (TEs) via DNA methylation. Here, the authors show that evolution of VANC, an Arabidopsis anti-silencing factor, and its target motifs allows sequence-specific demethylation, suggesting a way TEs can proliferate while minimizing damage to the host genome.

Abstract Background Hox gene clusters with at least 13 paralog group (PG) members are common in vertebrate genomes and in that of amphioxus. Ascidians, which belong to the subphylum Tunicata (Urochordata), are phylogenetically positioned between vertebrates and amphioxus, and traditionally divided into two groups: the Pleurogona and the Enterogona. An enterogonan ascidian, Ciona intestinalis (Ci), possesses nine Hox genes localized on two chromosomes; thus, the Hox gene cluster is disintegrated. We investigated the Hox gene cluster of a pleurogonan ascidian, Halocynthia roretzi (Hr) to investigate whether Hox gene cluster disintegration is common among ascidians, and if so, how such disintegration occurred during ascidian or tunicate evolution. Results Our phylogenetic analysis reveals that the Hr Hox gene complement comprises nine members, including one with a relatively divergent Hox homeodomain sequence. Eight of nine Hr Hox genes were orthologous to Ci-Hox1, 2, 3, 4, 5, 10, 12 and 13. Following the phylogenetic classification into 13 PGs, we designated Hr Hox genes as Hox1, 2, 3, 4, 5, 10, 11/12/13.a, 11/12/13.b and HoxX. To address the chromosomal arrangement of the nine Hox genes, we performed two-color chromosomal fluorescent in situ hybridization, which revealed that the nine Hox genes are localized on a single chromosome in Hr, distinct from their arrangement in Ci. We further examined the order of the nine Hox genes on the chromosome by chromosome/scaffold walking. This analysis suggested a gene order of Hox1, 11/12/13.b, 11/12/13.a, 10, 5, X, followed by either Hox4, 3, 2 or Hox2, 3, 4 on the chromosome. Based on the present results and those previously reported in Ci, we discuss the establishment of the Hox gene complement and disintegration of Hox gene clusters during the course of ascidian or tunicate evolution. Conclusions The Hox gene cluster and the genome must have experienced extensive reorganization during the course of evolution from the ancestral tunicate to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene arrangement on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to tunicates as well as later ascidian lineage-specific events.

Abstract Novel genomics-based approaches such as genome-wide association studies (GWAS) and genomic selection (GS) are expected to be useful in fruit tree breeding, which requires much time from the cross to the release of a cultivar because of the long generation time. In this study, a citrus parental population (111 varieties) and a breeding population (676 individuals from 35 full-sib families) were genotyped for 1,841 single nucleotide polymorphisms (SNPs) and phenotyped for 17 fruit quality traits. GWAS power and prediction accuracy were increased by combining the parental and breeding populations. A multi-kernel model considering both additive and dominance effects improved prediction accuracy for acidity and juiciness, implying that the effects of both types are important for these traits. Genomic best linear unbiased prediction (GBLUP) with linear ridge kernel regression (RR) was more robust and accurate than GBLUP with non-linear Gaussian kernel regression (GAUSS) in the tails of the phenotypic distribution. The results of this study suggest that both GWAS and GS are effective for genetic improvement of citrus fruit traits. Furthermore, the data collected from breeding populations are beneficial for increasing the detection power of GWAS and the prediction accuracy of GS.

The deposition of histone H3 variant CENP-A bound with histone H4 is a key feature designating the centromere region of a chromosome. Here the authors show acetylation on residues K5 and K12 in histone H4, mediated by the RbAp46/48-Hat1 complex, is required for deposition of CENP-A-H4 into centromeres.

Japanese morning glory (Ipomoea nil) has diverse flowering traits. Here, the authors describe the reference genome sequence of I. nil, annotations of genes and transposons, and compare evolution of the I. nilgenome to other Convolvulaceae and Solanales genomes.

Tardigrades are resistant to extreme environmental conditions including dehydration, radiation and the vacuum of space. Here the authors present a high-quality genome which displays minimal horizontal gene transfer, and identify the unique tardigrade protein Dsup which suppresses DNA damage.

Sex-determining regions (SDRs) or mating-type (MT) loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii. Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii. Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT–, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale–V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale. These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs.

The undifferentiated Gonium pectorale represents the initial transition to multicellularity. Here, Bradley Olson, Erik Hanschen and colleagues describe the genome of Gonium pectorale, demonstrating that co-option of the retinoblastoma cell cycle regulatory pathway was a key genetic change in the evolution of multicellularity.

