Search

Abstract Although Streptococcus suis has attracted public attention as a major swine and human pathogen, this bacterium has also been isolated from other animals, including ruminants. However, recent taxonomic studies revealed the existence of other species that were previously identified as S. suis, and some of these isolates were reclassified as the novel species Streptococcus ruminantium. In Japan, biochemically identified S. suis is frequently isolated from diseased ruminants; however, such isolates have not yet been identified accurately, and their aetiological importance in ruminants is unclear. Therefore, to understand the importance of S. suis and S. suis-like bacteria in ruminants, we reclassified S. suis isolates from ruminants according to the updated classification and investigated their genetic diversity. Although both S. suis and S. ruminantium were isolated from healthy and diseased ruminants, most of the isolates from diseased animals were S. ruminantium, implying that S. ruminantium is more likely to be associated with ruminant disease than S. suis. However, the ruminant S. suis and S. ruminantium isolates from diseased animals were classified into diverse genotypes rather than belonging to certain clonal groups. Genome sequence analysis of 20 S. ruminantium isolates provided information about the antibiotic resistance, potential virulence, and serological diversity of this species. We further developed an S. ruminantium-specific PCR assay to aid in the identification of this bacterium. The information obtained and the method established in this study will contribute to the accurate diagnosis of ruminant streptococcal infections.

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.

Our knowledge of DNA methylation systems in prokaryotes is mostly limited to those of culturable microbes. Here, Hiraoka et al. analyse DNA methylation patterns in metagenomic data from a microbial community, revealing new methylated motifs and experimentally validating the methyltransferases’ specificities.

Among Shiga toxin (Stx)–producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a β-glucuronidase–positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (β-glucuronidase–negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

Abstract A large variety of microbes are present in the human gut, some of which are considered to interact with each other. Most of these interactions involve bacterial metabolites. Phascolarctobacterium faecium hardly uses carbohydrates for growth and instead uses succinate as a substrate. This study investigated the growth behavior of the co‐culture of the succinate‐specific utilizer P. faecium and the succinogenic gut commensal Bacteroides thetaiotaomicron. Succinate production by B. thetaiotaomicron supported the growth of P. faecium and concomitant propionate production via the succinate pathway. The succinate produced was completely converted to propionate. This result was comparable with the monoculture of P. faecium in the medium supplemented with 1% (w/v) succinate. We analyzed the transcriptional response (RNA‐Seq) between the mono‐ and co‐culture of P. faecium and B. thetaiotaomicron. Comparison of the expression levels of genes of P. faecium between the mono‐ and co‐cultured conditions highlighted that the genes putatively involved in the transportation of succinate were notably expressed under the co‐cultured conditions. Differential expression analysis showed that the presence of P. faecium induced changes in the B. thetaiotaomicron transcriptional pattern, for example, expression changes in the genes for vitamin B12 transporters and reduced expression of glutamate‐dependent acid resistance system‐related genes. Also, transcriptome analysis of P. faecium suggested that glutamate and succinate might be used as sources of succinyl‐CoA, an intermediate in the succinate pathway. This study revealed some survival strategies of asaccharolytic bacteria, such as Phascolarctobacterium spp., in the human gut.

Epigenetic DNA base methylation plays important roles in gene expression regulation. We here describe a gene expression regulation network consisting of many DNA methyltransferases each frequently changing its target sequence-specificity. Our object Helicobacter pylori, a bacterium responsible for most incidence of stomach cancer, carries a large and variable repertoire of sequence-specific DNA methyltransferases. By creating a dozen of single-gene knockout strains for the methyltransferases, we revealed that they form a network controlling methylome, transcriptome and adaptive phenotype sets. The methyltransferases interact with each other in a hierarchical way, sometimes regulated positively by one methyltransferase but negatively with another. Motility, oxidative stress tolerance and DNA damage repair are likewise regulated by multiple methyltransferases. Their regulation sometimes involves translation start and stop codons suggesting coupling of methylation, transcription and translation. The methyltransferases frequently change their sequence-specificity through gene conversion of their target recognition domain and switch their target sets to remodel the network. The emerging picture of a metamorphosing gene regulation network, or firework, consisting of epigenetic systems ever-changing their specificity in search for adaptation, provides a new paradigm in understanding global gene regulation and adaptive evolution.

