The National BioResource Project has been organized and established to promote research activities using valuable bioresources. A total of twenty-eight bioresources for ten animals, nine plants and nine microorganisms/cell lines developed or collected in Japan were selected for the project. Resources are categorized into several different groups in the project; genetic resources, germplasm, genome resources and their information. Choices of how many resources must be preserved and maintained and in which categories are dependent on the status of the research community of each organism. These resources, if utilized systematically and intelligently, are powerful means for leading new scientific discoveries. Some examples can be seen in this paper. This paper reviews plant bioresources with the main focus on rice resource activities within the project.

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Primary and secondary trisomics as well as telotrisomics have been well characterized in rice morphologically and the transmission rates of extra chromosomes have been investigated. Primary trisomics have been utilized for associating genes and linkage groups with respective chromosomes. Secondary trisomics and telotrisomics were employed to associate genes with respective chromosome arms. As a result the positions of centromeres on the linkage maps could be pinpointed. Monosomic alien addition lines (MAALs) have a complete chromosome complement of Oryza sativa and a single chromosome of a wild species in genus Oryza. All the MAALs of O. officinalis and O. punctata have been produced. A partial set of MAALs of 5 other wild species of rice have also been produced. These aneuploid stocks of rice are useful not only for genetic study such as gene dosage analysis but also for rice breeding.

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The efficiency of N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells at the single-cell stage in rice (Oryza sativa L.) was about twice that of dry seeds. The proportion of recessive iso-chlorophyll mutants in the segregating progeny of M₁ plants decreased remarkably with treatment at later stages of embryonic development, perhaps because of the presence of diplontic selection and chimera formation when older, multicellular embryos were mutagenized. Panicle sterility of the M₁ plants showed a linear relationship with the frequency of chlorophyll mutations in the progeny. The mutagenicity of N-ethyl-N-nitrosourea was similar to that of MNU, but N-methyl-N′-nitro-N-nitrosoguanidine and N-ethyl-N′-nitro-N-nitrosoguanidine were less effective. Several thousand rice mutants affecting endosperm, culm length, heading date, and chlorophyll were obtained by MNU mutagenesis. The frequency of certain types of endosperm mutants seemed to differ according to the precise timing of treatment. The mutation rate was calculated as 7.4 × 10⁻⁶ per nucleotide, and the mutations were evenly distributed over the gene regions examined. These results indicate that our rice mutant library, generated by MNU mutagenesis, is a promising resource for identifying mutations in any rice gene.

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Genomic sequencing of rice followed by curated genome annotation predicted that the rice genome carries ~32,000 genes. To reveal functions of all the predicted genes is the next important challenge. One of the most effective ways to discover gene function is to analyze genetic mutations induced by insertion elements. Different insertion elements, such as T-DNA, Ds, dSpm, and the endogenous retrotransposon Tos17, have been utilized in rice and mutant lines carrying more than 1,500,000 insertions produced. In this article, features of Tos17, Tos17-induced mutant lines, related resources such as flanking sequence tags and phenome databases, and functional analysis of genes using these resources are reviewed.

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After the completion of its genomic sequencing, rice (Oryza sativa L.) has become firmly established as the premiere model plant among monocot crops. Various genetic resources have been developed for rice to accelerate the identification and characterization of rice genes with as-yet unknown functions. These resources include collections that carry natural variations and progeny derived from chemical and irradiation mutagenesis. Collections of rice strains with natural or artificial DNA insertions that create loss-of-function mutations have been of great importance for gene discovery and tagging. However, it is often difficult to observe mutant phenotypes for functionally duplicated genes and genes essential for plant growth and development. To overcome this disadvantage, gain-of-function strategies have been developed, including activation tagging and full-length cDNA overexpressing gene (FOX)-hunting systems. In this review, we summarize the current status and discuss the advantages and disadvantages of the gain-of-function approaches, and then, perspectives on accelerating discovery of rice genes.

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The destruction of natural habitats of wild plant species is accelerating due to environmental changes and various human activities. This situation requires sustained and extensive efforts to preserve and safeguard wild species. The National Institute of Genetics (NIG), Mishima, Japan, maintains 1,701 accessions of 20 wild species of the genus Oryza, which have been collected from tropical and subtropical areas all over the world. This paper reviews the history, biodiversity, ex-situ conservation, and utility of the NIG wild Oryza collection. In addition, we describe our recent activities to improve the reliability of the collection and our contributions as part of the National BioResource Project (NBRP) of Japan. This project aims to provide reliable and high-quality rice materials for researchers worldwide. As part of the NBRP, we have evaluated wild Oryza accessions for eighteen morphological or physiological traits of vegetative and reproductive organs and for resistance to bacterial blight disease, developed molecular markers to distinguish species and/or genome-species in the genus, and published the information on the Oryzabase database.

