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

Abstract

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.

Introduction

The National Bioresource Project (NBRP) of Japan is a set of government-supported programs run to collect, preserve and distribute bioresources that are essential to the life sciences (Kurata et al. 2010). One NBRP program is NBRP-RICE. Rice is an indispensable staple food crop that accompanies with human beings under the pressure of selection and breeding. Genetic resources of rice, such as cultivars, landraces, wild Oryza species, mutants and other experimental strains, are essential to both breeding and basic science. It is thought that there are over 350,000 cultivated lines of rice in the world (FAO 2010); many of them are stored and maintained in large-scale genetic resource centers such as the International Rice Research Institute (IRRI Philippines), the National Agriculture and Food Research Organization (NARO Japan) and other centers in rice-producing Asian countries. On the other hand, experimental strains and other genetic resources, such as mutants, wild species and other research materials, are maintained in relatively small institutes or universities (Eamens et al. 2004, Hsing et al. 2007, Jeon et al. 2000, Kim et al. 2004, Kolesnik et al. 2004, Kurata and Yamazaki 2006, Li et al. 2017, Miyao et al. 2003, Sallaud et al. 2004, van Enckevort et al. 2005). One such example is NBRP-RICE, operated by the National Institute of Genetics (NIG) and Kyushu University, Japan. NIG has charge of collecting, preserving and providing Oryza genetic resources. Kyushu University collects, preserves and provides experimental lines such as mutant strains, chromosomal segment substitution lines (CSSLs) and aneuploids derived from wild Oryza. In Japan, the Genebank project in NARO extensively collects landraces and cultivars and, thus, the collections of NBRP-RICE and NARO Genebank complement together to meet the request of resources used for both basic science and breeding.

In order to facilitate and promote the usage of genetic resources, it is important to provide genomic information. The Rice Annotation Project Database (RAP-DB, https://rapdb.dna.affrc.go.jp/) provides a comprehensive set of annotations for the genome sequence of rice, Oryza sativa, based on the Os-Nipponbare-reference-IRGSP-1.0 (IRGSP1.0), as well as transcriptome and SNP data of several cultivars of rice (Kawahara et al. 2013, Sakai et al. 2013). RAP-DB enables rice researchers to access genomic data of cultivated species. Oryzabase, a database constructed and operated by NBRP-RICE, focuses mainly on genomic and phenotypic information of wild Oryza resources in NBRP-RICE (Kurata and Yamazaki 2006, Yamazaki et al. 2010).

In this review, we introduce the bioresources accompanied with their biological attributes such as genome sequences and trait information, and examples of research conducted using those materials and the activities of NBRP-RICE to promote their usage.

Bioresources and activities in NBRP-RICE

(1)  Collection, preservation and distribution of wild Oryza species

NIG maintains more than 1700 wild Oryza species, which show a great degree of diversity in morphology (Fig. 1). NBRP-RICE follows the scientific names of wild Oryza species proposed by Vaughan and Morishima (2003), with respect to Drs. Oka and Morishima, who launched wild Oryza research at NIG. Within this system, NBRP-RICE wild accessions are classified into 21 wild species, among which the African species O. punctata is divided into diploid and tetraploid species under the same species name; O. rufipogon, the direct wild progenitor of cultivated rice O. sativa, includes both annual and perennial types; and annual O. nivara is distinguished from perennial O. rufipogon. NBRP-RICE holds 20 of the species except for O. schlechteri and their phylogenetic relationships is shown in Fig. 2 (Nonomura et al. 2010).

Fig. 1.

The incredible diversity of morphological characters in wild Oryza. (A) Gross morphologies of wild Oryza (AA, BBCC, CC, CCDD, EE and FF species). The text in brackets are accession numbers of NBRP-RICE wild Oryza resources. (B) Panicle architecture of wild Oryza (AA, BB, BBCC, CC, CCDD, EE and FF species). Panicle size, branch length and branch number have wide diversities. (C) Morphological diversities of grains among Oryza rufipogon accessions.

Fig. 2.

Phylogenetic relationships within the Oryza genus drawn by NJ method using polymorphisms against O. sativa Nipponbare chloroplast DNA. Leersia perrieri is used as an outgroup. Redrawn from Shenton et al. (2020).

Wild genetic resources are useful materials to search for genes or QTLs for resistance to biotic and abiotic stresses, yield and domestication related traits and others for the genetic improvement of cultivated species. Thus, the preservation and distribution of wild crop progenitors such as wild Oryza is beneficial for humankind. As environmental destruction and development of wild habitats is decreasing populations of wild Oryza, it is noteworthy that NBRP-RICE is dedicated to the ex situ conservation of wild Oryza. Seeds, genomic DNA and plantlets of many NBRP-RICE accessions are available via Oryzabase (https://shigen.nig.ac.jp/rice/oryzabase/locale/change?lang=en). The last section of this review gives detailed instructions on how to order these resources.