Horizontal gene transfer (HGT) has been widely suggested to play a critical role in the environmental adaptation of microbes; however, the number and origin of the genes in microbial genomes obtained through HGT remain unknown as the frequency of detected HGT events is generally underestimated, particularly in the absence of information on donor sequences. As an alternative to phylogeny-based methods that rely on sequence alignments, we have developed an alignment-free clustering method on the basis of an unsupervised neural network “Batch-Learning Self-Organizing Map (BLSOM)” in which sequence fragments are clustered based solely on oligonucleotide similarity without taxonomical information, to detect HGT candidates and their origin in entire genomes. By mapping the microbial genomic sequences on large-scale BLSOMs constructed with nearly all prokaryotic genomes, HGT candidates can be identified, and their origin assigned comprehensively, even for microbial genomes that exhibit high novelty. By focusing on two types of Alphaproteobacteria, specifically psychrotolerant Sphingomonas strains from an Antarctic lake, we detected HGT candidates using BLSOM and found higher proportions of HGT candidates from organisms belonging to Betaproteobacteria in the genomes of these two Antarctic strains compared with those of continental strains. Further, an origin difference was noted in the HGT candidates found in the two Antarctic strains. Although their origins were highly diversified, gene functions related to the cell wall or membrane biogenesis were shared among the HGT candidates. Moreover, analyses of amino acid frequency suggested that housekeeping genes and some HGT candidates of the Antarctic strains exhibited different characteristics to other continental strains. Lys, Ser, Thr, and Val were the amino acids found to be increased in the Antarctic strains, whereas Ala, Arg, Glu, and Leu were decreased. Our findings strongly suggest a low-temperature adaptation process for microbes that may have arisen convergently as an independent evolutionary strategy in each Antarctic strain. Hence, BLSOM analysis could serve as a powerful tool in not only detecting HGT candidates and their origins in entire genomes, but also in providing novel perspectives into the environmental adaptations of microbes.

While regulation of gene-enhancer interaction is intensively studied, its application remains limited. Here, we reconstituted arrays of CTCF-binding sites and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. In human induced pluripotent stem cells, STITCH inserted between MYC and the enhancer down-regulated MYC. Progressive mutagenesis of STITCH led to a preferential escalation of the gene-enhancer interaction, corroborating the strong insulation ability of STITCH. STITCH also altered epigenetic states around MYC. Time-course analysis by drug induction uncovered deposition and removal of H3K27me3 repressive marks follows and reflects, but does not precede and determine, the expression change. Finally, STITCH inserted near NEUROG2 impaired the gene activation in differentiating neural progenitor cells. Thus, STITCH should be broadly useful for functional genetic studies.

Summary: Targeted knockin mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knockin. Here, we demonstrate that tandem paired nicking, a method for targeted knockin involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knockin largely equivalent to that of the Cas9-nuclease-based approach. Tandem paired nicking seems to accomplish targeted knockin by DNA recombination analogous to Holliday’s model and creates intended genomic changes without introducing additional nucleotide changes, such as silent mutations. Targeted knockin through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering. : Hyodo et al. describe a method of precise genome editing using Cas9 nickases, which barely introduces unwanted insertions and deletions at the edited genomic loci. This method neither activates nor is inhibited by p53 signalling, allowing safe genome editing and suggesting promise for this method in clinical genome engineering. Keywords: CRISPR/Cas9, nickase, tandem paired nicking, knockin, Holliday’s model, crossover, non-crossover, p53, p21, tandem nicking

With the accumulation of knowledge on the relation between psychological stress and gut microbiota, there is growing interest in controlling stress and/or mood disorders via probiotic supplementation. We aimed to examine the effect of probiotic Bifidobacterium bifidum TMC3115 (TMC3115) supplementation using a sub-chronic and mild social defeat stress murine model in this study. TM3115 supplementation maintained body weight gain and alleviated a polydipsia-like symptom induced by the stress. In the analyses of fecal and cecal bacterial profiles, expansions of Proteobacteria in stressed mice and increases in Actinobacteria and Bifidobacterium in mice supplemented with TMC3115 were observed. There was no marked difference in the diversity of cecal bacteria between the tested mice. Elevated serum levels of inflammatory markers such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 were observed in the stressed mice, while TMC3115 only reduced the IL-6 level. These findings suggest that TMC3115 supplementation confers tolerance to psychosocial stress in the host through modulation of the gut microbiota and alleviation of stress-induced inflammatory responses. Furthermore, it may be expected to exert prevention and treatment of disorders related to peripheral IL-6, including depression.