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Genetic dissection of a wide range of naturally occurring variations in rice has significantly progressed by means of quantitative trait locus analysis. This genetic dissection has resulted in molecular cloning of genes and loci with biological and agronomic interest. The success of these analyses depends strongly on the plant materials used. In the last decade, many kinds of plant materials, and particularly advanced backcross populations, have been developed for the genetic analysis of traits of interest. Some of those materials have been deposited in the public domain in order to facilitate further analyses of rice genetics and molecular biology. In this review, we describe how such plant materials, including chromosome segment substitution lines (CSSLs) and introgression lines, could be used in genetic analysis, as well as the kinds of plant materials that could be developed and that are now available to the rice research community. Furthermore, we introduce our current activities related to large-scale development of CSSLs using diverse Asian rice accessions as donors.

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The genus Oryza has 24 species out of which two are cultivated (O. sativa and O. glaberrima) and 22 are wild species. Of the 22 wild species, six are in the primary gene pool of O. sativa complex and these wild species are easily crossable with the major cultivated species. These have the same AA genome as O. sativa. However, there are 10 wild species under O. officinalis complex having BB, CC, BBCC, CCDD, EE and FF genomes. The wild species of this complex are in the secondary gene pool and are cross incompatible with O. sativa. There are six most distantly related wild species with either diploids or tetraploids of GG, HHJJ and HHKK genomes and are highly cross incompatible with O. sativa. All the 22 wild species of Oryza are a vast reservoir of genes for biotic and abiotic stresses resistance. Some of the yield enhancing traits/genes from AA genome wild species have been identified and mapped with molecular markers for their integration into O. sativa genome. A broad-spectrum resistance gene for bacterial blight resistance (Xa21) has been identified in O. longistaminata and introduced into many rice cultivars. Advances in biotechnology have facilitated the development of interspecific hybrids between O. sativa and wild species of secondary and tertiary gene pools. Some important genes Pi40 and Bph18 for resistance to blast and brown planthopper, respectively, have been successfully transferred into elite cultivars from O. australiensis and the function of one blast resistance gene (Pi9) derived from O. minuta is elucidated. Many important genes from the most distantly related wild species such as O. alta, O. granulata, O. longiglumis and O. coarctata are expected to be transferred into cultivated rice in the future using the latest tools of molecular genetics and biotechnology.

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Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in eukaryotic genomes. SNPs may be functionally responsible for specific traits or phenotypes, or they may be informative for tracing the evolutionary history of a species or the pedigree of a variety. As genetic markers, SNPs are rapidly replacing simple sequence repeats (SSRs) because they are more abundant, stable, amenable to automation, efficient, and increasingly cost-effective. The integration of high throughput SNP genotyping capability promises to accelerate genetic gain in a breeding program, but also imposes a series of economic, organizational and technical hurdles. To begin to address these challenges, SNP-based resources are being developed and made publicly available for broad application in rice research. These resources include large SNP datasets, tools for identifying informative SNPs for targeted applications, and a suite of custom-designed SNP assays for use in marker-assisted and genomic selection, association and QTL mapping, positional cloning, pedigree analysis, variety identification and seed purity testing. SNP resources also make it possible for breeders to more efficiently evaluate and utilize the wealth of natural variation that exists in both wild and cultivated germplasm with the aim of improving the productivity and sustainability of agriculture.

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Rice was the first crop to have a high-quality reference genome sequence and is now at the forefront of intense functional and evolutionary research for two reasons—its central role in world food security, and its status as a model system for grasses. A thorough characterization of the rice genome cannot be accomplished without a deep understanding of its evolutionary history. The genus Oryza contains two cultivated and 22 wild rice species that represent 10 distinct genome types embedded within a robust phylogeny spanning a ~15 million year time span. The genus contains an untapped reservoir of agriculturally important traits and a historical record of genomic changes (especially those related to domestication, polyploidy, speciation and adaption).The two main objectives of the ‘Oryza Map Alignment Project’ (OMAP) were to functionally characterize the rice genome from a comparative standpoint and to provide essential tools to leverage the novel genetic diversity from wild relatives for rice improvement. The objective of this review is to summarize our efforts towards developing the most comprehensive genus-wide set of publicly available BAC resources for the genus Oryza, the first of its kind among plants (and perhaps higher eukaryotes), and their applications.

