No.44 (2005年/平成17年)

Transcription

No.44 (2005年/平成17年)

ISSN 0435-1096
Gamma Field Symposia
Number 44 (2005)
GENOME AND POST-GENOME RESEARCHES IN CROPS
AND MUTATION
Number 44
2005
INSTITUTE OF RADIATION BREEDING
NIAS
Hitachi-Omiya, Ibaraki-ken
Japan
GENOME AND POST-GENOME RESEARCHES IN CROPS AND MUTATION
Report of Symposium
held on
July 13-14, 2005
Institute of Radiation Breeding
NIAS
Hitachi-Omiya, Ibaraki-ken 319-2293
Japan
I
The lecturers and the members of the Symposium Committee
General discussion
II
List of Participants
(44th GF Symposium)
Abe Tomoko
Amano Etsuo
Chiba Bunya
Ebana Kaworu
Eguchi Hisashi
Emori Sumie
Fukatsu Eitaro
Fukuoka Hiroyuki
Fukuoka Syuichi
Fuwa Noritoshi
Goto Yoko
Hara Takashi
Hayashi Yoriko
Hidema Jyun
Hiraga Susumu
Hirai Yumi
Hirao Tomonori
Hirata Yutaka
Hirochika Hirohiko
Hiromichi Hara
Hoki Takehiro
Hori Yasuko
Huang Chao Feng
Iba Ryoichi
Ichida Hiroyuki
Iimure Takashi
Inafuku Masahito
Inoue Eiichi
Ishii Takuro
Ishikawa Tomoko
Isoda Keiya
Iwata Hiroyoshi
Kamio Akiko
Kataoka Hiromichi
Kawaguchi Masayoshi
Kinai Satomi
Kobayashi Toru
Kobayashi Shozo
Kondo Teijji
Kubota Motoshige
Kuboyama Tsutomu
Kusaba Makoto
RIKEN, FRS
Fukui Prefectural University
Miyagi Prefectural Furukawa Agricultural Experiment Station
National Institute of Agrobiological Sciences
Ministry of Agriculture,Forestry and Fisheries research Network
Tokita Seed Co., Ltd.
Forest Tree Breeding Center
National Agriculture and Bio-oriented Research Organization
Snow Brand Seed Co., Ltd.
Graduate School of Life and Environmental Sciences, University of Tsukuba
RIKEN, FRS
Touhoku University
National Institute of Crop Science
The Institute of Physical and Chemical Research
Graduate of Akita prefectural university
Tokyo University of Agriculture and Technology
College of Agriculture Ibaraki University
Sapporo Breweries LTD.
Graduate School of Agricultural Science, Tohoku University
Research Institute for Bioresources, Okayama University
Miyazak agricultural experiment station
RIKEN, FRS
Sapporo Breweries LTD.
National Institute of Livestock and Grassland Science
College of agriculture, Ibaraki university
Ibaraki Agricultural Center
Plant-Biotechnology Institute IBARAKI Agricultural Center
National Agricultural Research Center
Shizuoka Prefectural Citrus Experiment Station
The University of Tokyo
Hokkaido System Science Co., Ltd.
National Institute of Agrobiological Sciences, Institute of Radiation Breeding
National Institute of Fruit Tree Science
Ngano Agricultural Experiment Station
Ibaraki University
III
Ma Jian Feng
Maejima Shinichiro
Makino Takahiro
Manabe Toru
Mase Nobuko
Matsumoto Yuichi
Matsushita Shuji
Matsuyama Tomoki
Nishimura Minoru
Mi-Suk Seo
Miyao Akio
Miyasaka Hiroshi
Miyata Yuji
Morishita Toshikazu
Morita Ryouhei
Nagamura Yoshiaki
Nagato Yasuo
Nagatomi Shigeki
Nakagawa Hitoshi
Nishikawa Hiroshi
Nishimura Shigeo
Nishinaka Mio
Ogiwara Hitoshi
Ohashi Yoshi
Ohmiya Yasunori
Ohmura Mitsuo
Ohsugi Ryu
Ohta Satoshi
Ohta Kouki
Ohta Yuzo
Oka Seibi
Okamoto Kazuyuki
Okamura Masachika
Okano Katsunori
Oki Nobuhiko
Okumoto Yutaka
Onozaki Takashi
Saiki Yumi
Saito Minoru
Saito Hiroki
Saito Atsuo
Sano Yoshio
Sasaki Takuji
Sasaki Mai
Sato Yutaka
Seki Kousuke
Research Institute for Bioresources, Okayama University
Shizuoka Agriculturl Experiment Station
Shizuika Agriculturl Experiment Station
Hiroshima Prefecture Agricultural Research Center
RIKEN, FRS
Agricultural Technology Institute of Nagano
National institute of Agrobiological Sciences, Institute of Radiation Breeding
National institute of Agrobiological Sciences
Bio-oriented Technology Research Advancement Institution
Shizuoka Prefectural Tea Experiment Station
Tsukuba University
Graduated School of Agriculture, Kyoto University
Tokyo University of agriculture and technology
The University of Shizuoka
Graduate School of Agrucultural and Life Sciences, The University of Tokyo
Department of Agriculture, Shizuoka University
National Institute Agrobiological Sciences
Plant Lab. Kirin brewery Co.,Ltd
National Institute of Floricultural Science
Fukuoka Agricultural Research Center
Fukui Prefuctual Experimental Station
Takii Plant Breeding & Experiment Station
Hokkaido University
Shizuoka Agriculturl Experiment Station
Nagano Vegetable and Ornamental Crops Experiment Station
IV
Sekiguchi Fumihiko
Shimizu Akemi
Shinjo Yasuyo
Shirasawa Kenta
Suematsu Nobuhiko
Suzuki Kiyoshi
Suzuki katsuaki
Tabata Minako
Takada Norio
Takahara Manabu
Takahashi Makoto
Takahashi Masaki
Takamaru Kouichi
Takayasu Akio
Takeuchi Syunsuke
Takyu Toshio
Tamura Kanako
Tanaka Atsushi
Tanisaka Takatoshi
Toyota Kentaro
Tsubomura Miyoko
Tsukiyama Takuji
Tsutsumi Nobuhiro
Ukai Yasuo
Usui Noriko
Yamada Tetsuya
Yamaguchi Hiroyasu
Yamanouchi Hiroaki
Yamazaki Yukiko
Yokoyama tomosaburo
Yoshikawa Takanori
Yoshioka Terutaka
Yoshioka Toji
Yoshioka Yosuke
Yuri Murota
Japan Womenís University
Nakajima Yoshio Co., Ltd.
Shizuoka Agricultural Experiment Station
Fukui Prefectural University
Keisei Rose Nurseries, Inc.
Keisei Rose Nurseries, Inc.
Japan Atomic Energy Research Institute
Akita Prefectural University
Graduate School of Agriculture, Kyoto University
Iwate Agricultural Research Center
National Institute of Floricultural Science
National institute of Agrobiological Sciences, Institute of Radiation Breeding
National Institute of Genetics
Saitama Plant Promotion Center
Department of Agriculture in Kyoto University
University of Tsukuba
V
Foreword
The 1st Gamma Field symposium was held in 1962. During its 44-year history, we have selected various
themes related to mutation and breeding, and have invited leading scientists with expertise in these areas as lecturers
to provide information on a wide variety of related topics.
The 44th Gamma Field symposium entitled ìGenome and post-genome researches in crops and mutationî was
held July 13-14, 2005 in Mito, Ibaraki, Japan. The keynote address, ìImpact of the complete rice genome sequence
information on future basic and applied plant science researchîwas presented by Dr. Takuji Sasaki, National Institute
of Agrobiological Sciences. Eight lecturers were also invited to present results of their research: Dr. M. Kawaguchi
(University of Tokyo: Genome and post-genome researches in Lolium japonica), Dr. M, Omura (University of Tokyo: Genome analysis and breeding Citrus), Dr. Y. Hirai (The Institute of Physical and Chemical Research: Functional genomics based on the integration of metabolomics with transcriptomics ), Dr. Y. Yamazaki (National Institute
of Genetics: Advanced utilization of biological information), Dr. S. Fukuoka (NIAS: Natural variation and the study
for enhancing genetic diversity in rice), Dr. A. Miyao (NIAS: Reverse genetics for functional genomics of rice), Dr.
H. Fukuoka (National Agriculture and Bio-oriented Research Organization: Development and utilization of genome
information in vegetable crops), and Dr. M. Kusaba (University of Tokyo: Use of -ray-induced mutations in the genome era in rice).
Molecular genetics based on genome sequencing will be presumably be the most powerful tool for selecting
mutants of certain characteristics. This could change the mutation breeding dramatically, especially in rice, and expand its use into the other graminaceous crops which show genomic synteny to rice.
This publication includes the contributed papers from the invited lecturers and the discussions during the symposium (in Japanese). Without any doubt, genomic information on crops and mutation breeding will expand our
ability to develop superior varieties and enhance the plant breederís opportunities for plant breeder to develop such
varieties. The effectiveness of a mutation breeding approach will also become more apparent as the present array of
genome sequencing projects on various agriculturally important species is completed.
It is our sincere hope that the series of Gamma Field Symposia, including this issue, will help plant breeders
and researchers to realize the contribution that mutation breeding has made to the plant sciences.