Environmental adaptation is one of the most fundamental features of organisms. Modern genome science has identified some genes associated with adaptive traits of organisms, and has provided insights into environmental adaptation and evolution. However, how genes contribute to adaptive traits and how traits are selected under an environment in the course of evolution remain mostly unclear. To approach these issues, we utilize “Dark-fly”, a Drosophila melanogaster line maintained in constant dark conditions for more than 60 years. Our previous analysis identified 220,000 single nucleotide polymorphisms (SNPs) in the Dark-fly genome, but did not clarify which SNPs of Dark-fly are truly adaptive for living in the dark. We found here that Dark-fly dominated over the wild-type fly in a mixed population under dark conditions, and based on this domination we designed an experiment for genome reselection to identify adaptive genes of Dark-fly. For this experiment, large mixed populations of Dark-fly and the wild-type fly were maintained in light conditions or in dark conditions, and the frequencies of Dark-fly SNPs were compared between these populations across the whole genome. We thereby detected condition-dependent selections toward approximately 6% of the genome. In addition, we observed the time-course trajectory of SNP frequency in the mixed populations through generations 0, 22, and 49, which resulted in notable categorization of the selected SNPs into three types with different combinations of positive and negative selections. Our data provided a list of about 100 strong candidate genes associated with the adaptive traits of Dark-fly.

Speciation is a continuous process and analysis of species pairs at different stages of divergence provides insight into how it unfolds. Previous genomic studies on young species pairs have revealed peaks of divergence and heterogeneous genomic differentiation. Yet less known is how localised peaks of differentiation progress to genome-wide divergence during the later stages of speciation in the presence of persistent gene flow. Spanning the speciation continuum, stickleback species pairs are ideal for investigating how genomic divergence builds up during speciation. However, attention has largely focused on young postglacial species pairs, with little knowledge of the genomic signatures of divergence and introgression in older stickleback systems. The Japanese stickleback species pair, composed of the Pacific Ocean three-spined stickleback (Gasterosteus aculeatus) and the Japan Sea stickleback (G. nipponicus), which co-occur in the Japanese islands, is at a late stage of speciation. Divergence likely started well before the end of the last glacial period and crosses between Japan Sea females and Pacific Ocean males result in hybrid male sterility. Here we use coalescent analyses and Approximate Bayesian Computation to show that the two species split approximately 0.68-1 million years ago but that they have continued to exchange genes at a low rate throughout divergence. Population genomic data revealed that, despite gene flow, a high level of genomic differentiation is maintained across the majority of the genome. However, we identified multiple, small regions of introgression, occurring mainly in areas of low recombination rate. Our results demonstrate that a high level of genome-wide divergence can establish in the face of persistent introgression and that gene flow can be localized to small genomic regions at the later stages of speciation with gene flow.

Satsuma (Citrus unshiu Marc.) is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma (“Miyagawa Wase”) was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome.

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information.

There is increasing evidence that long non-coding RNAs (lncRNAs) are important for normal reproductive development, yet very few lncRNAs have been identified in phalluses so far. Unlike eutherians, phallus development in the marsupial tammar wallaby occurs post-natally, enabling manipulation not possible in eutherians in which differentiation occurs in utero. We treated with sex steroids to determine the effects of androgen and oestrogen on lncRNA expression during phallus development. Hormonal manipulations altered the coding and non-coding gene expression profile of phalluses. We identified several predicted co-regulatory lncRNAs that appear to be co-expressed with the hormone-responsive candidate genes regulating urethral closure and phallus growth, namely IGF1, AR and ESR1. Interestingly, more than 50% of AR-associated coding genes and lncRNAs were also associated with ESR1. In addition, we identified and validated three novel co-regulatory and hormone-responsive lncRNAs: lnc-BMP5, lnc-ZBTB16 and lncRSPO4. Lnc-BMP5 was detected in the urethral epithelium of male phalluses and was downregulated by oestrogen in males. Lnc-ZBTB16 was downregulated by oestrogen treatment in male phalluses at day 50 post-partum (pp). LncRSPO4 was downregulated by adiol treatment in female phalluses but increased in male phalluses after castration. Thus, the expression pattern and hormone responsiveness of these lncRNAs suggests a physiological role in the development of the phallus.

Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy-Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies.

