Nucleic Acids Research, 2006, Vol.

34, Database issue D745–D748

doi:10.1093/nar/gkj016

RMD: a rice mutant database for functional analysis

of the rice genome
Jianwei Zhang, Caishun Li, Changyin Wu, Lizhong Xiong, Guoxing Chen,
Qifa Zhang and Shiping Wang*

National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong

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Agricultural University, Wuhan 430070, China

Received June 28, 2005; Revised September 7, 2005; Accepted September 17, 2005

ABSTRACT moss (6). In rice, we have generated more than 129 thousand
independent transgenic lines with the enhancer trap construct
Rice Mutant Database (RMD, http://rmd.ncpgr.cn) is by Agrobacterium-mediated T-DNA insertion (7). The system
an archive for collecting, managing and searching has three built-in strategies for functional analysis of the rice
information of the T-DNA insertion mutants generated genome. First, T-DNA insertions cause gene mutations, pro-
by an enhancer trap system. We have generated viding an efficient approach for gene identification and isola-

129 000 rice mutant (enhancer trap) lines that are tion. Second, expression of the reporter gene indicates the
now being gathered in the database. Information col- presence of an enhancer element in the neighboring genomic
lected in RMD includes mutant phenotypes, reporter- region, which can be used for isolation and characterization
gene expression patterns, flanking sequences of of the enhancer. Third, the lines showing spatial- or temporal-
T-DNA insertional sites, seed availability and others, specific expression of the reporter gene can be used to drive
and can be searched by respective ID, keyword, ectopic expression of a transgene, thus useful for unveiling
latent functions of unknown or known genes. Large-scale
nucleotide sequence or protein sequence on the
screening and characterization of these rice mutant lines are
website. This database is both a mutant collection now in progress. Comprehensive information, including the
for identifying novel genes and regulatory elements phenotypes, reporter-gene expression patterns and flanking
and a pattern line collection for ectopic expression sequences of T-DNA insertional sites of these enhancer trap
of target gene in specific tissue or at specific growth lines has been generated. As there is an urgent need for pub-
stage. licly available rice mutant resources in functional genomic
studies by the rice research community (8), we undertook
the effort to establish the Rice Mutant Database (RMD,
INTRODUCTION http://rmd.ncpgr.cn) for public access. This database together
with other insertional rice mutant collections (8,9) should
Rice (Oryza sativa L.) has now become a model plant system serve as informative resources for accelerating functional
for genome research of the monocots, especially for cereal genomic studies of rice, as well as other monocots.
species. Although the draft and finished genome sequences
of both indica and japonica subspecies of rice have been
released, the function for majority of the genes in the rice
genome have not been experimentally studied (1,2). A com- ARCHITECTURE AND CONTENT OF
THE DATABASE
mon strategy for large-scale discovery of gene function is to
generate populations of DNA insertion mutants and identify RMD was constructed using a Redhat Linux system and main-
the genes by screening and characterizing the mutations. tained using MySQL, a relational database management
The enhancer trap system, based on the expression pattern system. The data collected in RMD are placed into three tables
of a reporter gene randomly integrated into the genome, (mutant lines, phenotypes and flanking sequences), that are
is regarded as a useful tool for identifying novel genes and linked via entry identifier (ID) (Figure 1). The general
regulatory elements. This technology was first applied in the information of current release 20050901 of RMD is summar-
bacterium (3) and now is widely used in animal research, ized in Table 1.
especially in Drosophila (4). There were also reports on The enhancer trap lines were generated using three japonica
the establishment of such system in Arabidopsis (5) and rice varieties (O.sativa ssp. japonica), Zhonghua 11 (Z11),

*To whom correspondence should be addressed. Tel: +86 27 87283009; Fax: +86 27 87287092; Email: swang@mail.hzau.edu.cn

 The Author 2006. Published by Oxford University Press. All rights reserved.

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D746 Nucleic Acids Research, 2006, Vol. 34, Database issue

SEARCH THE DATABASE

RMD can be freely accessed using a web browser at http://
rmd.ncpgr.cn. User can search information from the database
in two different ways as described below (Figure 2).

Keyword search
Keyword can be the entry ID of enhancer trap line, single word
or phrase. Each ID consists of eight characters, such as