We express our sincere thanks to the lecturers, chairpersons and attendees
On behalf of The Symposium Committee
Hitoshi Nakagawa, Chairperson
VI
PROGRAM
Opening address : H. NAKAGAWA
Congratulatory address : M. ISHIGE
Special lecture
Chairperson : Y. SANO
Impact of the complete rice genome sequence information on future basic and
applied plant science research…………………………………………………………………………… T. SASAKI
Session I
Chairperson : Y. NAGATO
Genome and post-genome researches in Lotus japonica…………………………………………… M. KAWAGUCHI
Session II
Chairperson : S. KOBAYASHI
Genome analysis and breeding in Citrus………………………………………………………………… M. OMURA
Session III
Chairperson : Y. UKAI
Functional genomics based on the integration of metabolomics with transcriptomics
………………………………………………………………………… M. Yokota H IRAI , K. SAITO
Session IV
Chairperson : Y. OKUMOTO
Advanced utilization of biological information………………………………………………………… Y. YAMAZAKI
Session V
Chairperson : T. TANISAKA
Natural variationation and the study for enhancing genetic diversity in rice…………………………… S. FUKUOKA
Session VI
Chairperson : A. TANAKA
Reverse genetics for functional genomics of rice………………………………………………………… A. MIYAO
Session VII
Chairperson : S. OKA
Development and utilization of genome information in vegetable crops ……………………………… H. FUKUOKA
Session VIII
Chairperson : T. ABE
Use of γ-ray-induced mutations in the genome era in rice ……………………………………………… M. KUSABA
Session IX
Chairperson : N. TSUTSUMI
General discussion
Closing address : Y. NAGATO
VII
CONTENTS
T. SASAKI
Impact of the Complete Rice Genome Sequence Information on Future Basic and
Applied Plant Science Research …………………………………………………………
1
M. KAWAGUCHI
Genome and post-genome researches in Lotus japonica ……………………………… 15
M. OMURA
Genome Analysis and Breeding in Citrus ……………………………………………… 25
M. Yokota HIRAI
K. SAITO
Functional Genomics Based on the Integration of Metabolomics with Transcriptomics …………………………………………………………………… 33
Y. YAMAZAKI
Advanced Utilization of Biological Information ……………………………………… 41
S. FUKUOKA
K. EBANA
Y. UGA
M. KAWASE
Natural Variationation and the Sstudy for Enhancing Genetic Diversity in Rice ……… 45
A. MIYAO
Reverse Genetics for Functional Genomics of Rice …………………………………… 55
H. FUKUOKA
Development and Utilization of Genome Information in Vegetable Crops …………… 63
M. KUSABA
Use of γ-ray-induced mutations in the genome era in rice …………………………… 69
General discussion (in Japanese) ……………………………………………………… 75
Gamma Field Symposia, No. 44, 2005 Institute of Radiation Breeding
NIAS, Japan
1
IMPACT OF THE COMPLETE RICE GENOME SEQUENCE INFORMATION
ON FUTURE BASIC AND APPLIED PLANT SCIENCE RESEARCH
Takuji SASAKI
National Institute of Agrobiological Sciences,
1-2, Kannondai 2-chome, Tsukuba, Ibaraki 305-8602
Introduction
Undoubtedly, rice has become the most widely
cultivated and most important staple food in the world
since the beginning of agriculture nearly 10,000 years
ago. The incorporation of wild rice in ancient agricultural systems and its subsequent domestication led to
the development of many rice varieties adapted to a
wide range of environmental conditions, varieties
suited to the cooking and eating preference of local
people, as well as varieties suited to various aspects of
local culture such as religion. These factors have
driven farmers, breeders and consumers to continuously demand for favorable rice strains and establish
cultivars with stable genetic background. At present,
about 600 million tons of rice is produced all over the
world to feed about 3 billion people mainly living in
Asia, Africa and Latin America (1).
In recent years, there is a shift in balance of rice
production and consumption in Japan primarily due to
overproduction. The Japanese government now compels some farmers to abstain from growing rice by
paying them the equivalent amount of the expected
yield for a cropping season. As a consequence, the
current Japanese rice breeding programs shifted its focus from developing new varieties with high yield to
varieties with highly desirable characteristics such as
good eating quality to satisfy the demand of Japanese
consumers. This is partly because rice has become
just one of the main staples as more Japanese rely on
other cereal crops as major source of carbohydrates.
The annual consumption of rice in Japan is nearly 60
kg per person (2). The rest of needed calories and nutrition are derived from other food sources such as
wheat in the form of bread and noodle, and meat products from cattle, pig and chicken normally fed with
corn. These cereals are mainly imported from foreign
countries. Currently, the energy based self-supply rate
in Japan is less than 40% (3). This means that the fragile food supply situation in Japan will be seriously affected if the global balance between production and
consumption collapsed due to serious biotic or abiotic
stresses to crop production or a rapid decline of agricultural output in countries with rapid population
growth. To overcome such a critical situation, we
must consider how to increase the efficiency of production of many types of crops, especially cereals, using the limited ground area for cultivation. So far,
conventional breeding technology based on selection
by observation of preferable appearance and growing
characters has produced many elite varieties adapted
to each local environment. However, this traditional
strategy has almost reached its limit in terms of generating new rice varieties with highly desirable characteristics. This situation is particularly serious in Japan
because of limited genetic resources for the japonica
type of rice widely cultivated here. Therefore we must
search for other sources of genetic variation that could
be useful in modifying the rice plant and breeding
novel rice varieties.
All information for maintenance of life is incorporated in the genome or the entire DNA composition
of every species. This genetic information which controls all biological processes involved in the life cycle
2
Takuji SASAKI
of the plant must be explored in future breeding programs to obtain promised and reproducible results. In
Japan, the Ministry of Agriculture, Forestry and Fisheries (MAFF) launched the Rice Genome Research
Program (RGP) in 1991 with the aim of fully decoding the rice genome in 3 phases over a 21-year period
(4). The RGP progressed at an unexpectedly fast rate
and a molecular genetic map with more than 3,000
DNA markers was completed by 1997. Preliminary
physical mapping using these DNA markers and advances in the development of sequencing technology
using automated capillary sequencers further encouraged the MAFF and RGP to proceed into the next laborious job of sequencing the entire rice genome
earlier than originally scheduled. Then in 1997, an international collaborative working group for rice genome sequencing, the International Rice Genome
Sequencing Project (IRGSP), was organized (5). With
the aim of accelerating the completion of genome sequencing, the 12 rice chromosome were shared
among the 10 participating countries and Japan became in-charge of chromosomes 1, 2, 6, 7, 8, and 9
(6). This review article describes the genome sequencing strategy of the international sequencing collaboration with emphasis on the analysis of the high-quality
rice genome sequence and its utilization in functional
and applied genomics.
IRGSP Sequencing Strategy
The IRGSP decided to use a conventional mapbased strategy for genome sequencing to obtain the
rice genome sequence (7). This relies on two important factors, a correct physical map and a highly accurate sequence data. A physical map targeted for
sequencing was constructed with minimally tiled
BAC or its equivalent clones. To satisfy this requirement, the BAC clones in a library were digested by a
few restriction enzymes and their banding patterns
were compared by computer software called FPC.
Each contig generated by FPC consisted of two or
more overlapping BACs. For contigs that could not be
assigned to its genetic position, another strategy of
grouping of BACs was used in ascertaining the genetic location and orientation of contigs (8). A total of
about 6,000 PCR-based DNA markers genetically
mapped or physically linked to the genetic map has
been developed and used for BAC library screening to
identify BACs located at each DNA marker (9). Thus,
two or more BACs could be assigned to the same
marker. To make a bridge between these two contigs,
the end-sequences of gap-flanking BACs were used to
design new PCR primers for screening new BACs containing the target sequences. This chromosome walking strategy could easily fill short gaps but not exactly
wide gaps particularly those located at regions with repeat sequences. In such case, the gaps were measured
cytogenetically using the fiber FISH technology. Rice
DNA was extended as a fiber and fixed on a slide
glass. Then each of two gap-flanked BACs was
stained by each of two different fluorescent dyes and
these BACs were hybridized with the fixed DNA fiber. This strategy could be used to measure gaps with
a maximum 100 kb distance. Gap sizes of more than
100 kb such as the gaps at centromere and telomere
were estimated by a conventional FISH technology
(10).
The IRGSP completed the construction of an accurate physical map with a total length of 370.7 Mb
which covers 95.3% of determined rice genome size,
388.8 Mb (Fig.1, Table 1) (11,12). The length of
physical map matches the sequenced length obtained
by deleting sequence overlap between adjacent
mapped clones. The genome size of sequenced japonica rice cultivar Nipponbare is nearly 400 Mb less
than that previously reported (13). This may be attributed to the relative measurement of genome size by
flow cytometry. The genome size obtained by IRGSP
is an absolute value and therefore represents the actual size of the rice genome. The quality and reliability of nucleotide sequence were confirmed using the
Phred-Phrap-Consed computer programs. Although a
99.99% accuracy of the sequence was set by the
IRGSP, almost all of the 370.7 Mb sequence was decoded with more than this accuracy. Genomic regions
rich in repeat of the same nucleotide or rich in trans-
3
Fig. 1 The physical map of rice, Oryza sativa ssp. japonica, cultivar Nipponbare. Left and right
vertical bars for each chromosome represent the genetic and physical map, respectively. Triangles along each physical map indicate the position of the centromere. The encircled centromere region corresponds to sequenced contigs. Telomeres marked with circles have been
confirmed to contain the telomere specific repeat, CCCTAAA.
