National BioResource Project Research Articles

Japan Biodiversity Information Initiative (JBIF)'s Efforts to Collect and Publish Biodiversity Information from Japan

The Japan Biodiversity Information Initiative (JBIF) was originally established in 2007 as the Global Biodiversity Information Facility (GBIF) Japan National Node to aggregate biodiversity data in Japan and conduct publications through GBIF. JBIF was later renamed after Japan became a GBIF observer, but activities including data publication through GBIF have continued to the present. JBIF operates with the support of the National BioResource Project (NBRP) by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), with collaboration from three institutions: the National Institute of Genetics (NIG), the National Institute for Environmental Studies (NIES), and the National Museum of Nature and Science (NMNS). The NBRP is a project that focuses on the collection, preservation, provision, and enhancement of bioresources. JBIF collects both observation and specimen data and publishes them through GBIF. For domestic data use, a search system for data published by JBIF is available on the JBIF website. Moreover, NMNS managed a museum network called the Science Museum Net (S-Net), and bilingual (Japanese and English) specimen data collected by S-Net is also available via the S-Net portal site. We are working to promote the biodiversity informatics field in Japan through a translation of the GBIF resources, including the website, important documents such as the GBIF Science Review, as well as organize workshops and conferences, primarily targeting students, researchers, museum curators, and local government officials, to facilitate the sharing of information and exchange of opinions on biodiversity information. To date, Japan has published 564 datasets and over 12 million occurrences to GBIF, making it the third-largest contributor of data to GBIF in Asia, following India and Taiwan. Moreover, regarding specimen-based occurrence data, Japan is the largest contributor in Asia. In this presentation, we will introduce JBIF's initiatives and future activities.

National Bioresource Project and Barley Genetic Resources

文部科学省の補助事業であるナショナルバイオリソースプロジェクトの中核機関の一つとして、岡山大学はオオムギ約2万系統を中心とするリソースの収集・保存・提供を実施している。リソースに関連する来歴、画像などの情報はデータベースとして公開しており、これらのリソースは国内外の研究・教育のために国内外に実費で提供されている。また、提供されたリソースを使用した研究成果は、データベースでの登録閲覧が可能となっている。保存系統はゲノム解読が進められており、配列情報の公開とともに、解読した系統の種子やDNAの入手も可能である。NBRPオオムギは最終的にすべての系統をゲノム解読し、使用したリソースを保存、提供するデジタルジーンバンク化を目指している。

A high-quality severe combined immunodeficiency (SCID) rat bioresource.

Immunodeficient animals are valuable models for the engraftment of exogenous tissues; they are widely used in many fields, including the creation of humanized animal models, as well as regenerative medicine and oncology. Compared with mice, laboratory rats have a larger body size and can more easily undergo transplantation of various tissues and organs. Considering the absence of high-quality resources of immunodeficient rats, we used the CRISPR/Cas9 genome editing system to knock out the interleukin-2 receptor gamma chain gene (Il2rg) in F344/Jcl rats-alone or together with recombination activating gene 2 (Rag2)-to create a high-quality bioresource that researchers can freely use: severe combined immunodeficiency (SCID) rats. We selected one founder rat with frame-shift mutations in both Il2rg (5-bp del) and Rag2 ([1-bp del+2-bp ins]/[7-bp del+2-bp ins]), then conducted mating to establish a line of immunodeficient rats. The immunodeficiency phenotype was preliminarily confirmed by the presence of severe thymic hypoplasia in Il2rg-single knockout (sKO) and Il2rg/Rag2-double knockout (dKO) rats. Assessment of blood cell counts in peripheral blood showed that the white blood cell count was significantly decreased in sKO and dKO rats, while the red blood cell count was unaffected. The decrease in white blood cell count was mainly caused by a decrease in lymphocytes. Furthermore, analyses of lymphocyte populations via flow cytometry showed that the numbers of B cells (CD3- CD45+) and natural killer cells (CD3- CD161+) were markedly reduced in both knockout rats. In contrast, T cells were markedly reduced but showed slightly different results between sKO and dKO rats. Notably, our immunodeficient rats do not exhibit growth retardation or gametogenesis defects. This high-quality SCID rat resource is now managed by the National BioResource Project in Japan. Our SCID rat model has been used in various research fields, demonstrating its importance as a bioresource.

