National Institute Of Agrobiological Sciences Research Articles

Development of 'Darumadattan', a semidwarf lodging-resistant Tartary buckwheat cultivar, using gamma-ray irradiation.

We developed a new cultivar of Tartary buckwheat (Fagopyrum tataricum Gaertn.), ‘Darumadattan’. This is the first semidwarf Tartary buckwheat cultivar to be developed by mutation breeding using gamma-ray irradiation. In 1999, 100 dry seeds of the leading Japanese cultivar, ‘Hokkai T8’ (known at that time as ‘Hokkei 1’), were gamma-ray-irradiated with a total dose of 500 Gy (25 Gy/h × 20 h) at the Institute of Radiation Breeding (IRB), National Institute of Agrobiological Sciences, Hitachiomiya, Ibaraki, Japan. The seeds were sown in August 1999 in a field at IRB, and M2 seeds were collected from the eight individual plants that survived. In August 2000, 240 M2 seeds were sown in a field, and one semidwarf plant was found. The line named ‘IRBFT-20’ developed from the selected plant was investigated for its semidwarf characteristic and genetic stability in 2001–2005. ‘IRBFT-20’ was submitted for registration in 2011 and registered as the cultivar ‘Darumadattan’ in 2013. This name was chosen because the plants resemble “Daruma dolls” and “dattan” means “Tartary” in Japanese. ‘Darumadattan’ is a highly lodging-resistant and high-yielding cultivar and is expected to be used as both a commercial cultivar and a crossing parent.

Comprehensive panicle phenotyping reveals that qSrn7/FZP influences higher-order branching

Rice grain number directly affects crop yield. Identifying alleles that improve panicle architecture would greatly aid the development of high-yield varieties. Here, we show that the quantitative trait locus qSrn7 contains rice FRIZZY PANICLE (FZP), a previously reported gene encoding an ERF transcription factor that promotes floral transition. Reduced expression of FZP in the reproductive stage increases the extent of higher order branching of the panicle, resulting in increased grain number. Genotype analysis of this gene in cultivars from the publicly available National Institute of Agrobiological Sciences (NIAS) Core Collection demonstrated that the extent of higher order branching, especially in the upper panicle, was increased in those cultivars carrying the FZP allele associated with qSrn7. Furthermore, chromosome segment substitution lines resulting from a cross between Koshihikari and Kasalath, the latter of which carries qSrn7/FZP, also showed that upper panicle higher order branching and grain yield were increased by qSrn7/FZP. Our findings indicate that qSrn7/FZP influences panicle branching pattern and is thus useful in the breeding of high-yield rice varieties.