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Oryzabase contains 16,000 biological resources, including wild rice, mutants, and experimental strains, together with 4000 trait genes collected from journal articles. The database also contains genetic and genomic maps, and 20,000 items from the published literature. Of these items, the most valuable are the biological resources, particularly the wild rice and mutant collections, because they have been thoroughly characterized by researchers and are accompanied by large quantities of data. The next most valuable content is the trait gene dictionary, which has been maintained continuously since 1995 and is based on published literature. Oryzabase also plays a role as an international site for the submission of rice genes. Besides maintaining original data, Oryzabase compiles genomic data that are systematically integrated with existing data holdings and provided with a user-friendly interface. Oryzabase can be accessed at http://www.shigen.nig.ac.jp/rice/oryzabase/.

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Bioinformatics tools and databases have become integral parts of scientific researches. With the global efforts in structural and functional characterization of the rice genome, rice is now at the forefront of cereal genomics with a robust informatics infrastracture that provides fundamental information for understanding the biology of rice and other cereal crops. A wide range of resources encompassing genome sequence data, genetic maps, molecular markers, insertional mutations, gene expression profiles, proteome, and the integration of these genomics data have become readily accessible in online databases providing various types of information that can be used for complete understanding of rice biology. The ultimate application of these genomics information and resources is widely reflected in efforts aimed at elucidating agronomic traits with important ramifications in breeding strategies.

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Rice is unique among plants in that it accumulates major amounts of two types of seed storage proteins, prolamines and glutelins, which along with starch, serve as sources of carbon and nitrogen for the post-germinative seedling. Here, we investigate the role of the small GTPase Rab5 in the biosynthesis of storage proteins through characterization of the loss-of-function mutant glup4. In situ RT-PCR and fluorescence microscopy revealed the glup4 mutant is impaired in glutelin mRNA localization to the cortical endoplasmic reticulum in developing endosperm cells. Furthermore, microarray and two-dimensional difference in gel electrophoresis analysis of transcript and protein abundance, respectively, identified a number of genes whose expression is affected by the loss of Rab5, including starch biosynthetic enzymes. Our results indicate Rab5 is important for RNA and protein sorting in rice seed and supports previous observations that the biosynthesis of starch and storage proteins may be interrelated.

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We had previously identified eight mutants, esp2 and g(G)lups1 to 7, which accumulated abnormally high amounts of proglutelin, the major storage protein in rice seeds. Analysis of their seed proteins by SDS-PAGE, their levels of the luminal chaperone BiP and gene-gene interactions indicated that these mutants fell into four classes. The most epistatic class consisted of esp2, which encodes a defective protein disulfide isomerase (PDI). A second class consisting of Glup1, glup2 and glup7 was hypostatic to esp2, and showed abnormally high levels of BiP, suggesting that maturation and export of proglutelins from the ER are inhibited in this class of mutants. The third class containing glup4, Glup5 and glup6 mutations was hypostatic to esp2, Glup1, glup2 and glup7. Since the glup4 allele encodes the small GTPase Rab5a, which participates in the trafficking of proglutelin from Golgi apparatus to the protein storage vacuole (PSV), this third class of mutants is likely affected in this process. Lastly, glup3, which encodes a vacuolar processing enzyme, which proteolytically processes proglutelin into acidic and basic subunits within the PSV, was hypostatic to the other mutants. Overall, these gene relationships are consistent with the sequential intracellular transport and processing of proglutelin and provide novel insights on the trafficking of proglutelin to the PSV.

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We applied the full-length cDNA overexpressor (FOX) gene-hunting system for systematic and genome-wide functional analysis of rice genes. In this study, we constructed a novel binary vector carrying the Gateway site-specific recombination cassette and then constructed rice FOX libraries containing a maximum of 13,823 independent, full-length cDNAs (fl-cDNAs) that correspond to approximately half the total number of rice fl-cDNA clones. By introducing the FOX libraries via Agrobacterium, we generated 2,586 FOX-rice lines exhibiting various visible alterations (e.g., plant height, tillers, leaves, and heading dates). The introduced fl-cDNAs, integrated into individual transgenic rice genomes, were amplified by genomic PCR and identified using sequencing analysis. The fl-cDNAs were PCR-amplified in 2,251 (94.2%) of the 2,389 FOX-rice lines that were examined, identifying 1,920 independent fl-cDNAs in the FOX lines. In addition to the previously generated FOX-rice plants, our new collection of FOX-rice lines produced through the Gateway system should be a useful tool for the efficient identification of gene functions in rice. Moreover, this Gateway-based technology should be applicable to other species in which a collection of fl-cDNA clones is available.