The harlequin ladybird beetle, Harmonia axyridis, has remarkable phenotypic diversity, with over 200 colour patterns. Here, Ando et al. show that this patterning is regulated by the transcription factor gene pannier and has diversified by repeated inversions and cis-regulatory modifications of pannier.

Abstract Background Contact domains of chromatin serve as a fundamental unit to regulate action of enhancers for target genes. Looping between a pair of CCCTC-binding factor (CTCF)-binding sites in convergent orientations underlies the formation of contact domains, while those in divergent orientations establish domain boundaries. However, every CTCF site is not necessarily engaged in loop or boundary structures, leaving functions of CTCF in varied genomic contexts still elusive. The locus containing Tfap2c and Bmp7 encompasses two contact domains separated by a region between the two genes, termed transition zone (TZ), characterized by two arrays of CTCF sites in divergent configuration. In this study, we created deletion and inversion alleles of these and other regions across the locus and investigated how they impinge on the conformation. Results Deletion of the whole two CTCF arrays with the CRISPR/Cas9 system resulted in impairment of blocking of chromatin contacts by the TZ, as assessed by the circular chromatin conformation capture assay (4C-seq). Deletion and inversion of either of the two arrays similarly, but less pronouncedly, led to reduction in the blocking activity. Thus, the divergent configuration provides the TZ with the strong boundary activity. Uniquely, we show the TZ harbors a 50-kb region within one of the two arrays that contacts broadly with the both flanking intervals, regardless of the presence or orientation of the other CTCF array. Further, we show the boundary CTCF array has little impact on intra-domain folding; instead, locally associating CTCF sites greatly affect it. Conclusions Our results show that the TZ not only separates the two domains, but also bears a wide interval that shows isotropic behavior of chromatin folding, indicating a potentially complex nature of actual boundaries in the genome. We also show that CTCF-binding sites inside a domain greatly contribute to the intra-domain folding of chromatin. Thus, the study reveals diverse and context-dependent roles of CTCF in organizing chromatin conformation at different levels.

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.

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 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.

Abstract In differentiated cells, chromosomes are packed inside the cell nucleus in an organised fashion. In contrast, little is known about how chromosomes are packed in undifferentiated cells and how nuclear organization changes during development. To assess changes in nuclear organization during the earliest stages of development, we quantified the mobility of a pair of homologous chromosomal loci in the interphase nuclei of Caenorhabditis elegans embryos. The distribution of distances between homologous loci was consistent with a random distribution up to the 8-cell stage but not at later stages. The mobility of the loci was significantly reduced from the 2-cell to the 48-cell stage. Nuclear foci corresponding to epigenetic marks as well as heterochromatin and the nucleolus also appeared around the 8-cell stage. We propose that the earliest global transformation in nuclear organization occurs at the 8-cell stage during C. elegans embryogenesis.

In animals, cellularization of a coenocyte is a specialized form of cytokinesis that results in the formation of a polarized epithelium during early embryonic development. It is characterized by coordinated assembly of an actomyosin network, which drives inward membrane invaginations. However, whether coordinated cellularization driven by membrane invagination exists outside animals is not known. To that end, we investigate cellularization in the ichthyosporean Sphaeroforma arctica, a close unicellular relative of animals. We show that the process of cellularization involves coordinated inward plasma membrane invaginations dependent on an actomyosin network and reveal the temporal order of its assembly. This leads to the formation of a polarized layer of cells resembling an epithelium. We show that this stage is associated with tightly regulated transcriptional activation of genes involved in cell adhesion. Hereby we demonstrate the presence of a self-organized, clonally-generated, polarized layer of cells in a unicellular relative of animals.

The prevalence of antibiotic resistance and the decrease in novel antibiotic discovery in recent years necessitates the identification of potentially novel microbial resources to produce natural products. Ktedonobacteria, a class of deeply branched bacterial lineage in the ancient phylum Chloroflexi, are ubiquitous in terrestrial environments and characterized by their large genome size and complex life cycle. These characteristics indicate Ktedonobacteria as a potential active producer of bioactive compounds. In this study, we observed the existence of a putative “megaplasmid,” multiple copies of ribosomal RNA operons, and high ratio of hypothetical proteins with unknown functions in the class Ktedonobacteria. Furthermore, a total of 104 antiSMASH-predicted putative biosynthetic gene clusters (BGCs) for secondary metabolites with high novelty and diversity were identified in nine Ktedonobacteria genomes. Our investigation of domain composition and organization of the non-ribosomal peptide synthetase and polyketide synthase BGCs further supports the concept that class Ktedonobacteria may produce compounds structurally different from known natural products. Furthermore, screening of bioactive compounds from representative Ktedonobacteria strains resulted in the identification of broad antimicrobial activities against both Gram-positive and Gram-negative tested bacterial strains. Based on these findings, we propose the ancient, ubiquitous, and spore-forming Ktedonobacteria as a versatile and promising microbial resource for natural product discovery.