DNA methylation of promoters is linked to transcriptional silencing of protein-coding genes, and its alteration plays important roles in cancer formation. For example, hypermethylation of tumor suppressor genes has been seen in some cancers. Alteration of methylation in the promoters of microRNAs (miRNAs) has also been linked to transcriptional changes in cancers; however, no systematic studies of methylation and transcription of miRNAs have been reported. In the present study, to clarify the relation between DNA methylation and transcription of miRNAs, next-generation sequencing and microarrays were used to analyze the methylation and expression of miRNAs, protein-coding genes, other non-coding RNAs (ncRNAs), and pseudogenes in the human breast cancer cell lines MCF7 and the adriamycin (ADR) resistant cell line MCF7/ADR. DNA methylation in the proximal promoter of miRNAs is tightly linked to transcriptional silencing, as it is with protein-coding genes. In protein-coding genes, highly expressed genes have CpG-rich proximal promoters whereas weakly expressed genes do not. This is only rarely observed in other gene categories, including miRNAs. The present study highlights the epigenetic similarities and differences between miRNA and protein-coding genes.

Adaptive divergence at the microgeographic scale has been generally disregarded because high gene flow is expected to disrupt local adaptation. Yet, growing number of studies reporting adaptive divergence at a small spatial scale highlight the importance of this process in evolutionary biology. To investigate the genetic basis of microgeographic local adaptation, we conducted a genome-wide scan among sets of continuously distributed populations of Arabidopsis halleri subsp. gemmifera that show altitudinal phenotypic divergence despite gene flow. Genomic comparisons were independently conducted in two distinct mountains where similar highland ecotypes are observed, presumably as a result of convergent evolution. Here, we established a de novo reference genome and employed an individual-based resequencing for a total of 56 individuals. Among 527,225 reliable SNP loci, we focused on those showing a unidirectional allele frequency shift across altitudes. Statistical tests on the screened genes showed that our microgeographic population genomic approach successfully retrieve genes with functional annotations that are in line with the known phenotypic and environmental differences between altitudes. Furthermore, comparison between the two distinct mountains enabled us to screen out those genes that are neutral or adaptive only in either mountain, and identify the genes involved in the convergent evolution. Our study demonstrates that the genomic comparison among a set of genetically connected populations, instead of the commonly-performed comparison between two isolated populations, can also offer an effective screening for the genetic basis of local adaptation.

Epigenetic variations of phenotypes, especially those associated with DNA methylation, are often inherited over multiple generations in plants. The active and inactive chromatin states are heritable and can be maintained or even be amplified by positive feedback in a transgenerational manner. However, mechanisms controlling the transgenerational DNA methylation dynamics are largely unknown. As an approach to understand the transgenerational dynamics, we examined long-term effect of impaired DNA methylation in Arabidopsis mutants of the chromatin remodeler gene DDM1 (Decrease in DNA Methylation 1) through whole genome DNA methylation sequencing. The ddm1 mutation induces a drastic decrease in DNA methylation of transposable elements (TEs) and repeats in the initial generation, while also inducing ectopic DNA methylation at hundreds of loci. Unexpectedly, this ectopic methylation can only be seen after repeated self-pollination. The ectopic cytosine methylation is found primarily in the non-CG context and starts from 3' regions within transcription units and spreads upstream. Remarkably, when chromosomes with reduced DNA methylation were introduced from a ddm1 mutant into a DDM1 wild-type background, the ddm1-derived chromosomes also induced analogous de novo accumulation of DNA methylation in trans. These results lead us to propose a model to explain the transgenerational DNA methylation redistribution by genome-wide negative feedback. The global negative feedback, together with local positive feedback, would ensure robust and balanced differentiation of chromatin states within the genome.

A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements.

Sex chromosomes turn over rapidly in some taxonomic groups, where closely related species have different sex chromosomes. Although there are many examples of sex chromosome turnover, we know little about the functional roles of sex chromosome turnover in phenotypic diversification and genomic evolution. The sympatric pair of Japanese threespine stickleback (Gasterosteus aculeatus) provides an excellent system to address these questions: the Japan Sea species has a neo-sex chromosome system resulting from a fusion between an ancestral Y chromosome and an autosome, while the sympatric Pacific Ocean species has a simple XY sex chromosome system. Furthermore, previous quantitative trait locus (QTL) mapping demonstrated that the Japan Sea neo-X chromosome contributes to phenotypic divergence and reproductive isolation between these sympatric species. To investigate the genomic basis for the accumulation of genes important for speciation on the neo-X chromosome, we conducted whole genome sequencing of males and females of both the Japan Sea and the Pacific Ocean species. No substantial degeneration has yet occurred on the neo-Y chromosome, but the nucleotide sequence of the neo-X and the neo-Y has started to diverge, particularly at regions near the fusion. The neo-sex chromosomes also harbor an excess of genes with sex-biased expression. Furthermore, genes on the neo-X chromosome showed higher non-synonymous substitution rates than autosomal genes in the Japan Sea lineage. Genomic regions of higher sequence divergence between species, genes with divergent expression between species, and QTL for inter-species phenotypic differences were found not only at the regions near the fusion site, but also at other regions along the neo-X chromosome. Neo-sex chromosomes can therefore accumulate substitutions causing species differences even in the absence of substantial neo-Y degeneration.