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Panicle branchiness is one of the key characters specifying rice yield. To understand the potential of panicle branchiness, the diversity of panicle branching patterns was investigated in 43 accessions of 19 wild Oryza species and 27 accessions of O. rufipogon, which is the wild ancestor of the Asian cultivated species O. sativa. All these accessions were selected from a core collection of wild relatives of rice maintained at the National Institute of Genetics (NIG), Mishima, Japan. We found that panicles of wild Oryza species closely related to O. sativa generated primary branches with homogeneously large numbers of secondary branching organs, resulting in a truncated conical type of branching pattern. In contrast, panicles of distantly related wild species had primary branches with acropetally declining numbers of secondary organs toward the distal end of panicles, resulting in a conical type of branching pattern. The main axis meristem, which was aborted in closely related species, was converted into a terminal spikelet in distantly related species. Our data suggested that the panicle branching pattern evolved from conical to truncated conical in the genus Oryza. The possible advantage of a truncated-conical type of branching pattern for rice yield will be discussed.

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To broaden the available rice genetic resources, we developed two populations of introgression lines of cultivated rice (Oryza sativa) carrying donor segments from the wild species Oryza glumaepatula and Oryza meridionalis. These lines contain overlapped introgressed donor segments that covered most parts of the genome of the two donors in the same of O. sativa genetic background (ssp. japonica, cv. Taichung 65). The introgression lines were developed through repeated backcrossing with Taichung 65 as a pollen parent and marker assisted selection. O. glumaepatula introgression lines consist of two sets of the lines (with O. glumaepatula and Taichung 65 cytoplasm, respectively): these comprise a total of 69 lines that cover 79.5 to 89.2% of the Oryza glumaepatula genome. The O. meridionalis introgression lines also consist of two sets of the lines (with O. meridionalis and Taichung 65 cytoplasm, respectively): these comprise a total of 78 lines covering the 81.5 to 98.0% of the Oryza meridionalis genome. These introgression lines significantly broaden rice genetic resources, and will facilitate analyses of the genetics of traits specific to the donor species.

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Wild relatives of cultivated rice (Oryza sativa L.) are useful sources of alleles with economic value for rice breeding programs. To effectively identify such alleles, three sets of introgression lines (ILs) carrying one or more chromosome segments derived from wild relatives with the A genome in the background of japonica rice cultivars Koshihikari and Itadaki were developed using marker-assisted backcrossing. The donor wild species include two accessions of O. rufipogon (IRGC Acc. 104814 and 104812) and one accession of O. glumaepatula (IRGC Acc. 100968) while 2 japonica rice cultivars, Koshihikari and Itadaki, were used as recurrent parents. The two sets of ILs from O. rufipogon in the Koshihikari background consisted of 40 and 47 lines, respectively. The average proportions of recurrent parental genome were 94.3% and 96.0% in the ILs derived from accessions 104814 and 104812, respectively. The ILs from O. glumaepatula in the Itadaki background consisted of 47 lines, with an average proportion of recurrent parental genome of 91.0%. To demonstrate the potential of these ILs in identifying useful alleles, the two sets of ILs were screened for resistance to rice blast (Magnaporthe grisea). By substitution mapping, two new loci for partial resistance to rice blast were detected on chromosomes 3 and 11 of O. rufipogon (104812).

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Oryza sativa and O. glaberrima are the cultivated forms of rice in Asia and Africa, respectively. While both possess important agricultural traits, genes that are unique to either species can be used to improve the other. To explore the genetic potential of the African rice as a genetic resource for the improvement of O. sativa, 34 chromosome segment substitution lines of O. glaberrima in the background of the elite japonica cultivar Koshihikari were developed and used to identify QTLs associated with 10 traits related to grain yield, plant stature and maturity. More than 90% of the entire genome of the donor parent was represented in contiguous or overlapping chromosome segments in the CSSL set. A total of 105 putative QTLs were identified, 84 of which had positive and 21 had negative effects on the traits examined. Of the 84 QTLs with positive effects, 64 were associated with increase in grain length, grain width, grain thickness, 100-grain weight, grain number per panicle and number of primary branches per panicle whereas 20 QTLs control plant stature. These results suggest that alleles from the African rice may be useful in improving traits related to grain yield and plant stature in existing cultivars of O. sativa.