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.

We generated a knock-in mouse line in which the gene encoding brain-derived neurotrophic factor (Bdnf) was replaced with a sequence for proBDNF containing human single nucleotide polymorphisms encoding arginines proximal to the cleavage site (R125M and R127L). The ratio of the mature form of BDNF (mBDNF) to precursor BDNF (proBDNF) in hippocampal tissue lysates was decreased in a manner dependent on the number of copies of the mutant gene, indicating that the mutations inhibited proteolytic conversion of proBDNF into mBDNF. Although homozygous mice had a proBDNF/mBDNF ratio of ~9:1, they survived until adulthood. The levels of mBDNF were reduced by 57% in heterozygous mutant mice, which exhibited a depressive-like behavior in the tail suspension test and weight gain when housed in social isolation, showing that impaired proBDNF cleavage contributes to stress-induced depressive-like phenotypes. Furthermore, socially isolated heterozygous mice displayed a pronounced deficit in daily nest-building behaviors. These findings suggest that the decreased production of mBDNF by impaired proBDNF cleavage disturbs daily activities in mice.

Feeding behavior is closely related to hypothalamic malonyl-CoA level in the brain and diet-induced obesity affects total CoA pools in liver. Herein, we performed a comprehensive analysis of the CoA pools formed in thirteen tissues of Zucker and Zucker diabetic fatty (ZDF) rats. Hypothalamic malonyl-CoA levels in obese rats remained low and were almost the same as those of lean rats, despite obese rats having much higher content of leptin, insulin, and glucose in their sera. Regardless of the fa-genotypes, larger total CoA pools were formed in the livers of ZDF rats and the size of hepatic total CoA pools in Zucker rats showed almost one tenth of the size of ZDF rats. The decreased total CoA pool sizes in Zucker rats was observed in the brown adipose tissues, while ZDF-fatty rats possessed 6% of total CoA pool in the lean rats in response to fa deficiency. This substantially lower CoA content in the obese rats would be disadvantageous to non-shivering thermogenesis. Thus, comparing the intracellular CoA behaviors between Zucker and ZDF rats, as well as the lean and fatty rats of each strain would help to elucidate features of obesity and type 2 diabetes in combination with result (s) of differential gene expression analysis and/or comparative genomics.

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.

Genomic analysis was performed on seven strains of Bifidobacterium thermacidophilum, a Sus-associated Bifidobacterium. Three strains from the feces of domestic pigs (Sus scrofa domesticus) and four strains from the rectal feces of free-range Japanese wild boars (S. s. scrofa) were compared. The phylogenetic position of these isolates suggested by genomic analyses were not concordant with that suggested by 16S rRNA sequence. There was biased distribution of genes for virulence, phage, metabolism of aromatic compounds, iron acquisition, cell division, and DNA metabolism. In particular four wild boar isolates harbored fiber-degrading enzymes, such as endoglucanase, while two of the pig isolates obtained from those grown under an intensive feeding practice with routine use of antimicrobials, particularly tetracycline harbored a tetracycline resistance gene, which was further proved functional by disk diffusion test. The tetW gene is associated with a serine recombinase of an apparently non-bifidobacterial origin. The insertion site of the tetW cassette was precisely defined by analyzing the corresponding genomic regions in the other tetracycline-susceptible isolates. The cassette may have been transferred from some other bacteria in the pig gut.

Objectives: Using ovariectomized rats, we examined the influence of combined exercise tolerance and natto intake on the bone loss inhibitory effect. Methods: We divided female Wistar rats into the following groups: Ovariectomy, Ovariectomy + Exercise, Ovariectomy + Natto Intake, Ovariectomy + Exercise + Natto Intake, and Pseudo-operative (Sham group). After conducting experiments on each group, we collected the tissues and performed morphological and molecular biological analyses. Results: In comparison with the Ovariectomy group, only in the Ovariectomy + Exercise group was there a significant bone loss inhibitory effect in the femoral cancellous bone. Although there was a tendency toward this trend seen in the Natto Intake and Exercise + Natto Intake groups, these differences were not significant. The increase in messenger RNA expression levels of alkaline phosphatase (osteoblast marker) in the bone marrow caused by ovariectomy was suppressed by individual factors, and by those in combination. However, messenger RNA expression levels of estrogen receptor alpha in the bone marrow showed a decreasing tendency with each factor, and decreased significantly with the combination, similar to the Sham group. Conclusion: This suggests that natto intake and exercise maintain bone mass by different molecular mechanisms and that these two factors do not simply act synergistically in combination to maintain bone mass.