(2)  Whole genome sequencing of wild Oryza species

Next-Generation Sequencing (NGS) technologies enable the acquisition of the whole genome sequences (WGSs) of any organisms. The availability of WGSs facilitates the evolutionary and functional analysis of genes. For this reason, NBRP-RICE has made efforts to obtain WGSs of wild Oryza species. NBRP-RICE provides WGSs of its resources through Oryzabase (https://shigen.nig.ac.jp/rice/oryzabase/) (Kajiya-Kanegae et al. 2021, Ohyanagi et al. 2016, Yamazaki et al. 2010), as well as its resources to international collaborative efforts on sequencing Oryza genomes (Huang et al. 2012, Shenton et al. 2020, Stein et al. 2018, Zhao et al. 2018).

Here, we outline the current landscape of NGS data of wild Oryza species available in public databases such as National Center for Biotechnology Information (NCBI) and DNA Data Bank of Japan (DDBJ). We identified 1629 WGS data sets of wild Oryza species in NCBI (Supplemental Table 1). The data were generated at 28 research institutes in China, Japan, the USA, France and Korea (Fig. 3A, 3B). The 89.5% (248/277) of NBRP-RICE/NIG data with a sequencing depth of >5× genome coverage enable high-confidence calling of sequence variations (Fig. 3B). Of all 1629 data sets, 96.6% were generated by short-read sequencing platforms and 3.3% by long-read platforms, with sequencing depths ranging from 0.04× to 178.43× genome coverages (Fig. 3C, 3D). WGS data of 794 (48.7%) and 202 (12.4%) wild Oryza strains available from NBRP-RICE/NIG and IRRI, respectively, were generated and deposited to public databases such as Oryzabase, DDBJ and NCBI. Thus, high sequencing depth and bioresource availability add further value to the sequence data and bioresources.

Fig. 3.

Summary of NGS data of wild Oryza deposited in public databases. (A) Countries, (B) major institutes and (C) sequencing platforms generating NGS data of wild Oryza. (D) Distribution of read depth of deposited data in each species. (E) Number of accessions generating NGS data in each species.

Short-read data have been extensively generated to call sequence polymorphisms such as SNPs and indels, and contribute greatly to reveal QTLs controlling agronomic traits (Huang et al. 2018, Wei et al. 2021, Wu et al. 2017), population structures and domestication history (Fawcett et al. 2013, Huang et al. 2012, Xu et al. 2011, Zhang et al. 2014). Most of the NGS data (75%) come from the wild progenitors of cultivated species: O. rufipogon, O. nivara and O. barthii (Fig. 3E). Imputed genome-wide SNPs obtained from low-coverage short reads of 446 O. rufipogon species published in Huang et al. (2012) are available at OryzaGenome 2.1 (http://viewer.shigen.info/oryzagenome21detail/index.xhtml) in Oryzabase (see the next section for details) (Kajiya-Kanegae et al. 2021). These sequences helped to reveal that O. rufipogon accessions can be divided into three subgroups, Or-I, -II and -III, and that japonica cultivars are most closely related to the Or-III population, which is distributed mainly in southern China (Huang et al. 2012).

Reference genomes of wild Oryza are important for studies of genome evolution and for the detection of genetic variations in regions that are highly polymorphic or absent in the genomes of cultivated species. To date, reference genomes of 26 accessions of 10 wild Oryza species have been deposited (Chen et al. 2013, Reuscher et al. 2018, Shenton et al. 2020, Stein et al. 2018, Wu et al. 2018, Zhao et al. 2018; Supplemental Table 2). Of these, 21 were based on short-read sequencing platforms and 5 on long-read platforms. Through the assembly of 13 O. rufipogon genomes, a pan-genome dataset of the O. sativa–O. rufipogon species complex was released (Zhao et al. 2018). Significant amounts of genome sequence data covering the entire Oryza genus have also been accumulated. These were generated in several recent studies focused on non-AA-genome species, and revealed important aspects of genome-size change, phylogenetic relationship, history of polyploidization and outstanding traits such as cold tolerance (Chen et al. 2013, Kitazumi et al. 2018, Reuscher et al. 2018, Shenton et al. 2020, Stein et al. 2018, Wu et al. 2018). Thus, the pan-genome dataset and genus-wide genome information will advance research toward understanding genome evolution and utilization of untapped genetic resources in the genus Oryza.