Clickable silk: novel silk materials containing azide reactive groups

Event Abstract Back to Event Clickable silk: novel silk materials containing azide reactive groups Hidetoshi Teramoto1, Mizuki Sasaki2, Yasushi Tamada2, Kenichi Nakajima1 and Katsura Kojima1* 1 National Institute of Agrobiological Sciences, Genetically Modified Organism Research Center, Japan 2 Shinshu University, Faculty of Textile Science and Technology, Japan Introduction: Silk fibroin protein from the domesticated silkworms, B. mori, has been studied as a candidate for biomaterials development due of its mechanical toughness, biocompatibility, and biodegradability[1]. In this study, a rapid, easy, and flexible method to modify silk fibroin was developed based on the unnatural amino acid mutagenesis methodology of proteins[2]. We previously succeeded in incorporating an unnatural amino acid, 4-azidophenylalanine (AzPhe) (Fig. 1), into silk fibroin by expressing a mutant of phenylalanyl-tRNA synthetase (PheRS) with expanded amino acid recognition capacity in silk glands[3]. The incorporation of AzPhe has made silk fibroin “clickable”: desired functional molecules could be attached to it by azide-specific click reactions[2]. In this paper, the fabrication of materials from clickable silk and their functionalization by click reactions were investigated. Photo-patternability of clickable silk materials was also investigated. Materials and Methods: Fig. 1 illustrates the preparation of clickable silk materials: AzPhe mixed in a commercially-available standard diet was administered to transgenic silkworms (5th instar) until they started to spin cocoons. The harvested cocoons were then processed into threads, films, and porous sponges with established techniques[4]. Results and Discussion: Clickable silk materials (threads, films, and porous sponges) were subjected to click reactions with carboxyrhodamine 110 alkyne (green fluorescence) or sulforhodamine B DBCO (red fluorescence) (Fig. 2). In any forms, the clickable silk materials were distinctly modified by the fluorescent molecules, thus demonstrating the high specificity of click reactions toward azide groups. We attributed the weak fluorescence observed for normal silk materials to non-specific binding of the reagents. Since azide groups in AzPhe are known to exhibit photolysis upon UV irradiation, we expected that the reactivity of azide groups would be geometrically controlled on clickable silk materials. Clickable silk films (cut to 7 × 7 mm2), irradiated with 365 nm UV light for 1 h through micropatterned photomasks, were reacted with carboxyrhodamine 110 alkyne (green fluorescence) or sulforhodamine B DBCO (red fluorescence) (Fig. 3). The micropatterns on the photomasks were successfully transferred to clickable silk films, demonstrating that desired functional molecules could be directly patterned onto clickable silk materials on a micrometer scale. Conclusions: Clickable silk materials with photo-patternability were created by the incorporation of an unnatural amino acid, AzPhe, into silk fibroin. They were easily modified by click reactions, suggesting that they could be a versatile platform on which to produce functionalized biomaterials for wide applications. This study also demonstrates the potential of the unnatural amino acid mutagenesis of proteins to generate novel types of protein-based biomaterials. This work was supported by JSPS KAKENHI Grant Numbers 24688008,15K07800.

A pregnancy case report of alagille syndrome; expression of NOTCH signaling in the placenta

A pregnancy case report of alagille syndrome; expression of NOTCH signaling in the placenta

Spikelet Sterility is Associated with a Lack of Assimilate in High‐Spikelet‐Number Rice

New rice (Oryza sativa L.) genotypes bearing a high spikelet number (HS) occasionally cannot show their high yield potentials due to the lower grain‐filling rate. Our objectives were to evaluate the effect of spikelet sterility on grain filling in HS cultivars and to determine whether the assimilation factor around anthesis is related to spikelet sterility. Two japonica × indica and one japonica × javanica HS cultivars were grown and compared with single‐standard indica and japonica rice cultivars. Lower spikelet fertility (higher sterility) was accompanied by a lower filling percentage of spikelets (observed/potential grain yield) across cultivars and within the HS cultivars bearing different spikelet numbers as a function of N fertilizer treatments. To increase assimilate supply into the whole or lower part of the panicle, plant density was halved at heading or the upper half of the panicle was removed during the middle‐heading period. The halved density, and the removal of the upper panicle in particular, significantly increased spikelet fertility by 1.3 to 1.5 times in the lower part of the panicle in the HS cultivars. The spikelet fertility in HS cultivars could easily be decreased under adverse conditions of assimilation around flowering. The removal of the upper panicle in the HS cultivars significantly increased by 1.3 times the pollen count on the pistil in the low panicle position. These results suggest that spikelet sterility in the lower part of the panicle lowers grain‐filling in the HS cultivars and that inadequate assimilate supply around flowering results in spikelet sterility through a fault in the pollination process.

Systems for making NIAS Core Collections, single-seed-derived germplasm, and plant photo images available to the research community

The National Institute of Agrobiological Sciences (NIAS) Genebank coordinates the conservation of plant, microorganism, and animal genetic resources related to food and agriculture in Japan. It also coordinates the distribution of genetic resources in the public domain for research, breeding, and educational purposes. To operate the NIAS Genebank efficiently, we have developed a genetic resources database, data management software, and web-based data retrieval systems to make the data available worldwide. This article describes the NIAS Genebank’s Core Collections of global and Japanese soybean (Glycine max (L.) Merr.), Japanese azuki bean (Vigna angularis (Willd.) Ohwi et Ohashi), and Japanese wheat (Triticum aestivum L. s. l.), all of which are available through the Genebank website. This article also describes new features of the NIAS Genebank database, such as the ability to select single-seed-derived germplasm of soybean in the plant search system and to download photographic data on accessions. By using the downloaded plant image PDF files, users can obtain detailed passport and agronomic information by clicking on the image of an accession of interest.