Table 1. Chromosome statistics based on analysis of sequence data and remaining gaps
chromosome
Sequenced
bases (bp)
1
2
3
4
5
6
7
8
9
10
11
12
Total
43260640
35954074
36189985
35489479
29733216
30731386
29643843
28434680
22692709
22683701
28357783
27561960
370733456
Gaps on arm regions
Total No.
5
3
4
3
6
1
1
1
4
4
4
0
36
Total length (Mb)
0.33
0.10
0.96
0.46
0.22
0.02
0.31
0.09
0.13
0.68
0.21
0.00
3.51
Telomere
gaps (Mb)
posons have been incorporated as phase 2 level low
quality sequence (14) and would re-analyzed as soon
as more reliable methods become available.
0.06
0.01
0.04
0.20
0.05
0.03
0.01
0.05
0.14
0.13
0.04
0.05
0.81
Centromere
gaps (Mb)
1.40
0.72
0.18
0.00
0.00
0.82
0.32
0.00
0.62
0.47
1.90
0.16
6.59
rDNA
(Mb)
6.95
0.25
7.20
Total length
(Mb)
45.05
36.78
37.37
36.15
30.00
31.60
30.28
28.57
30.53
23.96
30.76
27.77
388.82
Coverage
(%)
96.0
97.7
96.8
98.2
99.1
97.2
97.9
99.5
74.3
94.7
92.2
99.2
95.3
4
Takuji SASAKI
Characteristics of the Rice Genome Sequence
The decoded rice genome sequence was analyzed in terms of gene modeling, gene duplication
along each chromosome, chromosomal duplication,
distribution of class I simple sequence repeat (SSR),
distribution of transposable elements, insertion points
of rice endogenous retrotransposon Tos17, and insertion of chloroplast and mitochondria genome sequence (12). The gene prediction program FgeneSH
could predict a total of 37,644 genes excluding the
transposable elements. The averaged gene density is
9.9 kb per one gene. This value is about a half of that
reported in Arabidopsis thaliana, another fully sequenced plant species, mainly due to longer intron sequences in rice. Recently, detailed annotation of the
rice genome sequence was performed using rice fulllength cDNA sequences. As a result, about 30,000
rice genes could be identified with sufficient experimental evidence (15). This value however is much
lower than the number derived from gene prediction
using FgeneSH. In general, gene prediction programs
were designed to identify all probable genes in a sequence so that the number of predicted genes generated maybe more than the actual number of genes. In
addition, recent progress of molecular biology has
changed the concept on what a gene actually means.
A gene is defined conventionally as a DNA sequence
transcribed to RNA and then translated into protein.
However, not all of these RNAs are translated into
protein and some act as regulator of transcription of
other genes. Such RNAs are called non-protein coding RNAs and should also be classified as genes (16,
17).
The completed genome sequence provided a detailed characterization of SSR in the genome. SSR is
one of the most important sources of polymorphism
for breeding because of its high frequency in the genome. It can also be easily detected by PCR. Before
the completion of the genome sequence, searching for
SSR was time consuming and a very expensive task
involving the construction of a genomic library,
screening the library using a designed probe of a re-
peat sequence, sequencing the positive clones, and
confirmation of amplification by designed PCR primers flanking the SSR. The number of available SSR
prior to the elucidation of the genome sequence was
about 2,240 (18). This has increased several folds to
18,828 (12) after completion of the sequence. A comprehensive SSR information of the rice genome also
accelerates the detailed genetic mapping of target trait
for map-based cloning leading to successful identification of genes behind specific traits. SSR is also effectively used for tracing the evolution and/or
diversification of the genus Oryza.
The classification and distribution of transposons
within the rice genome were analyzed with the completion of the sequence. As in other eukaryotes, the
rice genome is characterized by a large content of
transposable elements. The insertion of transposons
into a genome results in the expansion of genome size
as shown by the sequence analysis of the Adh1 region
in maize (19). It is widely presumed that transposons
are biologically necessary in order to maintain the reproductive identity for each species. However, so far
there is no sufficient experimental data to support this
hypothesis. In total, both DNA and RNA type transposons occupy 35% of the rice genome (12). The
most abundant class 1 and class 2 transposons are
Ty3/gypsy and IS256/Mutator type, respectively. Class
1 also includes the most useful retrotransposon for
rice functional analysis, Tos17. Two copies of Tos17
have been identified in the rice genome. Although
Tos17 is inactive under normal conditions, it can be
activated during cell culture. The frequency of transposition depends on rice cultivars and varieties. In the
case of the japonica cultivar Nipponbare, transposition occurs at 10-15 loci during 3 months of cell culture (20). In another japonica variety Akitakomachi,
the frequency of transposition is 1.3-fold higher than
that observed in Nipponbare. Currently, 50,000 insertion mutant lines from cultivar Nipponbare have been
analyzed for phenotypes and among them, the flanking sequences of 20,000 lines have been identified
(21). Analysis of the flanking sequences for the remaining 30,000 lines is expected to be completed by
the end of 2007. The flanking sequences are easily referred to the genome sequence and the relationship between Tos17-inserted sequence and mutation is
immediately available. The data could indicate the
preference of insertion by Tos17 into an exon region
(12). Unfortunately, indica cultivar does not show
such transposition of Tos17 plausibly because of its inability to regenerate in cell culture.
Other characteristics of the genome such as gene
and segmental duplication along the 12 chromosomes,
nuclear and organelle genome sequence are described
in detail in the article published by the IRGSP (12).
Annotation of the Rice Genome Sequence
Annotation is a method of assigning biological
meaning to the genome sequence. To perform an accurate and reliable annotation, we need the information
on transcription of DNA to RNA. In case of Nipponbare, nearly 30,000 independent clusters of full-length
cDNA sequences are available (15). In addition to
these cDNAs, partial cDNA sequences (ESTs) of rice
and related plant species such as sorghum, wheat, barley and maize are available. In principle, an automated system was used to match cDNA to exons of
target gene (22). As a result, the number of protein
coding genes was estimated to be approximately
30,000. However, several practical problems such as
alternative splicing or error accidentally accompanied
with cDNA synthesis make this procedure complicated. These problems could be partially but not completely solved by manual curation of automated
annotation results. The Rice Annotation Project
(RAP) was organized to facilitate accurate annotation
of the sequence. So far, the functions of 19,969 annotated genes have been clarified, and 131 possible nonprotein RNAs have been identified. The annotation results are available at http://rapdb.dna.affrc.go.jp/ (23).
Accelerated Identification of Gene behind Phenotype
The most important and useful knowledge ob-
5
tained by the genome wide and extensive sequence information must be the identification of a gene
controlling a corresponding phenotype. Since the discovery of the genetic law by Gregor Mendel in 1865,
there was a strong interest in clarifying what directs a
phenotype and its difference in appearance as dominant/recessive character. It took more than one century since Mendelís time for the first success of
molecular genetic identification of responsible gene to
phenotype through the discovery of Huntington disease in 1993 (24). This success was realized by approval of indispensable utility of sequence
polymorphism to precise and detailed mapping of target phenotype. As stated above, accurately positionassigned and precisely decoded genome sequence is
the treasure box for finding and designing of molecular markers based on sequence polymorphism. The
first success of genetic identification of rice gene was
the case of disease resistance gene to rice bacterial
blight, Xa21 in 1995 (25). This was performed before
the launch of rice genome sequencing project, and after Xa21, several single Mendelian genes including another bacterial blight disease resistant gene Xa1 (26)
and dwarf gene d1 (27) were identified in 3-5 years.
Currently, single Mendelian rice genes are easily identified as those of Arabidopsis by both forward and reverse genetics.
In accordance with the progress of rice genome
sequencing and its data release, the required time for
genetic mapping has been very much shortened to a
period of 1-2 years. However, the time for preparation
of genetically pure segregating population cannot be
shortened without using some special facility for accelerating growth of plants. Recently, chromosome
segment substitution lines (CSSL) have been developed for detailed molecular genetic analysis of quantitative trait loci (QTL) by crossing japonica and indica
varieties followed by successive backcrossing with
one of the parents. It takes at least 5 years to establish
each CSSL, but once such CSSLs are developed, even
a gene involved in QTL could be identified within a
short period by using the genome sequence information (28). In fact, from year 2000 to present, many
6
Takuji SASAKI
genes involved in agriculturally important QTLs such
as Hd1 (29), Hd3a (30), and Hd6 (31) involved in
heading date, sh4 (32) and qSH1 (33) in shattering,
SKC1 (34) in salt tolerance, PSR1 (35) in regeneration
from callus, and Gn1a (36) in grain productivity have
been identified. Other important QTLs such as
drought tolerance are now being tackled by the Challenge Program of the Consultative Group on International Agricultural Research (CGIAR) to benefit poor
farmers using the fruits of genome research (37). A
list of identified rice genes is shown in Table 2.