(3)  OryzaGenome 2.1 as an archive of Oryza genome information

OryzaGenome 2.1 (http://viewer.shigen.info/oryzagenome21detail/index.xhtml) (Kajiya-Kanegae et al. 2021), within Oryzabase (https://shigen.nig.ac.jp/rice/oryzabase/), archives genome information of wild Oryza, supporting researchers using wild Oryza resources, mainly in NBRP-RICE. OryzaGenome 2.1 stores NGS genome information on 217 accessions in 19 wild Oryza species. In the Downloads section, information on accessions used for WGS can be retrieved by searching for various conditions such as species, genome type (AA, BB, CC, BBCC, CCDD, EE, FF, GG, etc.), growth habit, rank in the core collection and country of origin. Biological information of the accessions used for WGS can be obtained by clicking on accession numbers, which are linked to Oryzabase, and it is possible to request materials, depending on availability.

The Downloads section also provides polymorphic information on 33 O. rufipogon accessions against the reference genome sequence of cultivated rice, Nipponbare, in VCF file format. OryzaGenome 2.1 also provides a SNP viewer of 446 O. rufipogon accessions with low-coverage sequence reads (Huang et al. 2012) (http://viewer.shigen.info/oryzagenome21/mapview/MapView.do?action=ini&chromosomeNo=1) and a SNP effect table for 33 deep-sequenced O. rufipogon accessions (http://viewer.shigen.info/oryzagenome21detail/snptable/search.xhtml) (Kajiya-Kanegae et al. 2021).

(4)  Development of genetic transformation of wild Oryza which accelerates molecular genetic analysis and genome editing

Genetic transformation is an important technology for revealing, modulating and utilizing gene function. It is often used for molecular breeding, especially in genome editing. In addition, because of the difficulties posed by genetic crosses between distantly related species, transformation of wild Oryza accessions would be particularly valuable. Recently, we reported that a wide range of wild Oryza accessions, including those distantly related to cultivated species, can be genetically transformed by the immature-embryo method using Agrobacterium (Shimizu-Sato et al. 2020). By using 192 representative wild Oryza accessions covering 20 species in NBRP-RICE, we found that 90 accessions in 16 species form calluses from immature embryos and regenerate plantlets in tissue culture under several culture conditions (Hiei and Komari 2008), showing the feasibility of genetic transformation of wild Oryza. These accessions include AA, BB, CC, BBCC, CCDD, FF and HHJJ genome species. We also optimized the conditions of Agrobacterium infection for 51 accessions in 11 species. Many Oryza species could be transformed by the modified immature-embryo method. This method opens the door to genome editing, accelerating the study of wild Oryza genetic resources for molecular genetic analysis and future use in molecular breeding.

(5)  Collection of mutant strains generated by N-methyl-N-nitrosourea treatment of fertilized egg cells

The treatment of fertilized egg cells at the single cell stage by N-methyl-N-nitrosourea (MNU) is one of the most efficient methods to induce nucleotide substitutions with a low rate of chimeric mutants in rice (Satoh et al. 2010). It is empirically shown that the average number of mutations detected in 1 kb genic region is 7.4 single nucleotide polymorphisms (SNPs) using 1,000 M₂ mutant population made by this method (Suzuki et al. 2008). Thus, it is likely that researchers can obtain mutations in any gene of interest by both reverse and forward genetic screening of mutant pools made by this method and distributed from as NBRP-RICE described below. Mutation pools of rice cultivars ‘Kinmaze’ (3000 lines), ‘Taichung 65’ (3000 lines), ‘Kita-ake’ (1100 lines), ‘Yukihikari’ (1300 lines) and ‘IR64’ (1200 lines) are maintained by NBRP-RICE. ‘Kinmaze’ and ‘Taichung 65’ (T65) are standard japonica cultivars. The lower photoperiod sensitivity of ‘Kita-ake’ and ‘Yukihikari’, japonica cultivars adapted to northern Japan, makes it easier to grow them in plant incubators. ‘IR64’ is a widely cultivated indica cultivar. MNU-induced rice lines show a wide spectrum of morphological and physiological phenotypes, such as abnormalities in embryo and endosperm development, panicle architecture, plant height, leaf coloration, seed fertility and tillering. Seeds of most of these lines are available from NBRP-RICE. In addition, NBRP-RICE offers a system for TILLING (Targeting-Induced Local Lesions IN Genomes) analysis (McCallum et al. 2000), called TILLING Open Laboratory, where mutations in genes of interest can be screened by using facilities at Kyushu University.