Genebank data-management software incorporating seed-viability test results

The National Institute of Agrobiological Sciences' (NIAS) Genebank is the coordinating institute in Japan for the conservation of plant, microorganism and animal genetic resources related to food and agriculture. The NIAS Genebank manages this activity in collaboration with sub-banks located across Japan and acts as the central bank. It is important for genebanks to maintain the viability of genetic resources during conservation. Generally, seeds are stored at low temperature and low humidity. Germination percentage is used to monitor seed viability. We have developed data-management software to manage germination testing data in the NIAS Genebank. The software can monitor the pattern of the loss of seed viability of each accession and also support the selection of accessions that need regeneration of seeds. Web-based plant accessions search software displays the latest data on the availability and germination percentages of accessions.

Mapping and validation of QTLs for rice sheath blight resistance

Sheath blight, caused by Rhizoctonia solani, is one of the most serious diseases of rice. Among 33 rice accessions, mainly from National Institute of Agrobiological Sciences (NIAS) Core Collection, we found three landraces from the Himalayas—Jarjan, Nepal 555 and Nepal 8—with resistance to sheath blight in 3 years’ field testing. Backcrossed inbred lines (BILs) derived from a cross between Jarjan and the leading Japanese cultivar Koshihikari were used in QTL analyses. Since later-heading lines show fewer lesions, we used only earlier-heading BILs to avoid association with heading date. We detected eight QTLs; the Jarjan allele of three of these increased resistance. Only one QTL, on chromosome 9 (between markers Nag08KK18184 and Nag08KK18871), was detected in all 3 years. Chromosome segment substitution lines (CSSLs) carrying it showed resistance in field tests. Thirty F2 lines derived from a cross between Koshihikari and one CSSL supported the QTL.

New Sources of Resistance to Cadophora gregata f. sp. adzukicola and Fusarium oxysporum f. sp. adzukicola in Vigna spp.

Adzuki bean brown stem rot (BSR), caused by Cadophora gregata f. sp. adzukicola, and adzuki bean Fusarium wilt (AFW), caused by Fusarium oxysporum f. sp. adzukicola, are serious problems in Hokkaido, Japan, and have been managed using cultivars with multiple resistance. However, a limited number of adzuki bean varieties are resistant to these pathogens because of the frequent appearance of new races; thus, new sources of resistance have been sought in related Vigna spp., particularly in the section Angulares to which adzuki bean belongs. An analysis of selected Vigna accessions (JP81231 to JP235420) conserved in the Genebank of the National Institute of Agrobiological Sciences, Tsukuba, Japan, revealed wide variation in resistance spectra and resistance combinations, and eight disease response groups (A to H) were identified. Four of eight were newly detected response groups, suggesting the existence of novel resistance genes. Of 252 accessions from 26 species, 28 accessions in Vigna angularis var. nipponensis, V. hirtella, V. minima, and V. tenuicaulis (section Angulares) from group D, which were cross-compatible with adzuki bean, are expected to be potential sources of multiple resistance genes. They were resistant to all races of BSR and AFW pathogens examined.

Development of a Database of Plant Diseases in Japan and a System for Making Microorganism Genetic Resources and their DNA Sequence Data Available to the Research Community

A database of plant diseases reported in Japan and a Web-based data-retrieval system to search for information stored in the database have been developed. The retrieval system is linked with the database on microorganism genetic resources registered in the Genebank Project of the National Institute of Agrobiological Sciences (the NIAS Genebank), which provides access to detailed genetic information on strains of these microorganisms. The NIAS Genebank manages the registration of so-called “passport” data for these genetic resources and controls their storage to make samples available to researchers worldwide. Data management software has been developed to register the receipt, storage, testing, and multiplication of these genetic resources. Search software has also been developed to seek suitable genetic resources based on several search criteria to facilitate their distribution from the stocks stored in the NIAS Genebank. DNA sequence data for the D1/D2, 18S, and internally transcribed spacer ITS (ITS1-5.8S-ITS2) regions of rDNA and for the coding region of the β-tubulin gene for many of the conserved microorganisms can be accessed from the NIAS Genebank Web site. The database on Japanese plant diseases and the NIAS Genebank search system are expected to become increasingly useful tools for research and education related to microorganisms and their genetic data, as well as for improving plant protection and food security.