Table 2. Partial list of identified rice genes
Gene name
Phenotype
Xa1
resistance to X.oryzae pv.
oryzae (race1)
resistance ro X.oryzae pv.
oryzae (race 21)
resistance to X.oryzae pv.oryzae
resistance to M.grisea (race003)
resistance to M.grisea
spotted leaf (lesion-mimic)
spotted leaf (lesion-mimic)
dwarf (daikoku)
Xa21
Xa26
Pib
Pita
Spl7
Spl11
d1
ebisu dwarf (d2)
slr1
dwarf
slender
gid1
dwarf
gid2
sd1
dwarf
semi-dwarf (dee-geo-woo-gen,
IR8)
abnormal tillering
lax panicle
timekeeper of leaf initiation
slender glume
fertility restoration
moc1
LAX
PLASTOCHRON1
slg
Rf1
Hd1
Hd3a
Hd6
Ehd1
SKC1
PSR1
Gn1a
sh4
qSH1
dl
pair1
pair2
Lsi1
OsTPC1
QTL of flowering time
QTL of salt tolerance
QTL of shoot regeneration
QTL of grain productivity
QTL of shattering
QTL of shattering
drooping leaf
homologous pairing aberration
homologous pairing aberration
defective in silicon uptake
dwarf, dark green leaves, greening of roots
Characteristics of predicted gene
product
NBS-LRR type of plant R-gene
Chromosome
reference
4
26
receptor-LRR type of plant R-gene
11
25
NBS-LRR type of pant R-gene
heat stress transcription factor
U-box/armadillo repeat protein
alpha subunit of heterotrimeric GTPbinding protein
cytochrome P450(CYP90D2)
transcription factor with DELLA motif
unknown, homology with hormonesensitive lipase family
F-box protein
GA20 oxidase (GA20ox-2)
11
2
12
5
12
5
54
55
56
57
58
27
1
3
59
49
5
50
2
1
51
46
GRAS family nuclear protein
helix-loop-helix transcription factor
cytochrome P450(CYP78A11)
ubiquitin-related modifier
mitochondrially targeted pentatricopeptide repeat protein
transcription factor, CONSTANS family
FT family
casein kinase CK2 family
B-type response regulator
sodium transporter
ferredoxin-nitrite reductase
cytokinin oxidase/dehydrogenase
unknown
BEL1-type homeobox protein
YABBY gene family
unknown, coiled-coil motif
chromatin interacting HORMA domain protein
membrane protein similar to aquaporin
calcium transporter protein
6
1
10
7
10
60
61
62
63
64, 65
6
29
6
3
10
1
1
1
4
1
3
3
9
30
31
66
34
35
36
32
33
67
68
69
2
70
1
71
Application of the Rice Genome Information in
Other Grass Species
It has been well known that colinear alignment
of genes exist along chromosomes among grass species (38). This is called synteny and is thought as remnant of evolution from common ancestor. In fact,
paleontological study reveals that current grass species diverged from their ancestor about 70 million
years ago. The phylogeny tree of the grass species
based on combined data from chloroplast restriction
sites, sequences of chloroplast genes such as rbcL,
ndhF, and rpoC2, and sequences of several nuclear
genes such as phytochrome B and granule-bound
starch synthetase, and 18S ribosomal DNA, and morphology provides concrete evidence on the history of
evolution of major cereal crops (39). It is shown that
an Oryza ancestor diverged from maize and then the
Triticum family diverged from the Oryza ancestor.
During this long history of evolution, genome structure of each species has been rearranged and differed
in sizes to create an independent reproductive system
and to adopt itself to each geological ecosystem accidentally propagated from its origin of birth. Instead of
experience of such pressure of rearrangement, gene
alignments along chromosomes of each species are
common like a patchwork structure among grass species. The genome size of rice is 390-400 Mb and is
the smallest among agriculturally important cereal
crops. Although the reason why the rice genome is
small has yet unknown, less amount of transposons in
the rice genome is very advantageous to physically
identify a target gene and to sequence the identified
genic region. Also rice is so far the only one grass species successfully transformed by an align gene (40).
Utilization of the syntenic relationship between
rice and other cereal crops to identify and/or isolate
genes have been carried out for several traits. The
most extensively challenged case was the isolation of
barley stem rust resistance gene, Rpg1, located on
chromosome 1P (S) by referring to the corresponding
region in rice in the distal end of the short arm of chromosome 6 (41). Unfortunately, although both flanking
7
regions to Rpg1 conserve the colinear relationship
within barley and rice, physical mapping and genome
sequence analysis revealed that rice genome lacked
the corresponding region to Rpg1 (42). Through the
genetic mapping studies of plant specific disease resistance genes (R-genes) to cereal crops, it was shown
that R-genes were rarely observed in corresponding
genomic regions being judged as syntenic relationship
(43). The case of R-gene might be an exceptional one
because of its rapid evolution to respond to rapidly
evolving pathogen.
Unlike R-genes, successful cases of gene identification based on synteny were reported. The most wellknown and important case was the identification of
the gene involved in perception of gibberellin signal.
Dwarfness is one of the important agronomic traits because the effective uptake of fertilizer is realized by
preventing lodging and the effective accumulation of
carbohydrate in seeds is expected. This is the basic
concept of Green Revolution projects for rice by IRRI
(44) and wheat by CYMMIT (45). Presently, both responsible genes in rice and wheat to dwarf are identified and characterized for further physiological and
biochemical studies. Although both are different types
of gene, sd1 of rice is gibberellin 20 oxidase (46) and
Rht1 of wheat (47) is transcriptional factor integral to
plantís response to gibberellic acid, it is interesting to
realize that both are related to gibberellin, a plant hormone regulating plant height. It is known that not
only gibberellin but also brassinosteroids regulate
plant height (48), but the Green Revolution projects
only used the dwarf caused by aberration of gibberellin biosynthesis or signal transduction. The sd1 gene
was identified by purely genetic method using only
rice mutant line (46). On the other hand, the identification of Rht1 gene was succesful using both the genomic and genetic information of rice and
Arabidopsis (47). Firstly, the gene corresponding to
dwarf of Arabidopsis, AGI, was isolated. Because of
similar characteristics of this dwarfism to that of Rht1
of wheat such as recovery of height by gibberellin,
they tried to find out homologous gene to AGI in rice
EST catalogue that contained many data at late 90ís.
8
Takuji SASAKI
One such rice EST was found and was used to screen
wheat genomic library. A positive clone was isolated
and used for mapping to confirm its location on wheat
chromosome 4A, 4B, and 4D. This genomic region
was shown as colinear with rice chromosome 3 and
maize chromosome 1 containing the dwarf d8. This
example clearly shows the validity of synteny for homologous gene isolation.
A more interesting point for rice was the elucidation of the function of the RHT protein. Mutation of
the corresponding Rht1gene in rice has an opposite effect inducing dwarf or slender character (49). This is
because the DELLA motif known as gibberellin response domain in the RHT1 of wheat carries mutation
in the amino acid sequence which causes dwarfness.
On the other hand, SLR1 of rice that is homologous to
RHT1 has a normal amino acid sequence in the
DELLA motif, but has mutation in other sequence
which destroys the normal function of this protein.
These observations indicate that the wild-type
RHT1/SLR1 protein is a transcriptional factor regulating gibberellin signaling pathway (49). Further analysis of slr1 related rice dwarf mutants insensitive to
gibberellin, gid1 (50) and gid2 (51), have been very
useful in identification of gibberellin receptor in the
nucleus. This has been achieved with the application
of synteny among rice, wheat and maize (38).
Perspectives
The complete, map-based and accurate rice genome sequence information is a very important treasure for human beings. Since the beginning of
agriculture, man has continued to improve the cereal
crops by repeated crossing and indiscriminate selection resulting in many cultivated crops that provide
the major source of food. This continuous effort has
been benefited by a innovative approaches to optimize
expected output such as the introduction of chemical
fertilizer and pesticide, application of conventional genetics based on phenotype, or the utilization of nonconventional methods such as cell culture and regeneration. Molecular genetics based on genome se-
quence is presumably the most powerful tool for
breeding new type of plants that would even encompass the genetic boundaries among species. The challenge to combine the genetic composition of two
different species has been pursued in the last 20 years
by cell fusion strategy to generate a new plant species
with only advantageous characteristics. However, this
strategy has not been very successful and most results
were opposite to this expectation. In case of cereal
crops, a hybrid of rye and wheat, or Triticale has been
tried to obtain a better cereal crop (52). These studies
are now better facilitated by DNA markers which
could be used to check the genomic position where
chromosomal chimera occurs. Such innovations will
help to accelerate the production of novel plant varieties with highly desirable characteristics.
The availability of a well characterized cereal genome information will promote research in many
plant phenomena such as the mechanism of polyploidization (53). Polyploidy is widely used in cultivated plants such as bread wheat, banana, and sugar
cane to induce enlargement of plant size, increase of
grain size and improvement of other agronomic traits.
Some of the wild species of genus Oryza such as O.
minuta are tetraploid and could be used as a model for
understanding the mechanism underlying the conversion from diploid to tetraploid and vice versa. Although polyploidy has yet to be utilized in cultivated
rice, it could be a promising strategy to increase grain
yield by balancing plant size, culm diameter, culm
strength and whole panicle weight.
As a model plant, the accurate map-based sequence of the rice genome could be used to promote
ground breaking researches on diverse plant species.