(6)  Recombinant inbred lines, chromosome segment substitution lines (CSSL) and monosomic alien chromosome addition lines distributed by NBRP-RICE

To facilitate genetic analysis and characterization of quantitative trait loci (QTLs), NBRP-RICE developed and made available recombinant inbred lines (RIL) and chromosomal segment substitution lines (CSSLs) that cover the whole genomic region of donor accessions in the genetic background of recurrent parents (Table 1). Four RILs are derived from an intraspecific cross between subspecies japonica and indica. The CSSLs comprise 14 kinds of introgression lines and originate from six AA-genome Oryza species accessions with the genetic background of O. sativa T65: O. glaberrima, O. glumaepatula, O. meridionalis, O. rufipogon, O. nivara and O. longistaminata. The CSSLs include three pairs derived from reciprocal crossing of T65 and wild relatives (O. glaberrima, O. glumaepatula and O. meridionalis); each pair carries different cytoplasms with a similar nuclear genome composition, useful for studies of genetic interactions between nuclear and organellar genomes. NBRP-RICE also offers monosomic alien addition lines, called MAALs, which have a single chromosome of the wild Oryza punctata in addition to a complete complement of O. sativa chromosomes (Yasui and Iwata 1998).

Table 1. Recombinant inbred lines and chromosomal segment substitution lines distributed from NBRP-RICE

Type of population	Population identifier	Recurrent parent	Species classification (recurrent parent)	Donor	Species classification (donor)	Cytoplasm	Reference
Recombinant inbred lines	RIA	IR24	O. sativa indica type	Asominori	O. sativa japonica type	[Aso]	Tsunematsu et al. 1996
	RIB	Kinmaze	O. sativa japonica type	DV85	O. sativa indica type	[Kin]	Ikeda et al. 1998
	RIC	Taichung 65	O. sativa japonica type	DV85	O. sativa indica type	[T65]	Yasui et al. 2010
	RID	Taichung 65	O. sativa japonica type	ARC10313	O. sativa indica type	[T65]	Yasui et al. 2010
Chromosomal segment substitution lines	IAS	IR24	O. sativa indica type	Asominori	O. sativa japonica type	[IR24]	Kubo et al. 2002
	AIS	Asominori	O. sativa japonica type	IR24	O. sativa indica type	[Aso]	Kubo et al. 2002
	TACSSL	Taichung 65	O. sativa japonica type	ARC10313	O. sativa indica type	[T65]	Yasui et al. 2010
	TDCSSL	Taichung 65	O. sativa japonica type	DV85	O. sativa indica type	[T65]	Yasui et al. 2010
	GIL	Taichung 65	O. glaberrima	IRGC104038	O. glaberrima	[T65]	Doi et al. 1997
	IRGC103777[GILs]	Taichung 65	O. sativa japonica type	IRGC103777	O. glaberrima	[T65]	Yamagata et al. 2019
	IRGC103777[T65]	Taichung 65	O. sativa japonica type	IRGC103777	O. glaberrima	[gla]	Yamagata et al. 2019
	GLU-ILs[T65]	Taichung 65	O. sativa japonica type	IRGC105668	O. glumaepatula	[T65]	Yoshimura et al. 2010
	GLU-ILs[GLU]	Taichung 65	O. sativa japonica type	IRGC105668	O. glumaepatula	[glum]	Yoshimura et al. 2010
	MER-Ils[T65]	Taichung 65	O. sativa japonica type	W1625	O. meridionalis	[T65]	Yoshimura et al. 2010
	MER-IL[MER]	Taichung 65	O. sativa japonica type	W1625	O. meridionalis	[mer]	Yoshimura et al. 2010
	WK1962ILs	Taichung 65	O. sativa japonica type	W1962	O. rufipogon	[T65]	Yamagata et al. 2019
	IRGC105715ILs	Taichung 65	O. sativa japonica type	IRGC105715	O. rufipogon	[T65]	Yamagata et al. 2019
	W1508ILs	Taichung 65	O. sativa japonica type	W1508	O. longistaminata	[T65]	Ogami et al. 2019
	W1413ILs	Nipponbare	O. sativa japonica type	W1413	O. longistaminata	[Nip]	Thein et al. 2019