Classification of Genotypes of the Target Leaf Spot‐Resistant Gene (ds1) in a Sorghum Collection

Sorghum [Sorghum bicolor (L.) Moench] is the fifth most important cereal grown worldwide and is especially important in the semiarid tropics because of its tolerance of hot and dry environments. In addition, it has recently been used in biomass production due to its rapid summer growth in temperate zones. One sorghum disease called target leaf spot is caused by the necrotrophic fungus Bipolaris sorghicola (Lefebvre & Sherwin) Alcorn. Resistance to target leaf spot is controlled by a recently identified single recessive resistant gene (ds1). We studied the susceptibility of sorghum to target leaf spot and the polymorphism of ds1 and the geographic distribution of its alleles using selected specimens from a sorghum core collection. Among 86 lines in the National Institute of Agrobiological Sciences (NIAS) Genebank, 34 (39.6%) were susceptible to target leaf spot, each characterized by one of three alleles (S1, S2, and S3 types). The remaining 52 (60.4%) were resistant, with each characterized by one of four alleles (R1, R2, R3, and R4 types). Two new alleles of ds1, namely, the S3 type and the R4 type, were found in this study. The new alleles of ds1 both encoded mature protein. Both lines had an insertion in a promoter region, with one susceptible to target leaf spot and the other resistant, according to the assumption that the insertion altered transcriptional activity. Geographically, both susceptible and resistant alleles were found in African and South Asian lines. Only resistant alleles were found in the lines from East Asia, suggesting either a bottleneck effect or strong selective pressure by target leaf spot in East Asia. With these DNA markers, the susceptibility of sorghum to target leaf spot can be easily classified.

Nucleotide sequence variation in mitochondrial COI gene among 147 silkworm (Bombyx mori) strains from Japanese, Chinese, European and moltinism classes

We characterized the nucleotide sequences of PCR-amplified mitochondrial COI fragments of 147 silkworm (Bombyx mori) strains that have been maintained in the National Institute of Agrobiological Sciences. Coding sequences (714 bp) of the 147 COI fragments were classified into eight haplotypes based on nucleotide differences at eight segregating sites. No length variation was identified in this region. The 5'-noncoding region showed different features, wherein changes in the number of Ts in the T-stretch, together with two base substitutions, were observed. As a result, the 147 COI noncoding sequences were classified into six haplotypes. Combining the coding and noncoding regions, we identified 14 haplotypes. One of the 14 haplotypes, Hap1A was exclusively abundant in the Japanese native strain class, while this haplotype was less frequent in the other three native strain classes. This finding suggests that the Japanese strain class underwent significant genetic differentiation from the Chinese, European, and moltinism classes, when the each class is regarded as a population. Comparison of the nucleotide sequences to those of B. mandarina (which inhabits Japan) revealed changes that are significantly larger than those within either B. mori or B. mandarina. Furthermore, we detected no common haplotypes between them, which suggests the concept of suppressed gene flow between the two species.

DDBJ progress report

The DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp) provides a nucleotide sequence archive database and accompanying database tools for sequence submission, entry retrieval and annotation analysis. The DDBJ collected and released 3 637 446 entries/2 272 231 889 bases between July 2009 and June 2010. A highlight of the released data was archive datasets from next-generation sequencing reads of Japanese rice cultivar, Koshihikari submitted by the National Institute of Agrobiological Sciences. In this period, we started a new archive for quantitative genomics data, the DDBJ Omics aRchive (DOR). The DOR stores quantitative data both from the microarray and high-throughput new sequencing platforms. Moreover, we improved the content of the DDBJ patent sequence, released a new submission tool of the DDBJ Sequence Read Archive (DRA) which archives massive raw sequencing reads, and enhanced a cloud computing-based analytical system from sequencing reads, the DDBJ Read Annotation Pipeline. In this article, we describe these new functions of the DDBJ databases and support tools.