Distinction of Paramecium strains by a combination method of RAPD analysis and multiplex PCR

Paramecium is employed as a valuable model organism in various research fields since a large number of strains with different characteristics of size, morphology, degree of aging, and type of conjugation can be obtained. It is necessary to determine a method for the classification and simple identification of strains to increase their utility as a research tool. This study attempted to establish a polymerase chain reaction (PCR)-based method to differentiate strains of the same species. Genomic DNA was purified from several strains of P. caudatum, P. tetraurelia, and P. bursaria used for comparison by the random amplified polymorphic DNA (RAPD)-PCR method. In P. tetraurelia and P. bursaria, it was sufficiently possible to distinguish specific strains depending on the pattern of random primers and amplification characteristics. For the classification of P. caudatum, based on the sequence data obtained by RAPD-PCR analysis, 5 specific primer sets were designed and a multiplex PCR method was developed. The comparative analysis of 2 standard strains, 12 recommended strains, and 12 other strains of P. caudatum provided by the National BioResource Project was conducted, and specific strains were identified. This multiplex PCR method would be an effective tool for the simple identification of environmental isolates or the management of Paramecium strains.

Collection, preservation and distribution of <i>Oryza</i> genetic resources by the National Bioresource Project RICE (NBRP-RICE)

Biological resources are the basic infrastructure of bioscience research. Rice (Oryza sativa L.) is a good experimental model for research in cereal crops and monocots and includes important genetic materials used in breeding. The availability of genetic materials, including mutants, is important for rice research. In addition, Oryza species are attractive to researchers for both finding useful genes for breeding and for understanding the mechanism of genome evolution that enables wild plants to adapt to their own habitats. NBRP-RICE contributes to rice research by promoting the usage of genetic materials, especially wild Oryza accessions and mutant lines. Our activity includes collection, preservation and distribution of those materials and the provision of basic information on them, such as morphological and physiological traits and genomic information. In this review paper, we introduce the activities of NBRP-RICE and our database, Oryzabase, which facilitates the access to NBRP-RICE resources and their genomic sequences as well as the current situation of wild Oryza genome sequencing efforts by NBRP-RICE and other institutes.

The macroalgal culture collection in Kobe University (KU-MACC) and a comprehensive molecular phylogeny of macroalgae based on the culture strains

ABSTRACT The Kobe University Macroalgal Culture Collection (KU-MACC), as a section of the Kobe University Research Center for Inland Seas, distributes cultures of macroalgae to the public under the umbrella of the National BioResource Project of Ministry of Education, Culture, Sports, Science and Technology and Japan Agency for Medical Research and Development, in collaboration with the Microbial Culture Collection of the National Institute of Environmental Studies. KU-MACC principally holds macroalgae (Phaeophyceae, Rhodophyta and Ulvophyceae), but also holds some important related taxa of Schizocladiophyceae and Charophyceae. They are unialgal strains maintained as vegetative thalli and by cryo-preservation. Stock cultures are grown in culture chambers under various temperature conditions in transparent polypropylene vials containing Provasoli’s Enriched Seawater (PES) or PESI medium. The collection includes the “genome strains” of Ectocarpus siliculosus, Chondrus crispus, Chara braunii and their related stains. Depending on the phylogenetic group, DNA sequences of the nuclear rDNA ITS region, chloroplast rbcL, and mitochondrial cox3 genes are determined and used for taxonomy and as genetic tags. Based on the genetic data, molecular phylogenies of Ulvophyceae, Phaeophyceae and Rhodophyta are presented. In the rbcL molecular phylogeny of Ulvophyceae, due to rather limited coverage of culture strains, only members of Ulothricales, Ulvales and Bryopsidales are represented by KU-MACC strains, whereas 10 orders are recognized in the class. The molecular phylogeny of Phaeophyceae based on the cox3 gene showed a tree topology similar to the published multigene molecular phylogeny except for the position of Ishige okamurae, although the bootstrap supports of most nodes were low: Ishigeales branched near the base of Phaeophyceae next to Discosporangiales in the multigene molecular phylogeny, but in the cox3 tree, it showed a long branch and was sister to Fucales. In the molecular phylogeny of Rhodophyta, 14 orders are represented by KU-MACC strains covering about half of currently recognized Bangiophycean and Florideophycean orders.

