Masahiko Kumagai

Genetic diversity and evolutionary relationships in genus Oryza revealed by using highly variable regions of chloroplast DNA.

We studied the phylogeny of the genus Oryza using chloroplast DNA sequences. To identify regions containing sufficient variation for elucidating the relationship of closely related species with fine resolution and high reliability, we first compared the complete chloroplast sequences of Oryza sativa japonica, O. sativa indica, and O. nivara, and identified regions containing many variant sites. Nucleotide sequences of the variant regions were newly determined in 19 Oryza species including 58 cultivated and wild strains. An in silico pre-analysis of the whole chloroplast genome and subsequent nucleotide sequencing of the regions with the greatest number of variant sites, which were disclosed to be hot spots by the in silico pre-analysis, enabled us to examine genetic diversity in the genus Oryza with excellent resolution. Based on phylogenetic trees constructed using highly diverged regions in the chloroplast genome, we discuss the maternal relationships among Oryza species.

TASUKE: a web-based visualization program for large-scale resequencing data

Summary: Because an enormous amount of sequence data is being collected, a method to effectively display sequence variation information is urgently needed. tasuke is a web application that visualizes large-scale resequencing data generated by next-generation sequencing technologies and is suitable for rapid data release to the public on the web. The variation and read depths of multiple genomes, as well as annotations, can be shown simultaneously at various scales. We demonstrate the use of TASUKE by applying it to 50 rice and 100 human genome resequencing datasets. Availability and implementation: The tasuke program package and user manual are available from http://tasuke.dna.affrc.go.jp/. Contact: taitoh@affrc.go.jp

Whole-Genome Analysis of Herbicide-Tolerant Mutant Rice Generated by Agrobacterium-Mediated Gene Targeting

Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20-100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing.

Complete fusion of a transposon and herpesvirus created the Teratorn mobile element in medaka fish

Mobile genetic elements (e.g., transposable elements and viruses) display significant diversity with various life cycles, but how novel elements emerge remains obscure. Here, we report a giant (180-kb long) transposon, Teratorn, originally identified in the genome of medaka, Oryzias latipes. Teratorn belongs to the piggyBac superfamily and retains the transposition activity. Remarkably, Teratorn is largely derived from a herpesvirus of the Alloherpesviridae family that could infect fish and amphibians. Genomic survey of Teratorn-like elements reveals that some of them exist as a fused form between piggyBac transposon and herpesvirus genome in teleosts, implying the generality of transposon-herpesvirus fusion. We propose that Teratorn was created by a unique fusion of DNA transposon and herpesvirus, leading to life cycle shift. Our study supports the idea that recombination is the key event in generation of novel mobile genetic elements.Teratorn is a large mobile genetic element originally identified in the small teleost fish medaka. Here, the authors show that Teratorn is derived from the fusion of a piggyBac superfamily DNA transposon and an alloherpesvirus and that it is widely found across teleost fish.

Centromere evolution and CpG methylation during vertebrate speciation

Centromeres and large-scale structural variants evolve and contribute to genome diversity during vertebrate speciation. Here, we perform de novo long-read genome assembly of three inbred medaka strains that are derived from geographically isolated subpopulations and undergo speciation. Using single-molecule real-time (SMRT) sequencing, we obtain three chromosome-mapped genomes of length ~734, ~678, and ~744Mbp with a resource of twenty-two centromeric regions of length 20–345kbp. Centromeres are positionally conserved among the three strains and even between four pairs of chromosomes that were duplicated by the teleost-specific whole-genome duplication 320–350 million years ago. The centromeres do not all evolve at a similar pace; rather, centromeric monomers in non-acrocentric chromosomes evolve significantly faster than those in acrocentric chromosomes. Using methylation sensitive SMRT reads, we uncover centromeres are mostly hypermethylated but have hypomethylated sub-regions that acquire unique sequence compositions independently. These findings reveal the potential of non-acrocentric centromere evolution to contribute to speciation.Centromeres and large-scale structural variants evolve and contribute to genome diversity during vertebrate speciation. Here Ichikawa et al perform de novo long-read genome assembly of three inbred medaka strains, and report long-range structure of centromeres and their methylation as well as correlation of structural variants with differential gene expression.

Hypomethylated domain-enriched DNA motifs prepattern the accessible nucleosome organization in teleosts

BackgroundGene promoters in vertebrate genomes show distinct chromatin features such as stably positioned nucleosome array and DNA hypomethylation. The nucleosomes are known to have certain sequence preferences, and the prediction of nucleosome positioning from DNA sequence has been successful in some organisms such as yeast. However, at gene promoters where nucleosomes are much more stably positioned than in other regions, the sequence-based model has failed to work well, and sequence-independent mechanisms have been proposed.ResultsUsing DNase I-seq in medaka embryos, we demonstrated that hypomethylated domains (HMDs) specifically possess accessible nucleosome organization with longer linkers, and we reassessed the DNA sequence preference for nucleosome positioning in these specific regions. Remarkably, we found with a supervised machine learning algorithm, k-mer SVM, that nucleosome positioning in HMDs is accurately predictable from DNA sequence alone. Specific short sequences (6-mers) that contribute to the prediction are specifically enriched in HMDs and distribute periodically with approximately 200-bp intervals which prepattern the position of accessible linkers. Surprisingly, the sequence preference of the nucleosome and linker in HMDs is opposite from that reported previously. Furthermore, the periodicity of specific motifs at hypomethylated promoters was conserved in zebrafish.ConclusionThis study reveals strong link between nucleosome positioning and DNA sequence at vertebrate promoters, and we propose hypomethylated DNA-specific regulation of nucleosome positioning.