NIASGBdb: NIAS Genebank databases for genetic resources and plant disease information

The National Institute of Agrobiological Sciences (NIAS) is implementing the NIAS Genebank Project for conservation and promotion of agrobiological genetic resources to contribute to the development and utilization of agriculture and agricultural products. The project’s databases (NIASGBdb; http://www.gene.affrc.go.jp/databases_en.php) consist of a genetic resource database and a plant diseases database, linked by a web retrieval database. The genetic resources database has plant and microorganism search systems to provide information on research materials, including passport and evaluation data for genetic resources with the desired properties. To facilitate genetic diversity research, several NIAS Core Collections have been developed. The NIAS Rice (Oryza sativa) Core Collection of Japanese Landraces contains information on simple sequence repeat (SSR) polymorphisms. SSR marker information for azuki bean (Vigna angularis) and black gram (V. mungo) and DNA sequence data from some selected Japanese strains of the genus Fusarium are also available. A database of plant diseases in Japan has been developed based on the listing of common names of plant diseases compiled by the Phytopathological Society of Japan. Relevant plant and microorganism genetic resources are associated with the plant disease names by the web retrieval database and can be obtained from the NIAS Genebank for research or educational purposes.

2D-DIGE analysis of rice proteins from different cultivars

The 2D-DIGE (2-Dimensional Fluorescence Difference Gel Electrophoresis) method was applied to proteomic phenotyping of natural variants in 10 varieties of rice (Nipponbare, Koshihikari, Sasanishiki, Akitakomachi, Hitomebore, Hinohikari, Kasalath, Rexark, Bleiyo, Cho-ko) from the world rice collection (WRC) in the GenBank of the National Institute of Agrobiological Sciences (NIAS), Japan. Salt-soluble protein extracts of Nipponbare brown rice were labeled with Cy2 fluorochrome and used as an internal standard. Protein extracts from nine other rice varieties were labeled with Cy3 or Cy5 fluorochrome and applied to 2D-PAGE (13 cm gel length) analysis. Approximately 700 spots of rice proteins were observed. Fluorescence intensities of each of these spots for the nine rice varieties were expressed as relative ratios to that of Nipponbare. Statistical analysis revealed the spot numbers of five Japanese rice varieties above threshold five (relative ratio of each variety to Nipponbare exceeded 5-fold or was less than 1/5) to be less than three, while those of four varieties from other countries were more than five (especially, Kasalath which was 29 and Bleiyo which was 23). The 2D-DIGE method seems to be useful for analyzing natural varieties of different cultivars and also for comparing the expression of allergen proteins.

Consequences of ex situ Conservation on the Genetic Integrity of Germplasm Held at Different Gene Banks: A Case Study of Bread Wheat Collected in Pakistan

ABSTRACTGenetic diversity and genetic integrity were tested for wheat (Triticum aestivum L.) landraces conserved in two gene banks with considerably different germplasm management systems. We identified two sets of 17 wheat accessions derived from identical sources collected in Pakistan, which were later deposited at the National Institute of Agrobiological Sciences, Japan (NIAS) and the Plant Genetic Resources Program, Pakistan (PGRP). Regeneration of the conserved germplasm was infrequent at both gene banks. Amplified fragment length polymorphism (AFLP) profiles did not indicate prominent changes in the banding patterns of the wheat landraces conserved at the two gene banks. No significant differences were observed in allele frequency of the detected loci in comparison to the original collections (materials preserved without regeneration and multiplication), except in one accession at NIAS. We conclude that genetic diversity was conserved at these two gene banks with different management systems. However, among the 161 scored loci, 26 AFLP bands disappeared at the same loci in NIAS and PGRP accessions, four disappeared only in NIAS accessions, and 28 only in PGRP accessions. At one particular locus, the band disappeared in 12 PGRP accessions but not in NIAS accessions. The results indicate that unique selection might occur at PGRP and that regeneration strategies to reduce it will be needed in the future.