De novo microbial genome sequencing reached a turning point with third-generation sequencing (TGS) platforms, and several microbial genomes have been improved by TGS long reads. Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and it has a function in the production of the traditional Japanese fermented food "natto." The B. subtilis natto BEST195 genome was previously sequenced with short reads, but it included some incomplete regions. We resequenced the BEST195 genome using a PacBio RS sequencer, and we successfully obtained a complete genome sequence from one scaffold without any gaps, and we also applied Illumina MiSeq short reads to enhance quality. Compared with the previous BEST195 draft genome and Marburg 168 genome, we found that incomplete regions in the previous genome sequence were attributed to GC-bias and repetitive sequences, and we also identified some novel genes that are found only in the new genome.

Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVC-induced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m(2) of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy.

Volvocalean green algae have among the most diverse mitochondrial and plastid DNAs (mtDNAs and ptDNAs) from the eukaryotic domain. However, nearly all of the organelle genome data from this group are restricted to unicellular species, like Chlamydomonas reinhardtii, and presently only one multicellular species, the ∼4,000-celled Volvox carteri, has had its organelle DNAs sequenced. The V. carteri organelle genomes are repeat rich, and the ptDNA is the largest plastome ever sequenced. Here, we present the complete mtDNA and ptDNA of the colonial volvocalean Gonium pectorale, which is comprised of ∼16 cells and occupies a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line. The mtDNA and ptDNA of G. pectorale are circular-mapping AT-rich molecules with respective lengths and coding densities of 16 and 222.6 kilobases and 73 and 44%. They share some features with the organelle DNAs of V. carteri, including palindromic repeats within the plastid compartment, but show more similarities with those of C. reinhardtii, such as a compact mtDNA architecture and relatively low organelle DNA intron contents. Overall, the G. pectorale organelle genomes raise several interesting questions about the origin of linear mitochondrial chromosomes within the Volvocales and the relationship between multicellularity and organelle genome expansion.

Organisms are remarkably adapted to diverse environments by specialized metabolisms, morphology, or behaviors. To address the molecular mechanisms underlying environmental adaptation, we have utilized a Drosophila melanogaster line, termed "Dark-fly", which has been maintained in constant dark conditions for 57 years (1400 generations). We found that Dark-fly exhibited higher fecundity in dark than in light conditions, indicating that Dark-fly possesses some traits advantageous in darkness. Using next-generation sequencing technology, we determined the whole genome sequence of Dark-fly and identified approximately 220,000 single nucleotide polymorphisms (SNPs) and 4,700 insertions or deletions (InDels) in the Dark-fly genome compared to the genome of the Oregon-R-S strain, a control strain. 1.8% of SNPs were classified as non-synonymous SNPs (nsSNPs: i.e., they alter the amino acid sequence of gene products). Among them, we detected 28 nonsense mutations (i.e., they produce a stop codon in the protein sequence) in the Dark-fly genome. These included genes encoding an olfactory receptor and a light receptor. We also searched runs of homozygosity (ROH) regions as putative regions selected during the population history, and found 21 ROH regions in the Dark-fly genome. We identified 241 genes carrying nsSNPs or InDels in the ROH regions. These include a cluster of alpha-esterase genes that are involved in detoxification processes. Furthermore, analysis of structural variants in the Dark-fly genome showed the deletion of a gene related to fatty acid metabolism. Our results revealed unique features of the Dark-fly genome and provided a list of potential candidate genes involved in environmental adaptation.

Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

Moyamoya disease is an idiopathic vascular disorder of intracranial arteries. Its susceptibility locus has been mapped to 17q25.3 in Japanese families, but the susceptibility gene is unknown.Genome-wide linkage analysis in eight three-generation families with moyamoya disease revealed linkage to 17q25.3 (P