Ongoing comparative studies on other Oryza species
will facilitate exchange and interchange all of the secrets of diversification in the genus Oryza. Eventually,
a novel Oryza species with all the favorable characteristics of an ideal cereal crop may be generated based
on the complete knowledge of all biological processes
involved in the life cycle of the rice plant. A new paradigm of breeding based on genomics could be the
most effective approach to catch up with the events
that occurred during the past 10,000 years in the
course of natural evolution. The next 10-20 years
could be the turning point in understanding many biological phenomena based on this new paradigm.
16.
Acknowledgement
17.
I am grateful to Dr. Baltazar Antonio of Genome
Resource Center of NIAS for his useful comments
and English revision.
18.
19.
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Takuji SASAKI
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12
Takuji SASAKI
イネゲノム全塩基配列解読のインパクト
佐々木卓治
農業生物資源研究所
イネ品種「日本晴」の全ゲノム塩基配列解読が
国際協調体制（IRGSP）の下に２
０
０
４年末に完了し
た。この成功は，いうまでもなくイネ・コメに対
する世界中の多くの人々の関心の高さを示してい
る。コメは世界人口の半数が常食としており，副
食としての利用を加えればその需要度はコムギと
双壁をなす。むしろ，今後予測される人口増加地
域がコメを常食しているアジア地域であることを
考えれば，その生産量を増加させる戦略は地球規
模の協力により練られることが望まれる。その基
盤となるのが究極の遺伝情報の集積であるゲノム
塩基配列である。イネの全遺伝情報は，１
２本の染
色体に格納されている。IRGSPは，約３，
５
０
０個の
BAC/PACを染色体に沿って，由来した個所に正し
く整列再配置した地図を作製した。その結果得ら
れた全ゲノム長は３億９千万塩基対であり，その
９
５％にあたる３億７千万塩基対を９
９．
９
９％の精度
で解読した。完全解読された配列にはどのような
種類の遺伝情報が書き込まれているのであろう
か。基本的には「イネの全遺伝情報」が書き込ま
れているはずだが，現在の情報解読技術ではまだ
全てを手中にすることはできない。基本的なイネ
遺伝子数は，約４万個と予測される。これら予測
遺伝子と実際の転写産物（完全長cDNAおよび
EST）を照合し，存在の裏付けがとれるものは約
３万個である。これらについては文献データベー
ス検索等により機能を探り，約１万個については
既存の機能情報を付加することが行われている。
この成果は２
０
０
４年１
２月に開催された国際イネアノ
テーション会議で得られたものであり，公開され
ている。
塩基配列解読プロジェクトと並行して，各種イ
ネ表現形質に対応する遺伝子の同定を行うプロ
ジェクトが推進されている。ひとつは，遺伝子破
壊による逆遺伝学的方法，もうひとつは，
「日本
晴」の正確な塩基配列を基準にして，他のイネ品
種との詳細な多型情報を得て，遺伝解析を精密に
行う遺伝学的方法である。前者では，遺伝子破壊
因子として，イネゲノム中に本来存在するレトロ
トランスポゾンTos17の，細胞培養時の転移現象
を利用することで，安定的な挿入配列の保持を実
現しており，現在までに５万系統を作出してお
り，表現型データベースと破壊遺伝子の照合が進
めば，多くの未知遺伝子あるいは既知遺伝子で
あってもそれらの新たな機能が決定できるものと
期待される。一方，遺伝学的方法においては，ゲ
ノム塩基配列情報により従来よりも格段に迅速か
つ正確に，例えば戻し交配集団の中から必要なゲ
ノム断片を有する個体を選抜し，さらには遺伝子
が存在する候補領域を１０∼２
０kbにまで狭めること
が可能になった。複数個の遺伝子が関与する表現
型の場合でも，戻し交配集団からの正確な必要後
代選抜をゲノム情報に依存して行うことで，各々
を１遺伝子座支配によるものと分割して取り扱う
ことができる。出穂期やいもち病圃場抵抗性な
ど，多くの実例が示されつつある。
標準塩基配列の新たな利用面としては，
「比較」
ゲノム解析が挙げられよう。例えば，Oryza属内
で，多様な栽培イネ間の特性（表現型）の違いと
塩基配列多型の相関，選抜の繰り返しの結果起
こったであろう連鎖不平衡と対応する表現型，近
縁野生イネと栽培イネのゲノム構造比較による挿
入・欠落配列の特性と進化との関連，種間の生殖
的隔離の分子機構，等々の比較が容易になる。一
方，イネ科穀類間で遺伝子単離をシンテニーに基
づいて試みた例はいくつかある。成功例としては
Rht１/D８/slendarの例が挙げられる。これらはい
ずれも相同遺伝子の変異によって起こる表現型で
あるが，変異が起こる個所でこの遺伝子のジベレ
リンのシグナル伝達に関する機能に相違があるた
13
めに，異なる結果を生じている。今後は基盤的研
究と共に，主食料確保とその品質向上に向けた応
用研究に高精度イネゲノム全塩基配列情報が一層
利用されると期待される。
14
Takuji SASAKI
質疑応答
中川：先日，私は牧草類の分子育種にかかわる国
際会議に出ました。そのときに，やはりイネの
ゲノム情報を用いたcomparative mappingといい
ますか，牧草の場合には他殖性であるとか，あ
るいは倍数性が多いといった問題点があって，
そのもの自体をマッピングしていくのは非常に
困難があるのですが，このイネのゲノムを使う
と非常に簡単にできるということで，かなり広
く使われています。これはとりもなおさず農業
生物資源研究所の貢献だと思うのですが，これ
を牧草などが利用するときに，何か注意をした
らいい点がありましたら教えていただきたい。
佐々木：いや，むしろそれは私の方が教わらなけ
ればいけないことで，他殖性については非常に
悩ましいのです。今日話をしたほとんどは全部
自殖性の植物で，トウモロコシは他殖といって
も，実は自殖に近いものです。今おっしゃった
のは恐らくマーカーとして特に遺伝子として使
われると有効だということですから，注意して
ほしいのは重複等が結構ありますので，でき上
がったものが必ずしもピンポイントにはなって
いないということは，頭に留めていただけると
ありがたいかと思います。ただ，そのようにお
役に立っているという情報が非常にありがたい
わけです。
また，チャレンジプログラムといって，世界
を見渡して多くの作物種，特に発展途上国のた
めの食料確保に向けて，イネに限らず，キャッ
サバやバナナも含めて，CGIARの中でのプログ
ラムが今進んでいます。そこで私もバナナにか
かわっています。バナナは単子葉ではあるので
すが，イネとは遠いためにイネの情報が使える
可能性を探るということで少し協力させても
らっています。バナナが面白いのはpolyploidな
のです。しかも自然にpolyploid，三倍体になっ
ている。そういうメカニズム等はやはり，もち
ろん単純に染色体を倍数にしたらいいというも
のではないですが，そういったものも将来収量
を増やすときに一つ考えなければいけないファ
クターではないかと思って興味を強く持ってお
ります。
NIAS, Japan
15
GENOME AND POST-GENOME RESEARCHES IN LOTUS JAPONICUS
Masayoshi KAWAGUCHI
Department of Biological Sciences, Graduate School of Science, University of Tokyo
Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0033
Introduction
Legumes (Fabaceae) constitute the third largest
family of the angiosperms and compose of three subfamilies, 730 genera and more than 19,400 species
(Gepts et al. 2005; Levis et al. 2005). Subfamily Caesalpinioideae includes species such as Tamarindus indica, Saraca asoca, Cercis chinensis, Caesalpinia
decarpetala var. japonica, C. sappan, Gleditsia japonica, Chamaecrista nomame, Copaifera pubiflora. Subfamily Mimosoideae includes Acacia xanthophloea,
Albizia julibrissin, Leucaena leucocephala, Entada
phaseoloides, Mimosa pudica, Samanea saman
known as a rain tree. Papilionoideae forms the largest
subfamily and includes Glycine max, Pueraria lobata,
Amphicarpaea bracteata edgeworthii var. japonica,
Erythrina variegata, Apios fortunei, Strongylodon
macrobotrys, Flemingia prostrata, Cajanus cajan,
Clitoria ternatea, Vigna unguiculata, Vigna angularis,
Vigna radiata, Phaseolus vulgaris, Canavalia lineata,
Lespedeza buergeri, Lespedeza cuneata, Kummerowia
striata, Desmodium podocarpum ssp. oxyphyllum,
Arachis hypogaea ssp. hypogaea, Zornia cantoniensis, Hedysarum vicioides ssp. japonicum, Astragalus
sinicus, Oxytropis japonica, Glycyrrhiza uralensis, Indigofera pseudorinctoria, Crotalaria sessiliflora, Wisteria floribunda, Cytisus scoparius, Lupinus luteus,
Trifolium repens, Medicago sativa, Vicia faba, Lathyrus odoratus, Pisum sativum, Cicer arietinum, Robinia pseudocacia, Sophora flavescens, Pterocarpus
indicus. The remarkable diversity ranges from phenotypes, seed proteins, secondary metabolites to biologi-
cal interactions such as symbiotic nitrogen fixation
with rhizobia. Legumes also include economically important crops commencing with soybean and contribute a great variety of the uses, for example, food,
forage, fat, oil, green manure, medical drug, dyestuff,
rubber, beer, resin, cork, sand control tree, ornamental
plant and so on. Leguminous plants such as pea
(Pisum sativum), bean (Phaseolus vulgaris) and soybean have also contributed as an experimental plant in
the field of genetics and plant physiology. However,
due to their relatively large genome size, tetraploidy,
self-incompatibiliy, and the low transformation and regeneration frequencies, some important aspects of legume biology cannot be addressed at molecular level
especially.