Research examples using genetic resources in NBRP-RICE/NIG/Kyushu University

The Oryza genetic resources held in NBRP-RICE have accelerated surveys of key genes responsible for the domestication of rice cultivars, agronomically important traits and adaptation of wild relatives to natural environments. For example, Ishii et al. (2013) showed that humans might have selected the closed panicle trait of ancestral O. rufipogon during rice domestication, which promotes the retention of seeds on panicles and self-pollination, consequently increasing yields. Using nearly isogenic lines (NILs) with NBRP-RICE O. rufipogon accession W0630 as the recurrent parent, they revealed that interspecific variations in the noncoding regulatory region upstream of the OsLIGULELESS coding region determined the opened or closed shape of panicles. Wu et al. (2018) identified a multi-gene locus in African rice cultivars that changes the plant habit from prostrate to erect in an F₂ population of O. glaberrima × NBRP-RICE accession O. barthii W1411, prompting an idea that convergent evolution in plant architecture occurred independently in Asian and African rice cultivars. Using deepwater rice accessions, Hattori et al. (2009) identified SNORKEL1 and SNORKEL2, which encode ethylene response factors that trigger the deepwater response. The SEMIDWARF1 (SD1) allele of a landrace with deepwater response, which differs from the major SD1 allele present in modern cultivars, was selected from O. rufipogon for deepwater rice cultivation in Bangladesh (Kuroha et al. 2018). Similarly, using deepwater accessions, Nagai et al. (2020) identified a mechanism regulating internode elongation, in which the antagonistic function of ACCELERATER of ELONGATION 1 (ACE1) and DECELERATER of ELONGATION 1 (DEC1) operates after and before deepwater-induced internode elongation, respectively, and discussed their contributions in domestication by comparing their alleles from O. rufipogon.

Approximately, 30 loci that cause abnormal starch content or endosperm development have been characterized (Satoh et al. 2003). The waxy mutant doesn’t have amylose, which is synthesized by granule-bound starch synthase. dull mutants have a low amylose content, and five loci are known: du1, du2, du3, du4 and du5 (Yano et al. 1988). The sugary1 (sug1) and sugary2 (sug2) mutants accumulate phytoglycogen-type water-soluble polysaccharides instead of starch. Wrinkled-endosperm mutants are typified by sug1, sug2, shrunken1 and shrunken2 (Nakagami et al. 2017, Yano et al. 1984). More that 10 causal genes for these 30 mutant loci have been identified (Isshiki et al. 2000, Kawagoe et al. 2005, Kubo et al. 2005, Nakagami et al. 2017, Ohdan et al. 2005, Tuncel et al. 2014).

CSSLs distributed by NBRP-RICE find extensive use in exploiting and isolating QTLs for various traits unique to wild accessions. A QTL for resistance to green rice leafhopper was delimited within a 31.2-kbp region by using CSSLs derived from T65 × O. rufipogon, WK1962ILs (Phi et al. 2019). To reveal the genetic basis for promoting outcrossing, Ogami et al. (2019) analyzed anther length in O. longistaminata, which has the longest anther length among AA genome species, by using CSSLs derived from T65 × O. longistaminata, W1508ILs, and identified several QTLs that explain the longer length.

These are only a few examples of research supported by NBRP-RICE resources. Over 300 reports based on NBRP-RICE resources have been published since 2002 (https://rrc.nbrp.jp/projects/12?lang=en).

How to request Oryza genetic resources in NBRP-RICE

Oryzabase (https://shigen.nig.ac.jp/rice/oryzabase/) (Yamazaki et al. 2010) is an integrated database for resources distributed by NBRP-RICE, and a portal for ordering the bioresources from NBRP-RICE. Researchers can search by accession number or species name, and can gain access to a series of core collections covering the wide genetic diversity in Oryza. By clicking on accession numbers, basic information on accessions of interest can be obtained. Users can then order resources through the “Add” button.

Author Contribution Statement

Y.S., K.T., Y.Ya., H.M., Y.Yo., K.N.T., T.S., M.N.-T., T.K., K.-I.N., H.Y. and T.K. contributed to the collection, preservation and distribution of resources. H.K.-K. and S.K. contributed to the construction of the database. Y.S., T.K., A.A. and S.S.-S. analyzed genome and phenotype data, and contributed to design and production of Figures and Tables. Y.S., K.T., Y.Y., A.A., S.S.-S., T.K., K.-I.N., Y.H. and T.K. wrote the manuscript.

Acknowledgments

We thank Mr Furuumi and Ms Miyabayashi in NIG for their extensive long-term efforts in maintaining the NBRP-RICE resources. This work was supported by a Japan Society for the Promotion of Science Grant-in-Aid A for Scientific Research [20H00424 to Y.S.]; the National BioResource Project (NBRP) to Y.S.; and the National Institute of Genetics (NIG)-JOINT [88B2018 to H.K.-K.]. Computations were performed on the NIG supercomputer at the ROIS National Institute of Genetics.

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