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02Z15DJ04 and 03Z11BO64. The first two digits of each
ID represent the year in which the line was generated (‘02’
for year 2002, ‘03’ for year 2003 and so on). The next three
characters are the abbreviation of rice variety with which the
mutant line was generated (Table 1). The last four characters
represent the order of the line produced. For quick searching
the database, user can use the ID as query word and the query-
ing result displays all details for the entry, including its mor-
Figure 1. The composition of Rice Mutant Database. The component in dotted
phological mutation, traits screened under stress conditions,
frame will be integrated into the database. expression pattern of the reporter gene in different tissues at
different developmental stages, validated flanking sequence(s)
and seed availability. If one does not know the ID, any key-
Table 1. General information of the Rice Mutant Database, release 20050901 words, especially the words or phrases listed in Supplementary
Table 1 or the suggested keywords provided on the website,
Data type Number of entries can be used for searching. The querying result will show a list
Total enhancer trap line 128 560 of entries that are linked to the individual subclass pages.
From Zhonghua 11 (Z11) 113 262 Three Boolean operators, ‘AND’, ‘OR’ and ‘NOT’ with all
From Zhonghua 15 (Z15) 14 197 letters entered in UPPERCASE, can be used in combination
From Nipponbare (NPB) 1101 with keywords for refining the search statement.
Mutant phenotypea 23 844
Expression pattern 5511
A wizard is provided for advanced search (Figure 2).
Flanking sequenceb 13 738 Two advanced search pages for Microsoft Internet Explorer
Mutant photograph 733 (IE) browser and non-IE browsers are designed, respectively.
Seed available 53 432 The system will provide the appropriate page automatically
a
The information indicates the lines that had been screened for at least one
when the user clicks the ‘Advanced search’ button. First, user
type of phenotypes (including abiotic and biotic stress screening) listed in can choose one of the four screening types for mutant cate-
Table 2. gories from the selectable menu for IE browser or radio but-
b
In total 11 910 mutant lines have one flanking sequence for each, 753 lines two tons for non-IE browsers. Second, type of traits screened can
sequences for each, 67 lines three sequences for each, 16 lines four sequences for be selected from a clickable menu for IE browser or radio
each, 9 lines five sequences for each and 2 lines six sequences for each.
buttons for non-IE browsers. The last step is to select the
phenotype from a clickable list for IE browser or checkboxes
for non-IE browsers. For more targeted search, one or more of
Zhonghua 15 (Z15) and Nipponbare (NPB). The data of the four checkboxes for developmental stages can be used for
mutant phenotypes, which refer to novel genes controlling the search. The querying results will display one or a list of
the phenotypes, are collected by screening types falling into entries that are linked to the individual subclass pages.
the three categories: morphology under normal growth con-
ditions, abiotic stress and biotic stress (Table 2 and Supple- BLAST search
mentary Table 1). Each of the screening types involves two or The BLAST program (10), including BLASTN, TBLASTN
more attributes referred to as ‘traits’. Phenotypes were scored and TBLASTX, is integrated into RMD. User can use either
for each of the traits at different developmental stages. The nucleotide sequence or protein sequence as query to search the
phenotype data for each trait were routinely collected based on flanking sequences of T-DNA insertional sites. The querying
20 T1 (in some cases T2) plants per entry, from which segre- results will display the corresponding ID of mutant line as well
gation ratios of the traits were also scored. The expression as all the alignments of homolog sequences.
patterns of the reporter gene, which refer to the regulatory
elements, are also embodied into the category of ‘Screening
type’ (Table 2 and Supplementary Table 1). The expression
patterns were examined for five different tissues of T0 (in some FUTURE PLANS
cases T1) plants at various developmental stages. Majority RMD is a new database. The majority of data in this release
of the flanking sequences of T-DNA insertional sites were (20050901) is generated by one of the three groups, Wuhan
obtained by thermal asymmetric interlaced-PCR and a group, in the National Special Key Program on Rice Func-
small proportion of the flanking sequences were obtained tional Genomics of China. The complete screening with
by plasmid rescue. All of these sequences are anchored on screening traits listed in Table 2 or Supplementary Table 1
rice chromosomes. and collecting flanking sequences from
129 000 independent
 Nucleic Acids Research, 2006, Vol. 34, Database issue D747

Table 2. Mutation phenotypes in RMDa

Screening type Traitb Number of phenotype in trait Developmental stagec

Seedling Tillering Heading Ripening
p p
Morphology under normal growth conditions Leaf 25 —
p —
Flag leaf 8 —
p — —
Root 8 p —
p — —
Culm 11 —
p —
Spikelet 6 — — p —
Panicle 10 — — —
p

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Seed 7 —
p — —
Abiotic stress Abscisic acid 3 p — — —
Drought 3 p — — —
Low nitrogen 3 p — — —
Low phosphorus 3 —
p — —
Biotic stress Bacterial blight 2 —
p p — —
Blast 2 p p —
p —
Expression Leaf 7 p —
Root 7 —
p —
p —
Culm 2 — p —
Flower 11 — — —
p
Seed 4 — — —
a
Supplementary Table 1 for detail phenotypes of this table.
b
Abscisic acid: plants treated with 30-80 mM of abscisic acid; Drought stress: stopping watering at 3-4 leaf stage and counting the number of days to leaf rolling; Low
nitrogen: plants cultured in solution containing 1/10 of normal (1.428 mM NH4NO3) nitrogen; Low phosphorus: plants cultured in solution containing 1/30 of normal
(0.323 mM NaH2PO4) phosphorus; Bacterial blight: inoculation with Xanthomonas oryzae pv. oryzae; Blast: inoculation with Pyricularia grisea Sacc.
c
The symbol ‘H’ indicates the stage at which screening was conducted.

SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS
We thank Professors Liyuan He, Zhuqing Zhao and Deming Jin
as well as the students of Huazhong Agricultural University
and professor Xuebiao Pan and his students of Yangzhou
University who have contributed to the screening of rice
mutants. This work was supported by grants from the
National Special Key Program on Rice Functional Genomics
of China and the National Natural Science Foundation of
China. Funding to pay the Open Access publication charges
Figure 2. Query of data in Rice Mutant Database. for this article was provided by the Ministry of Science and
Technology of China.
enhancer trap lines generated by Wuhan group are expected to
be finished within next few years. In addition, data collected Conflict of interest statement. None declared.
by other two (Beijing and Shanghai) groups of the same pro-
gram will give additional information for RMD and enhance
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