Lotus japonicus
In 1992, Handberg and Stougaard in Aarhus University, Denmark, reported that L. japonicus (Regel)
Larsen naturally growing in Japan has a characteristic
suitable for molecular genetics (Handberg and Stougaard 1992) (Figure 1). L. japonicus is diploid
(2n=12), self-fertile perennial legume and capable of
stable transformation. L. japonicus has six chromosomes and small genome size (442 Mb per haploid of
an ecotype Gifu B-129) (Ito et al. 2000). The generation time is 3 months and up to 6,000 seeds can be
harvested from one plant. An accession Gifu B-129
derived from Gifu prefecture has been used widely
and a large number of the symbiotic mutants deficient
in nodulation and mycorrhization have been isolated
Masayoshi KAWAGUCHI
16
Fig. 1 Lotus japonicus
by EMS and ion-beam mutagenesis. Transposon tagging enabling fast gene identification from tagged
lines is feasible by introducing maize Ac transposable
element in L. japonicus (Schauser et al. 1999; Zhang
et al. 2003). Medicago truncatula closely related to alfalfa is also known as another model legume for reverse and forward genetics (Cook 1999, May and
Dixon 2004). M. truncatula has also been used by a
growing number of researchers mainly on France and
USA.
Miyakojima MG-20
Miyakojima MG-20 is an accession introduced
in 1998 in order to facilitate molecular genetic analysis in L. japonicus (Kawaguchi 2000). The origin of
Miyakojima is a Miyakojima island of Okinawa prefecture, the southernmost island of Japan. Miyakojima plants show the earliest flowering trait among
accessions of L. japonicus collected, and set many
flowers and pods under fluorescent light. The germplasm was established by self-crossing 7 times in an
insect-free small Biotron at Komaba campus in University of Tokyo. The generation time (from a seed to
the next generation of seeds) of Miyakojima is two
months under a light intensity of 150 Es -1m-2. Even under lower light condition (57 Es-1m-2), the plant set its
first flowers in 53 days and set pods within 3 months
after germination. These features indicate that Miyakojima is suitable for indoor handling and genetics.
DNA polymorphisms by amplified fragment length
polymorphism (AFLP) were evaluated among 15 accessions and the different species derived from Algeria, L. filicaulis (Kawaguchi et al. 2001). As a result,
Miyakojima was found to have 4.5% polymorphism
compared to Gifu, which is approximately two times
larger than the combination of Gifu and other accessions. On the other hand, L. filicaulis was found to
have 36.7% polymorphism, indicating that Miyakojima has moderate for DNA polymorphism with Gifu.
Then in order to examine the genetic analysis, Miyakojima was reciprocally crossed with Gifu and the
symbiotic mutants. F1 plants grew well and set a number of pods as their parents. Red stem color and trichomes observed in Gifu were inherited dominantly
when Miyakojima was crossed as a female partner. In
addition, four different kinds of symbiotic mutants isolated from Gifu (Ljsym70 [nodulation], Ljsym72
[nodulation- mycorrhization], alb1-1 [cooperative
histogenesis], and har1-5 [hypernodulation]) were
crossed with Miyakojima. F2 populations demonstrated that these mutant phenotypes were segregated
in a recessive and monogenic manner following Mendelian inheritance. These findings indicate that Miyakojima is suitable as a crossing partner of Gifu.
Today, Miyakojima has been used for a large-scale expressed sequence tags (ESTs) and whole genome sequencing analyses in Kazusa DNA Research Institute.
The seeds of Miyakojima can be obtained together
with Gifu from National Bio-resource Project, Legume Base
http://www.shigen.nig.ac.jp/legume/legumebase/index.jsp.
Construction of molecular linkage map
Many researchers and postgraduates interested in
molecular genetics in legume moved in concert to develop a molecular linkage map of L. japonicus based
on amplified fragment length polymorphism (AFLP),
simple sequence repeat (SSR) polymorphism and derived cleaved amplified polymorphic sequence
(dCAPS). The F2 population derived from a cross between Gifu B-129 and Miyakojima MG-20 was used
for mapping. The resulting linkage groups correspond
to the six pairs of chromosomes of L. japonicus and
consist of 564 markers with nearly 480 cM length
(Hayashi et al. 2001). Using the same cross combination, Kazusa DNA Research Institute placed 1,310
TAC (transformation-competent artificial chromosome)
clones on the genetic map using 691 SSR and 80
cleaved amplified polymorphic sequence (CAPS)
makers (Young et al. 2005). On the other hand, Danish group constructed the molecular linkage map with
a total of 524 AFLP markers, 3 RAPD markers, 39
gene-specific markers, 33 SSR markers with 367 cM
length based on an inter-specific cross of L. japonicus
and L. filicaulis (Sandal et al. 2001). These markers
associated with sequence information provide enormous value in positional cloning in L. japonicus. Actually positional cloning using them enabled to
identify the symbiotic genes such as SymRK essential
for both mycorrhization and nodulation (Stracke et al.
2002), Har1 involved in autoregulation of nodulation
via long-distance signaling (Krusell et al. 2002;
Nishimura et al. 2002), Nfr1 and Nfr5 encoding possible Nod factor receptors (Madsen et al. 2003,
Radutoiu et al. 2003), Castor and Pollux encoding putative ion channel crucial for fungal and rhizobial symbiosis (Imaizumi-Anraku, Takeda et al. 2005) and
Sst1 indispensable for symbiotic nitrogen fixation
(Krusell et al. 2005). For mapping and quantitative
trait loci (QTLs) analysis, recombinant inbred lines
(RILs) have been made from intra and inter-specific
crosses of Lotus (Udvardi et al. 2005).
17
Sciences analyzed gene expression profiles during
early stages of formation of symbiotic nitrogen-fixing
nodules by means of macroarray analysis (Kouchi et
al. 2005). They found that expression of a total of
1,076 genes which expression was significantly activated during the stages from bacterial infection to nodule organogenesis involving the onset of symbiotic
nitrogen fixation. These genes include a number of
novel nodule-specific genes or enhanced genes that
are related to defense responses, phytohormone synthesis, signal transduction, membrane transport, cell
wall synthesis and transcriptional regulation.
To obtain an unprecedented overview of the
metabolic and signaling differentiation accompanied
with nodule development, Udvardi et al. in Max
Planck Institute for Molecular Plant Physiology in
Golm, Germany, generated more than 5,000 nodule
cDNA clones and identified approximately 860 genes
using the array that were activated in nodules at transcriptional level (Colebatch et al. 2004). One third of
these are involved in metabolism and transport, and
over 100 genes encode proteins that are likely to be involved in signal transduction and transcriptional regulation. Regarding reproductive organ development,
Watanabe et al in Iwate University used a cDNA microarray derived from flower buds of L. japonicus
(Endo et al. 2002). Cluster analysis allowed them to
identify 132 independent cDNA groups that were predominantly expressed in immature and mature anthers.
RNA interference
ESTs and transcriptomics
To know the comprehensive feature of genes expressed in legume, Kazusa DNA Research Institute
generated a total 74,472 3' -end expressed sequence
tags (ESTs) from cDNA libraries produced from nodulated roots, seedlings, flowers and pods of L. japonicus (Asamizu et al. 2004). Clustering of the ESTs
revealed 20,457 non-redundant sequences composed
of 8,503 contigs and 11,954 sigletons. Using the
18,144 non-redundant ESTs among these sequences,
Kouchi et al. in National Institute of Agrobiological
As of 4th February 2005, L. japonicus EST projects have generated 111,471 ESTs derived from a variety of cDNA libraries, and the sequences are
available in public database (Udvardi et al. 2005).
These ESTs clustered into 28,460 unique sequence
contigs. To dissect the gene function in legume-specific phenamena, RNA interference (RNAi) is one of
a potent means of eliminating gene expression in
plants and animals (Plasterk and Ketting 2000). To
demonstrate whether RNAi works in L. japonicus,
two groups generated RNAi constructs against leghea-
Masayoshi KAWAGUCHI
18
moglobin genes that are predominantly expressed in
root nodules, and introduced into roots via
Agrobacterium-mediated transformation (Kumagai
and Kouchi 2003; Ott et al. 2005). As a result, the
transcript levels could be significantly reduced
enough to develop ineffective nodules containing high
levels of free oxygen in independent RNAi lines.
RNAi should be applied to clarity gene function related to legume-specific phenomena such as symbiosis, second matabolites and storage of proteins in
future.
TILLING
TILLING (Target induced Local Legions in Genomes) is a reverse genetics tool that identifies individuals having point mutations in genes of interest in
a population of EMS-mutagenized M2 plants (McCallum et al. 2000a, b). Labelled gene-specific primers
are used to amplify a functional domain of the gene.
The PCR products are subjected to form heteroduplexes between wild type and a mutant line that are
then treated with a endonuclease derived from plant
celery, CEL1. This enzyme specifically cleaves the
products at the sites of mismatch that allow us to identify the plants containing a one base mutation in the
gene of interest.