Rice Varieties in Archaic East Asia: Reduction of Its Diversity from Past to Present Times

The Asian cultivated rice, Oryza sativa, is one of the most important crops feeding more than a third of global population. In spite of the studies for several decades, the origin and domestication history of rice varietal groups, japonica and indica, have not been fully unveiled. Genetic information of ancient rice remains is essential for direct and exclusive insight into the domestication history of rice. We performed ancient DNA analysis of 950- to 2,800-year-old rice remains excavated from Japan and Korea. We found the presence of both japonica- and indica-type varieties in the Yayoi period and the middle ages of Japan and the middle part of Korea Peninsula 2,000 years ago. It is popularly considered that japonica has been exclusively cultivated in northern part of East Asia including Japan and Korea. Our result disclosed unexpectedly wide diversity of rice varieties in archaic East Asia. The present results from ancient rice DNA reveal an exclusive insight for the domestication history of rice which is not provided as far as contemporary rice.

Fusion of piggyBac -like transposons and herpesviruses occurs frequently in teleosts

BackgroundEndogenous viral elements play important roles in eukaryotic evolution by giving rise to genetic novelties. Herpesviruses are a large family of DNA viruses, most of which do not have the ability to endogenize into host genomes. Recently, we identified a novel type of endogenous herpesvirus, which we named “Teratorn”, from the medaka (Oryzias latipes) genome, in which the herpesvirus is fused with a piggyBac-like DNA transposon, forming a novel mobile element. Teratorn is a unique herpesvirus that retains its viral genes intact and has acquired the endogenized lifestyle by hijacking the transposon system. However, it is unclear how this novel element evolved in the teleost lineage and whether fusion of two mobile elements is a general phenomenon in vertebrates.ResultsHere we performed a comprehensive genomic survey searching for Teratorn-like viruses in publicly available genome data and found that they are widely distributed in teleosts, forming a clade within Alloherpesviridae. Importantly, at least half of the identified Teratorn-like viruses contain piggyBac-like transposase genes, suggesting the generality of the transposon-herpesvirus fusion in teleosts. Phylogenetic tree topologies between the piggyBac-like transposase gene and herpesvirus-like genes are nearly identical, supporting the idea of a long-term evolutionary relationship between them.ConclusionWe propose that piggyBac-like elements and Teratorn-like viruses have co-existed for a long time, and that fusion of the two mobile genetic elements occurred frequently in teleosts.

Unlinking the methylome pattern from nucleotide sequence, revealed by large-scale in vivo genome engineering and methylome editing in medaka fish

The heavily methylated vertebrate genomes are punctuated by stretches of poorly methylated DNA sequences that usually mark gene regulatory regions. It is known that the methylation state of these regions confers transcriptional control over their associated genes. Given its governance on the transcriptome, cellular functions and identity, genome-wide DNA methylation pattern is tightly regulated and evidently predefined. However, how is the methylation pattern determined in vivo remains enigmatic. Based on in silico and in vitro evidence, recent studies proposed that the regional hypomethylated state is primarily determined by local DNA sequence, e.g., high CpG density and presence of specific transcription factor binding sites. Nonetheless, the dependency of DNA methylation on nucleotide sequence has not been carefully validated in vertebrates in vivo. Herein, with the use of medaka (Oryzias latipes) as a model, the sequence dependency of DNA methylation was intensively tested in vivo. Our statistical modeling confirmed the strong statistical association between nucleotide sequence pattern and methylation state in the medaka genome. However, by manipulating the methylation state of a number of genomic sequences and reintegrating them into medaka embryos, we demonstrated that artificially conferred DNA methylation states were predominantly and robustly maintained in vivo, regardless of their sequences and endogenous states. This feature was also observed in the medaka transgene that had passed across generations. Thus, despite the observed statistical association, nucleotide sequence was unable to autonomously determine its own methylation state in medaka in vivo. Our results apparently argue against the notion of the governance on the DNA methylation by nucleotide sequence, but instead suggest the involvement of other epigenetic factors in defining and maintaining the DNA methylation landscape. Further investigation in other vertebrate models in vivo will be needed for the generalization of our observations made in medaka.

Establishment of the three-dimensional genome structure in vertebrate early embryos

Global DNA demethylation is a hallmark of embryonic epigenetic reprogramming. Yet, mammalian embryos engage non-canonical DNA methylation-maintenance mechanisms to ensure inheritance of exceptional epigenetic germline features to the soma. Besides the paradigmatic genomic imprints these exceptions remain ill-defined and the mechanisms ensuring demethylation resistance in the light of global reprogramming remain poorly understood. Here we will report how the TRIM28 complex protects genomic imprints from aberrant demethylation during early epigenetic reprogramming. We will further describe how other, non-imprinted genes use this machinery to resist DNA demethylation. In particular, we show that the Y-linked gene Rbmy1a1 is highly methylated in mature sperm and resists DNA demethylation post-fertilization. Aberrant hypomethylation of the Rbmy1a1 promoter results in its ectopic activation causing male-specific periimplantation lethality. Rbmy1a1 is thus a novel target of the TRIM28 complex, which is required to protect its repressive epigenetic state during embryonic epigenetic reprogramming.