Development of introgression lines of AA genome Oryza species, O. glaberrima, O. rufipogon, and O. nivara, in the genetic background of O. sativa L. cv. Taichung 65.

To evaluate and utilize potentially valuable quantitative trait loci or genes of wild relatives in the genetic background of domesticated crop species, chromosome segment substitution lines (CSSLs) are a valuable tool. CSSLs can be constructed through the exchange of chromosome segments of AA genome species of the genus Oryza with cultivated rice, Oryza sativa L. Here we report the development of three sets of CSSLs carrying segments of AA genome species closely related to Oryza sativa—O. glaberrima (IRGC 103777 from Mali), O. rufipogon (W1962 from China), and O. nivara (IRGC 105715 from Cambodia)—in the genetic background of ssp. japonica cultivar Taichung 65 through the use of 101 to 121 simple-sequence-repeat markers in whole-genome genotyping and marker-assisted selection. The materials are available via the National Bioresource Project (Rice) Oryzabase Web page.

Genetic diversity of 21 experimental chicken lines with diverse origins and genetic backgrounds.

The genetic characteristics and diversity of 21 experimental chicken lines registered with the National BioResource Project of Japan were examined using mitochondrial D-loop sequences and 54 microsatellite DNA markers. A total of 12 haplotypes were detected in the 500-bp mitochondrial DNA sequences of the hypervariable segment I for 349 individuals of 21 lines. The 12 haplotypes belonged to three (A, D, and E) haplogroups, out of the eight (A‒H) common haplogroups in domestic chickens and red junglefowls. The haplogroups A and D were widely represented in indigenous chickens in the Asian and Pacific regions, and the haplogroup E was the most prevalent in domestic chickens. Genetic clustering by discriminant analysis of principal components with microsatellite markers divided 681 individuals of 21 lines into three groups that consisted of Fayoumi-, European-, and Asian- derived lines. In each of the cladograms constructed with Nei’s genetic distances based on allele frequencies and the membership coefficients provided by STRUCTURE and with the genetic distance based on the proportion of shared alleles, the genetic relationships coincided well with the breeding histories of the lines. Microsatellite markers showed remarkably lower genetic heterozygosities (less than 0.1 observed heterozygosity) for eight lines (GSP, GSN/1, YL, PNP, BM-C, WL-G, BL-E, and #413), which have been maintained as closed colonies for more than 40 years (except for #413), indicating their usefulness as experimental chicken lines in laboratory animal science research.

A sexually dimorphic peptidergic system in the lower spinal cord controlling penile function in non-human primates.

Experimental animal study. Although a population of gastrin-releasing peptide (GRP) neurons in the lumbar spinal cord has an important role in erection and ejaculation in rats, little information exists on this GRP system in primates. To identify the male-specific GRP system in the primate spinal cord, we studied the lumbosacral cord in macaque monkeys as a non-human primate model. University laboratory in Japan. To determine the gene sequence of GRP precursors, the rhesus macaque monkey genomic sequence data were searched, followed by phylogenetic analysis. Subsequently, immunocytochemical analysis for GRP was performed in the monkey spinal cord. We have used bioinformatics to identify the ortholog gene for GRP precursor in macaque monkeys. Phylogenetic analysis suggested that primate prepro-GRP is separated from that of other mammalian species and clustered to an independent branch as primates. Immunocytochemistry for GRP further demonstrated that male-dominant sexual dimorphism was found in the spinal GRP system in monkeys as in rodents. We have demonstrated in macaque monkeys that the GRP system in the lower spinal cord shows male-specific dimorphism and may have an important role in penile functions not only in rodents but also in primates. Tissues of Nihonzaru (Japanese macaque monkeys) were provided in part by National Institutes of Natural Sciences (NINS) through the National Bio-Resource Project (NBRP) of the MEXT, Japan. This work was supported in part by KAKENHI from the Japan Society for the Promotion of Science (JSPS) (to KT; 15KK0343, 15J40220 and HS; 15K15202, 15KK0257, 15H05724).