A large population of M2 L. japonicus seeds (approx. 76,000) from approx. 5,000 EMS-mutagenized
seeds were grown by Trevor Wang, John Innes Centre, and J. Perry, Martin Parniske, Sainsbury laboratory (present position, Munich University) (Perry et
al. 2003). One plant from each family was selected
and the genomic DNA was extracted from each plant
to provide the TILLING population. They have
TILLed both the pre-selected nodule mutant population and the general population for sucrose synthase
(SUS1) mutants. Sucrose synthase catalyses the cleavage of sucrose to fructose and UDP-glucose, enabling
plant tissue to metabolize the starch. In total, they
have TILLed 17 different gene fragments in their general TILLING population and have identified 93 mutations, of which 1% are truncations, 64% result in
missense changes and 35% cause silent changes. The
average mutation frequency is 6 mutations per 2.3
Mb, indicating the M2 population bears 1,300 mutations per Lotus genome.
Genome analysis
L. japonicus is a target of large-scale genome sequencing projects, together with M. truncatula. Miyakojima has been used for the whole genome
sequencing project by KAZUSA DNA Research Institute. As of January 2005, approximately 165 Mb of
the genome sequence in L. japonicus (45 Mb, phase 3
(finished); 120 Mb, phase 1 (still in draft)) is available
from public data base (Young et al. 2005). Gene density is estimated 1 gene per 6.3 kb basen on Fgenesh
predictions. Sequencing a total of 300 Mb will cover
almost all gene-rich region. Sequencing of the generich regions will be scheduled for completion by the
end of 2006. Information about the L. japonicus genome sequence and ESTs can be accessed through
Web sites
(www.kazusa.or.jp/lotus/).
The web site includes information on DNA markers,
genetic linkage maps, recombinant inbred (RI) lines,
nucleotide sequences TAC and BAC clones, annotation of predicted genes.
Synteny
Microsynteny refers to conserved gene content
and order over a short, physically aligned DNA contig
between interspecies. In 2000, I and colleagues found
a microsynteny between Lotus and soybean during the
process of positional cloning of har1 hypernodulating
mutant. har1 mutants (Wopereis et al. 2000; Kawaguchi et al. 2002), like soybean nts1 hypernodulating
mutants (Carroll et al. 1985; Akao and Kouchi 1992),
are unable to produce autoregulation signal from the
shoots (Delves et al. 1986). Assuming microsynteny
with the soybean nts1 genomic region, L. japonicus
orthologues of soybean RFLP makers expected to be
located in the vicinity of the Nts1 gene were searched
for a database of L. japonicus ESTs and used for link-
age analysis. In this way LjSUT1, an orthologue of
Gm221 (sugar transporter), was shown to be tightly
linked to the har1 gene at a distance of 0.8 cM. A
TAC clone, LjT25N16, carrying the LjSUT1 gene,
also contains the gene for fructose-bisphosphate aldolase whose soybean orthologue Gm036 has been
mapped to the vicinity of nts1 indicative of presence
of microsyntheny between Lotus Har1 and soybean
Nts1 regions. A map-based cloning strategy showed
that the Har1 gene encodes a receptor-like kinase
with leucine-rich repeats in extracellular domain
(Krusell et al. 2002; Nishimura et al. 2002). Interestingly, Har1 showed the highest level of identity with
CLAVATA1 (CLV1) of all Arabidopsis receptor-like kinases (Clark et al. 1997). CLV1 is known to negatively regulate formation of the shoot apical meristem
via cell-cell communication. Sequencing of a soybean
orthologue, GmCLV1B showed that the hypernodulating EMS-induced mutant En6500 that is allelic to
nts1 mutant has a nonsense mutation near the transmembrane domain (Nishimura et al. 2002), indicating
that molecular genetics of L. japonicus could make a
contribution to identify the genes responsible for soybean mutant phenotype.
Today, in addition to L. japonicus and soybean,
synteny can be recognized in various leguminous species such as M. truncatula, alfalfa, pea, chickpea,
mungbean and common bean. Simplified synteny
maps for these 8 species have been constructed in legume for the first time (Choi et al. 2004; Zhu et al.
2005). Using the simplified consensus map as well as
microsyntheny, identification of the genes for QTL is
on going in pea and soybean.
Towards functional analysis of genes that form a
tandem cluster
Genomic sequencing in L. japonicus has revealed a number of genomic regions with tandem cluster. For example, L. japonicus has at least four
chalcone isomerase (CHI) genes that are fundamental
in the generation of ecophysiollogically active flavonoids and that they form a tandem cluster within 15
19
kb (Shimada et al. 2003). Five of six dihydroflavonol
4-reductase (DFR) genes that are responsible for the
anthocyanin and condensed tannin synthesis also form
a cluster within a 38 kb region in the L. japonicus genome (Shimada et al. 2005). Leghemoglobin genes
are also tandem arranged in the relatively narrow genomic region (Uchiumi et al. 2002). Given that these
genes are functionally equivalent, EMS mutagenesis
that introduces point mutations is thought to be ineffective to define the gene function. TILLING would
enable to identify the individuals having a point mutation, but the double or triple mutants in genes that
form a tandem cluster would be hardly obtained due
to the low recombination frequency among the genes.
In order to clarify the function of genes forming
a tandem cluster and perform saturation mutagenesis
in L. japonicus, mutational approach involving large
deletion in the genome would be promising in future.
It is known that ion-beam and gamma-ray irradiations
often create large deletion in the genome although
they involve chromosomal arrangement such as inversion and translocation (Shikazono et al. 2005). Whole
genome analysis using microarrary and/or oligo chips
made referring to L. japonicus ESTs and genome sequence would allow us to rapidly identify the genomic regions including large deletion in the beamirradiated individuals. Mutagenesis involving genomic deletion would also be beneficial for functional
analysis of genes encoding microRNAs and short peptides that regulate plant growth and development.
L. japonicus opens the way to the future research
Through concentrated development of infrastructure based on the cooperation and genome sequencing
project by Kazusa DNA Research Institute, L. japonicus is getting the remarkable results in the field of
symbiosis with rhizobia and arbuscular micorrhizal
fungi. To clarify the all gene function, mutagenesis by
ion-beam and gamma-ray irradiations that delete a tandem gene cluster will be required. In addition,
through the integrated analysis of microsynteny
among model and crop legumes mainly on the most
Masayoshi KAWAGUCHI
20
important crop, soybean, a large number of the genes
leading to usefulness such as oil production, pathogen
and insect resistance will be identified. Extension of
the microsyntheny analysis from Papilionoideae to
Caesalpinioideae and Mimosoideae would make a
large contribution towards understanding of the remarkable biodiversity at molecular level. Legume issues in Plant Physiology in 2003 and 2005 are telling
the arrival of the new era of the extensive and profound research in legume.
9.
10.
11.
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23
ミヤコグサのゲノム・ポストゲノム研究
川口正代司
東京大学大学院理学系研究科生物科学専攻
質疑応答
長戸：ミヤコグサをモデル植物として考えられた
きっかけは，多分，共生が主要なターゲットだ
と思うのですが，それ以外のものについても，
やはりマメ科と例えばアブラナ科はだいぶ形態
的にも違いますから，その辺の話，あるいはそ
の辺の変異体の蓄積とか，いかがでしょうか。
川口：確かにマメは根粒バクテリアと共生し，窒
素固定をするので，当初その分子機構に関心を
持つ研究者が多かったのですが，その後，マメ
独自の形態をターゲットにした研究が徐々にで
すが，始まりつつあります。
一つは葉の形態で，例えばミヤコグサの葉は
複葉で，シロイヌナズナやイネにはそれはあり
ません。葉の作りが違うので，その仕組みがど
うなっているのかとか，蝶形の花も，放射相称
ではなく，左右相称なので，それがどのように
して形成されるのかとかです。
今回は紹介しませんでしたが，種の数という
点から植物の中で最も種のバリエーションに富
む属がマメのファミリーにあって，それはAstragalusというレンゲの仲間です。レンゲの仲
間が２，
０００種ほどあって，植物全体の１％ぐら
いの非常に大きなファミリーを形成していま
す。最近ではAstragalusの分子系統などの研究
も進みつつあります。ファミリーの中の多様性
や，形質の違い，それらと遺伝子をつなげてい
こうという研究が徐々にですが，始まりつつあ
ります。
ただ，遺伝子情報は出てくるのですが，それ
らの機能ということになると，まだかなり距離
があります。ミヤコグサの遺伝子の機能解析は
やりやすくなりましたが，ほかのマメでどれほ
ど遺伝子の機能解析ができるかということにな
ると，まだこれから形質転換の系を開発してい
く必要があります。
長戸：CLAVATA1の話しですが，イネでは基本
的にシロイヌナズナと同じことをやっていて，
HAR1は多分全然別のものです。CLAVATA1の
オーソログ（ortholog）はあれしかないのですか。
川口：それはよく聞かれる質問なのですが，植物
にとってメリステム（meristem）は非常に重要
ですので，マメにもCLAVATA1に相当するもの
があると思っていて，ずっと探しているわけで
すが，今のところ見つかっていません。実はミ
ヤコグサでメリステムに影響する新しい変異体
を見つけています。そこからCLAVATA1ではな
いものがマメのメリステムを制御している可能
性が示唆されています。ですので，ナズナやイ
ネとは違うものが，マメのメリステムを制御し
ているのではないかと考えています。
長戸：それでちょっと思い出したのは，例えば
LEAFYもシロイヌナズナのLEAFYとエンドウ
のLEAFYは多分全然機能が違いますよね。イ
ネのLEAFYは多分シロイヌナズナのLEAFYと
大体似ている。ということは，マメ科というの
はめちゃくちゃ変なのですか。
川口：そうかもしれません。最近私が思うのは，
マメ科のゲノムでかなり変なことが起きたとい
うことです。今言われたように，LEAFYのよう
な遺伝子というのはもっと広く植物で共通で
あってもいいのですが，実際マメでLEAFYの遺
伝子がつぶれると，異常な花芽が作られる他に
複葉が１枚の葉っぱになってしまうのです。進
化プロセスで遺伝子機能がどのように変わって
きたかというのはまだよく分からないわけです
24
Masayoshi KAWAGUCHI
が，そうした例が今後一つ一つ分かってくるで
しょう。ひょっとしたらマメの内部で一部シス
テムの転換みたいなことがあったかもしれない
のですが，そうしたことが将来分かっていくの
ではないかと思っています。
NIAS, Japan
25
GENOME ANALYSIS AND BREEDING IN CITRUS
Mitsuo OMURA
Shizuoka University
Ohya 836, Suruga-ku, Shizuoka 422-8529
Introduction
Citrus fruits are among the most popular fruits
cultivated in Japan and around the world. The long
history of citrus culture has generated many types of
citrus, including Satsuma mandarin (C. unshiu),
Clementine (C. clementina), sweet orange (C. sinensis), grapefruit (C. paradisi), lemon (C. limon), and
yuzu (C. junos). These species show a wide diversity
in fruit characteristics, such as fragrance, pigments,
size and shape of juice sacs, oil glands, and developmental patterns of the fruit rind. Citrus fruits contain
various bio-functional and health-promoting compounds, including carotenoids, limonoids, terpenoids,
alkaloids, flavonoids, coumarins, and major vitamins
and nutrients. The wide diversity in citrus provides
the source to generate new cultivars by breeding. Hybridization has generated inter-specific hybrids such
as tangor and tangelo, and new hybrids are screened
by individual selection. However, the long duration of
the juvenile phase until flowering and the large tree
size of citrus holds back the development of a systematic breeding program based on genetic information.