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.

The thymus is a lymphoid organ unique to vertebrates, and it provides a unique microenvironment that facilitates the differentiation of immature hematopoietic precursors into mature T cells. We subjected the evolutionary trajectory of the thymic microenvironment to experimental analysis. A hypothetical primordial form of the thymus was established in mice by replacing FOXN1, the vertebrate-specific master regulator of thymic epithelial cell function, with its metazoan ancestor, FOXN4, thereby resetting the regulatory and coding changes that have occurred since the divergence of these two paralogs. FOXN4 exhibited substantial thymopoietic activity. Unexpectedly, histological changes and a functional imbalance between the lymphopoietic cytokine IL7 and the T cell specification factor DLL4 within the reconstructed thymus resulted in coincident but spatially segregated T and B cell development. Our results identify an evolutionary mechanism underlying the conversion of a general lymphopoietic organ to a site of exclusive T cell generation.

We performed mitogenome analysis of Japanese firefly Luciola cruciata (Coleoptera: Lampyridae), which is unique species in Japan. It is classified into six haplotypes based on the difference of COII sequence on mitochondrial DNA. The complete mitogenome sequence of Tohoku group has been registered so far, we newly analysed West Japan groups which belong to different haplotype. The total length of analysed mitogenome was 15,990 bp, being one base longer than the case of Tohoku’s firefly. The base substitution was found at 273 positions over the whole mitochondrial sequence, while amino acid substitution accompanying it was observed at only 11 positions.

We will report the complete mitochondrial genome sequence of Japanese firefly ‘Heike Botaru’, Luciola lateralis (Coleoptera: Lampyridae). Total length of this mitogenome was 16,719 bp and the composition of each base was A (42.50%), C (9.01%), G (14.16%), T (34.33%), respectively. The obtained sequence fulfils general mitogenome composition of metazoan (13 protein coding sequences (CDSs), 22 tRNA genes, two rRNA subunits, and an AT-rich region). From the phylogenetic tree analysis using 25 kinds of insect mitogenome including firefly family was found that L. lateralis is the closest to the genus Aquatica.

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.

In this paper, we report the development, evaluation, and application of ultra-small low-power wireless sensor nodes for advancing animal husbandry, as well as for innovation of medical technologies. A radio frequency identification (RFID) chip with hybrid interface and neglectable power consumption was introduced to enable switching of ON/OFF and measurement mode after implantation. A wireless power transmission system with a maximum efficiency of 70% and an access distance of up to 5 cm was developed to allow the sensor node to survive for a duration of several weeks from a few minutes’ remote charge. The results of field tests using laboratory mice and a cow indicated the high accuracy of the collected biological data and bio-compatibility of the package. As a result of extensive application of the above technologies, a fully solid wireless pH sensor and a surgical navigation system using artificial magnetic field and a 3D MEMS magnetic sensor are introduced in this paper, and the preliminary experimental results are presented and discussed.

Experiments were conducted to investigate the effect of the Bacillus subtilis var. natto fermented soybean (FS) on growth performance, microbial activity in the caeca and cytokine gene expression in spleen of domestic meat type chickens. Chicks were fed commercial diets supplemented with 0 (control), 1 or 2% FS for 80 days from 0 to 80 days of age, and a fourth group of chicks were fed control diet from 0 to 27 days of age, and then fed a 2% FS diet from 28 to 80 days of age. Supplementation with FS did not decrease feed intake compared to chicks fed the control diet, and there were no significant differences in body weight gain and feed efficiency due to dietary treatment. FS supplementation had no effect on carcass weight, breast and thigh meat composition, or abdominal fat deposition. There were no significant differences in pH, NH3-N concentration, or number of microorganisms in the cecum content in each group; however, total acetic acid concentration increased (P

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.

Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

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.

Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed Xist, an imprinted gene, has been considered as a single cis-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than Xist expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs-miR-374-5p and miR-421-3p-mapped adjacent to Xist that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: Jpx, Ftx and Zcchc13. To our knowledge, this is the first identified cluster of imprinted genes in the cis-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.