CRISPR/Cas9-mediated mutagenesis of the dihydroflavonol-4-reductase-B (DFR-B) locus in the Japanese morning glory Ipomoea (Pharbitis) nil

CRISPR/Cas9 technology is a versatile tool for targeted mutagenesis in many organisms, including plants. However, this technique has not been applied to the Japanese morning glory (Ipomoea [Pharbitis] nil), a traditional garden plant chosen for the National BioResource Project in Japan. We selected dihydroflavonol-4-reductase-B (DFR-B) of I. nil, encoding an anthocyanin biosynthesis enzyme, as the target gene, and changes in the stem colour were observed during the early stages of plant tissue culture by Rhizobium [Agrobacterium]-mediated transformation. Twenty-four of the 32 (75%) transgenic plants bore anthocyanin-less white flowers with bi-allelic mutations at the Cas9 cleavage site in DFR-B, exhibiting a single base insertion or deletions of more than two bases. Thus, these results demonstrate that CRISPR/Cas9 technology enables the exploration of gene functions in this model horticultural plant. To our knowledge, this report is the first concerning flower colour changes in higher plants using CRISPR/Cas9 technology.

ナショナルバイオリソースプロジェクト「ニホンザル」の現状と課題

ナショナルバイオリソースプロジェクト「ニホンザル」の現状と課題

Rat Genome and Model Resources.

Rats remain a major model for studying disease mechanisms and discovery, validation, and testing of new compounds to improve human health. The rat’s value continues to grow as indicated by the more than 1.4 million publications (second to human) at PubMed documenting important discoveries using this model. Advanced sequencing technologies, genome modification techniques, and the development of embryonic stem cell protocols ensure the rat remains an important mammalian model for disease studies. The 2004 release of the reference genome has been followed by the production of complete genomes for more than two dozen individual strains utilizing NextGen sequencing technologies; their analyses have identified over 80 million variants. This explosion in genomic data has been accompanied by the ability to selectively edit the rat genome, leading to hundreds of new strains through multiple technologies. A number of resources have been developed to provide investigators with access to precision rat models, comprehensive datasets, and sophisticated software tools necessary for their research. Those profiled here include the Rat Genome Database, PhenoGen, Gene Editing Rat Resource Center, Rat Resource and Research Center, and the National BioResource Project for the Rat in Japan.

Design and application of a target capture sequencing of exons and conserved non-coding sequences for the rat.

BackgroundTarget capture sequencing is an efficient approach to directly identify the causative mutations of genetic disorders. To apply this strategy to laboratory rats exhibiting various phenotypes, we developed a novel target capture probe set, TargetEC (target capture for exons and conserved non-coding sequences), which can identify mutations not only in exonic regions but also in conserved non-coding sequences and thus can detect regulatory mutations.ResultsTargetEC covers 1,078,129 regions spanning 146.8 Mb of the genome. We applied TargetEC to four inbred rat strains (WTC/Kyo, WTC-swh/Kyo, PVG/Seac, and KFRS4/Kyo) maintained by the National BioResource Project for the Rat in Japan, and successfully identified mutations associated with these phenotypes, including one mutation detected in a conserved non-coding sequence.ConclusionsThe method developed in this study can be used to efficiently identify regulatory mutations, which cannot be detected using conventional exome sequencing, and will help to deepen our understanding of the relationships between regulatory mutations and associated phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2975-9) contains supplementary material, which is available to authorized users.

Transposons (transposable elements), discovered in maize by Barbara McClintock in 1948, are DNA sequences that can change their positions within the genome. Transposition of transposons can introduce mutations in the host genome. Actually, there was a close and astonishing relationship between transposons and the culture of the Edo era. In late Edo, a large number of mutant Asagaos (Japanese morning

Genes to CellsVolume 20, Issue 8 p. i-i Cover ImageFree Access Transposons (transposable elements), discovered in maize by Barbara McClintock in 1948, are DNA sequences that can change their positions within the genome. Transposition of transposons can introduce mutations in the host genome. Actually, there was a close and astonishing relationship between transposons and the culture of the Edo era. In late Edo, a large number of mutant Asagaos (Japanese morning glory) were isolated, and it became popular to cultivate them; in fact, many mutations were caused by transposons. These mutant Asagaos have been collected by researchers and are currently maintained by a group led mainly by Kyushu University. See the website of National BioResource Project (NBRP) “Asagao” (http://www.shigen.nig.ac.jp/asagao/) for further details. Designed by TRAIS Co., Ltd. (Kobe, Japan) First published: 03 August 2015 https://doi.org/10.1111/gtc.12270AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume20, Issue8August 2015Pages i-i RelatedInformation

Inbreeding Ratio and Genetic Relationships among Strains of the Western Clawed Frog, Xenopus tropicalis.