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We have developed two sets of chromosome segment substitution lines (CSSLs) of cultivated rice (Oryza sativa) carrying donor segments from indica cultivars DV85 and ARC10313. The lines in each set contain chromosomal segments that cover most of the donor genome in a uniform genetic background (ssp. japonica cv. Taichung 65). The starting materials were several recombinant inbred lines derived from the crosses Taichung 65 × DV85 and Taichung 65 × ARC10313. The CSSLs were generated by repeated backcrossing to Taichung 65 (pollen parent), with marker-assisted selection applied at several marker loci. The CSSLs of DV85 (TD-CSSLs) comprise 45 lines that cover 76.6% of the DV85 genome, and the CSSLs of ARC10313 (TA-CSSLs) comprise 44 lines that cover 74.4% of the ARC10313 genome. We investigated the genetic control of days-to-heading in both sets of CSSLs and demonstrated the genetic contribution of several chromosome regions. These CSSLs provide a valuable tool for rice germplasm enhancement, and we expect them to reveal the genetic basis of traits specific to the donor cultivars.

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Twenty near-isogenic lines (NILs) of rice (Oryza sativa L.) containing 11 blast resistance genes (Pib, Piz-5, Pi9, Pi3, Pia, Pik-s, Pik, Pik-h, Pi7(t), Pita and Pita-2) were developed by recurrent backcrossing of 19 donor varieties to a susceptible Japonica-type variety, Lijiangxintuanheigu (LTH). The resistant plants were selected in each backcross generation using a specific avirulent blast isolate for each targeted resistance gene. The NILs showed similar reaction patterns to those of the corresponding resistance-containing monogenic lines (MLs) when challenged with 20 standard blast isolates from the Philippines, suggesting that the target resistance genes were successfully introduced. The introgression of the targeted resistance genes was further confirmed by SSR markers that were located within the chromosome regions where the resistance genes had previously been mapped. Genome surveys using SSR markers showed that the NILs had genetic compositions more similar to that of the recurrent parent LTH than did the MLs, which had been derived using fewer backcrosses to LTH. The morphological characteristics of each NIL were generally similar to those of LTH. These NILs, with the genetic background of a Japonica-type variety, are expected to be valuable genetic tools for research and breeding programs for blast resistance.

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The brown planthopper [BPH; Nilaparvata lugens (Stål.)] is one of the most destructive insect pests in Asian rice-growing areas. Two genes conferring resistance to BPH, BPH25(t) and BPH26(t) derived from a BPH-resistant indica rice cultivar, Oryza sativa ADR52, have been identified. However, they are linked to genes conferring late heading and hybrid spikelet sterility. To eliminate these unfavorable traits (linkage drag), we generated BC₆F₁ populations carrying BPH25(t) or BPH26(t) in a BPH-susceptible japonica cultivar, Taichung 65, through marker-assisted selection. We selected three near-isogenic lines (NILs) carrying BPH25(t) without late heading date and one NIL carrying BPH26(t) without spikelet sterility from BC₆F₂ progeny that showed between 96.3 and 99.8% of the Taichung 65 genetic background through whole-genome survey. In antibiosis testing, the rates of surviving insects and of females with swollen abdomens were lower on the NILs than on Taichung 65, indicating that bph25(t) and Bph26(t) alleles from ADR52 controlled the resistance to BPH. The NILs will serve as useful resources for (1) monitoring BPH virulence and for (2) increasing resistance to BPH.

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To analyze genetic features in the Japanese rice population, which is mainly composed of closely related accessions, a core set of single-nucleotide polymorphisms (SNPs) was selected from SNP resources based on Japanese rice cultivars. A total of 25,199 SNPs were newly detected from the comparison of genomic sequences between two cultivars (Eiko and Rikuu132) and Nipponbare as a reference. A total of 81,499 non-redundant SNPs, including 67,051 SNPs of Koshihikari detected in a previous study, were used as candidates to select the core SNPs. Across the entire genome, 3379 SNPs were selected based on the chromosomal position of each SNP and were investigated each allele of 92 Japanese rice accessions and 3 from outside of Japan. As a result, 2551 SNPs were found to be informative for at least 91 cultivars for reducing the potential risks of genotyping error. The Japanese rice accessions were classified into three groups (upland, lowland Hokkaido, and other lowland) using all 2551 SNPs. In addition, a core set of 768 SNPs was selected to provide an even distribution among the chromosomes. Comparison of dendrograms generated by all 2551 SNPs and the core set of 768 SNPs demonstrated that the core SNPs could be used efficiently and reliably in the classification of the Japanese rice population. The core SNPs can be used for diversity analysis and for genetic analysis of the biparental populations of Japanese rice accessions.

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