Retrospective Dosimetry Using Violet Thermoluminescence from Natural Quartz in Soil

The violet thermoluminescence (VTL)-retrospective dosimetry was developed and was then confirmed to be effectual method in emergency situation using quartz extracts from surface soil collected around tokai, Ibaraki, Japan. All of the measurements were undertaken by VTL measurement condition using modified single aliquot regenerative-dose protocol.An on-line automatic spectrometric system installed with a small spectrometer and a CCD camera unit was developed for weak and changeable TL-spectrum measurement with rising temperature for application to natural quartz. From spectrometry of the TL, two emission peaks, consisting of 400 nm and 620 nm, were revealed. In this paper, 400 nm were only investigated.The accumulated γ-doses of quartz extracts from surface soils which were 10 m from radiation source at gamma field at the Institution of Radiation Breeding (IRB), National Institute of Agrobiological Sciences (NIAS), were evaluated to be 0.8~0.9 Gy using VTL measurement, concordant with the value measured by the commercial pocket dosimeter. In addition, the dose of quartz extracted from surface soils set at 20 m was evaluated to be 0.2 Gy, agreeable to the value calculated from the pocket dosimeter’s value. Conclusively, the VTL-retrospective dosimetry was recommended for the quartz extracts from surface soils in the emergency situation.

A Novel NAC Transcription Factor, IDEF2, That Recognizes the Iron Deficiency-responsive Element 2 Regulates the Genes Involved in Iron Homeostasis in Plants

Iron is essential for most living organisms, and thus iron deficiency poses a major abiotic stress in crop production. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified a novel transcription factor of rice and barley, IDEF2, which specifically binds to the iron deficiency-responsive cis-acting element 2 (IDE2) by yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets experiment revealed that IDEF2 predominantly recognized CA(A/C)G(T/C)(T/C/A)(T/C/A) within IDE2 as the core-binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by the RNA interference (RNAi) technique and chimeric repressor gene-silencing technology (CRES-T) caused aberrant iron homeostasis in rice. Several genes up-regulated by iron deficiency, including the Fe(II)-nicotianamine transporter gene OsYSL2, were less induced by iron deficiency in the RNAi rice of IDEF2, suggesting that IDEF2 is involved in the regulation of these genes. Many genes with repressed expression in IDEF2 RNAi rice possessed the IDEF2-binding core sites in their promoters, and the flanking sequences were also highly homologous to IDE2. IDEF2 bound to OsYSL2 promoter region containing the binding core site, suggesting direct regulation of OsYSL2 expression. These results reveal novel cis-element/trans-factor interactions functionally associated with iron homeostasis.

Development of mini core collection of Japanese rice landrace

A mini core collection of Japanese rice landrace was developed based on a genome-wide SSR polymorphism survey of 236 accessions. The accessions used in the initial survey were selected based on the historical record and passport data from the collection maintained at the Genebank of the National Institute of Agrobiological Sciences (NIAS). These accessions were analyzed using 32 SSR markers. A total of 247 alleles were detected and the number of alleles per locus ranged from 3 to 15 (mean 7.7). Principal coordinate analysis using SSR data enabled classification of the accessions into three major groups, two Japonica groups and one Indica group. To develop a Japanese landrace mini core collection, the SSR data on the 236 accessions were subjected to cluster analysis and 50 groups were recognized at a similarity index of 0.88. A single accession from each of the 50 groups was selected. These 50 accessions retained 87.5% of the alleles detected in the original 236 accessions, and covered the variation of the initial set of accessions in terms of several agro-morphological traits.

Evaluation of genotypic variation in leaf photosynthetic rate and its associated factors by using rice diversity research set of germplasm

In order to evaluate genotypic variation, we measured leaf photosynthetic rate (Pn) and its associated factors for the rice diversity research set of germplasm (RDRS) selected from the Genebank in National Institute of Agrobiological Sciences (NIAS). Pn showed large genotypic variation from 11.9 to 32.1 micromol m(-2 )s(-1). The variation in stomatal conductance to CO2 (Gs) explained about 50% of that in Pn, while that in nitrogen concentration (N) in leaves explained about 35%. The genotype group which mainly consists of aus type indica tended to have higher Gs, and the genotype group which corresponds to japonica had a higher nitrogen concentration (N) in leaves. The relationships of Pn with Gs and N were not significantly different among genotype groups, suggesting photosynthetic efficiencies are similar among genotype groups.