The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB), Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

ナショナルバイオリソースプロジェクト「ニホンザル」の活動について

ナショナルバイオリソースプロジェクト「ニホンザル」の活動について

SABRE2: A Database Connecting Plant EST/Full-Length cDNA Clones with Arabidopsis Information

The SABRE (Systematic consolidation of Arabidopsis and other Botanical REsources) database cross-searches plant genetic resources through publicly available Arabidopsis information. In SABRE, plant expressed sequence tag (EST)/cDNA clones are related to TAIR (The Arabidoposis Information Resource) gene models and their annotations through sequence similarity. By entering a keyword, SABRE searches and retrieves TAIR gene models and annotations, together with homologous gene clones from various plant species. SABRE thus facilitates using TAIR annotations of Arabidopsis genes for research on homologous genes from other model plants. To expand the application range of SABRE to crop breeding, we have recently upgraded SABRE to SABRE2 (http://sabre.epd.brc.riken.jp/SABRE2.html), by newly adding six model plants (including the major crops barley, soybean, tomato and wheat), and by improving the retrieval interface. The present version has integrated information on >1.5 million plant EST/cDNA clones from the National BioResource Project (NBRP) of Japan. All clones are actual experimental resources from 14 plant species (Arabidoposis, barley, cassava, Chinese cabbage, lotus, morning glory, poplar, Physcomitrella patens, Striga hermonthica, soybean, Thellungiella halophila, tobacco, tomato and wheat), and are available from the core facilities of the NBRP. SABRE2 is thus a useful tool that can contribute towards the improvement of important crop breeds by connecting basic research and crop breeding.

126 Establishment of a live young mouse production system using unfertilized oocytes cryopreserved via a simple vitrification method

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97 % of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32% to 49 % of the embryos. Source of funding: This study was supported by a Grant-in-aid for National BioResource Project (NBRP) Fundamental Technologies Upgrading Program. No additional external funding was received for this study. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest: None declared. nakagata@gpo.kumamoto-u.ac.jp

ナショナルバイオリソースプロジェクト「ニホンザル」現状と第3期の展望

ナショナルバイオリソースプロジェクト「ニホンザル」現状と第3期の展望

Genetic and phenotypic characterization of a Japanese wild-derived DOB/Oda rat strain

Wild-derived rat strains can provide novel genome resources that are not available in standard laboratory strains. Genetic backgrounds of wild-derived strains can facilitate effective genetic linkage analyses and often modulate the expression of mutant phenotypes. Here we describe the development and characterization of a new inbred rat strain, DOB/Oda, from wild rats (Rattus norvegicus) captured in Shitara, Aichi, Japan. Phenotype analysis of 109 parameters revealed that the DOB/Oda rats had small body weight, preference for darkness, and high locomotor activity compared with the rat strains in the National BioResource Project for the Rat (NBRP-Rat) database. Genome analysis with 357 SSLP markers identified DOB/Oda-specific alleles in 70 markers. The percentage of SSLP markers that showed polymorphism between the DOB/Oda strain and any of 132 laboratory strains from NBRP-Rat varied from 89 to 95%. The polymorphic rate (average of the values of the percentage) for the DOB/Oda strain was 91.6%, much higher than the rates for available wild-derived strains such as the Brown Norway rat. A phylogenic tree constructed with DOB/Oda and all the strains in NBRP-Rat showed that the DOB/Oda strain localized within the wild rat groups, apparently separate from the laboratory strains. Together, these findings indicated that the DOB/Oda rat has a unique genome that is not available in the laboratory strains. Therefore, the new DOB/Oda strain will provide an important genome resource that will be useful for designing genetic experiments and for the discovery of genes that modulate mutant phenotypes.