The Citrus Genome Analysis Team (CGAT) of
the National Institute of Fruit Tree Science (NIFTS)
has started genome analysis to obtain genetic and
physiological information on citrus fruit production
and quality. CGAT has focused on EST cataloging
and its application in breeding since 1993. cDNA libraries derived from fruits and flowers at different developmental stages were constructed to obtain a wide
set of genes involved in fruit development and quality,
affecting features such as such as seedlessness, peel-
ing, sugar content, and accumulation of bio-functional
compounds. By 2005, 33 108 EST clones had been
analyzed with the support of the STAFF Institute (Table 1).
EST clones and sequences are used as multi-purpose tools for the study of fruit physiology, molecular
biology, and breeding (OMURA et al. 2000). They
have been used to research fragrance (SHIMADA et al.
2004) and in the induction of precocious flowering for
the assay of gene function in fruit ( ENDO et al. 2005).
Recently, a prototype microarray with 2213 spots
( SHIMADA et al. 2005a) and a revised GeneChip with
21 500 UniGene-derived oligo DNAs have been produced to promote the molecular analysis of fruit development and quality. In the manuscript, recent study
on Citrus genome mapping by EST based DNA markers and their application to breeding will be introduced.
Linkage map by CAPS markers based on ESTs
1) Generation of CAPS markers in Citrus
The EST sequences were used to generate CAPS
markers (KONIECZNY and AUSUBEL , 1993) for construction of genetic map of Citrus. Approximately
40% of primer pairs based on the ESTs produced
polymorphic PCR fragments after digestion by recognition enzymes ( UEDA et al. 2003). A linkage map of
the core mapping population of the cross between Miyagawa wase (Citrus unshiu) and Kiyomi (MiyaTrovita orange [C. sinensis]) was
gawawase)
constructed with 394 CAPS markers covering 800 cM
(OMURA et al. 2003). The CAPS markers were also
Mitsuo OMURA
26
Table 1. The sources of EST catalogs analyzed in CGAT.
Library
Originating cultivar
Tissue & stage
VSS
FRI
FRM
ALM
OVA
ALP
WFY
BFC
FBI
GSA
RGP
SLG
YJS
PCC
EIC
STG
ANT
LLL
EGJ
FOHK8S
FOSK8S
SHGA1
Valencia orange
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Miyagawa wase
Valencia orange
Miyagawa wase
Miyagawa wase
Lisbon lemon
Kinokuni
Kinokuni (Hira)
Seedless kinokuni
C. tamurana
Young seed
Fruit pulp, developing
Fruit pulp, maturation
Albedo, maturation
Ovary, flowering
Albedo, peeling initiation
Whole fruit, young
Rind, coloring
Flower bud, 30 days before flowering
Seed, imbibition 4 days
Root, seedling 2 weeks
Shoot, seedling 2 weeks
Juice sac, 60 days after flowering1
Callus, proliferating
Callus, embryogenesis
Stigma, flowering
Anther, flowering
Leaf, young
Ovule 60-70 DAF
Ovule 56 DAF
Ovule 56 DAF
Style, flowering
used to construct linkage maps of 4 other mapping
populations of Citrus.
2) Mapping of the breeding objective traits in Citrus
Seedlessness is one of the most important traits
in citrus breeding. Some sources are available for
breeding ( YAMAMOTO et al. 1995, 1997). A practical
seedlessness gene derived from Satsuma mandarin
was mapped on linkage group 6 of the core mapping
population at a major QTL related to seed number
( OMURA et al. 2003). A second source of seedlessness was derived from Mukaku-kishu (C. kinokuni);
the seedlessness is regulated by a single dominant
gene ( YOSHIDA et al. 2005). The locus was located on
linkage group 9 through the genetic analysis of a
population of 94 hybrid individuals of a cross between Okitsu-46 , an early maturing hybrid, and NO5í, a seedless hybrid of derived from Mukaku-kishu í(C.
kinokuni). Male sterility also contributes to the production of seedless fruit. The male sterility of Kiyomi
tangor was regulated by 3 genes ( NAKANO et al.
2001), 2 of which were mapped on linkage group 8 in
No. of clones
577
1,054
386
622
827
941
1,688
1,654
2,367
1,920
960
1,920
920
960
1,152
3,552
2,600
2,016
2,112
980
980
1,920
the third mapping population of a cross between Kiyomií and Okitsu-41 ( NAKANO et al. 2003). The loci involved in seedlessness from 3 different sources were
shown in the integrated CAPS linkage map (Fig.
1)( OMURA et al. 2002a).
In addition, the traits related to fruit qualities
such as sugar and acid contents, peel thickness, rind
and pulp color, and carotenoid contents were also analyzed and mapped on the CAPS linkage maps as
QTLs (OMURA et al 2002b).
3) Generation of DNA markers for early selection
Citrus breeding is costly in terms of land, labor,
and facilities because of the long juvenile period and
large tree size. Therefore, breeders have pursued early
selection to allow preliminary screening of unsuitable
individuals at the seedling phase before field planting.
Seedlessness, resistance to diseases such as citrus tristeza virus (CTV), and higher contents of health-promoting substances are important targets for breeding
by DNA marker screening. If seed-bearing individuals
could be removed early from the breeding population,
27
Fig. 1. An integrated CAPS linkage map. The photographs show the loci related to seed characteristics, polyembryony on linkage group K-01, embryo color on K-03, seed number (major QTL) derived from S
ë atsumaí
on K-06, male sterility on K-08, and seedlessness derived from ëMukaku kishuíon K-09..
the field performance of breeding would be greatly
improved. Markers tightly linked to the seedlessness
are the most desired for breeding programs. However,
there has been no a wide-use markers to select the
seedlessness from Mukaku kishu , so far. Mukaku
kishuí had common restriction site on the CAPS
marker linked to seedlessness with the majority of
breeding source cultivars, and the rare restriction site
linked to it were derived from the other parent cultivar, Lee, of NO-5. Therefore, to generate new markers widely applicable to selection of the seedlessness,
the analysis of genomic sequence near the locus for
seedlessness has been started ( YAMAMOTO et al.
2005) by using a BAC library which was constructed
from Satsuma mandarin Miyagawa wase; it consists
of 36 864 clones of 130 kb on average, covering 13.8
¹the genome ( SHIMADA et al. 2005b).
4) Typing of citrus cultivars by DNA markers
Most recent commercial cultivars have been derived from hybridization among a limited range of species and cultivars ( NESUMI et al. 2003). CAPS
markers designed from citrus ESTs were applied to
genotyping of citrus species and hybrid cultivars
( UEDA et al. 2003). A set of CAPS markers could provide the molecular proofing to identify cultivars.
To visually display the genetic variation among
citrus cultivars, CGAT analyzed cultivar genotypes
throughout the genome. Fig. 2 compares the genotypes of Kiyomi and Tsunokaori (Kiyomi Satsuma).
They have similar characteristics but can be distinguished in only 5 regions with different graphical
genotypes among loci in all the linkage groups. On
those regions, QTLs for rind thickness, carotenoid
content, and locule number, and other traits in which
28
Mitsuo OMURA
the two cultivars differ were detected in the mapping
population between Kiyomi and Satsuma ( OMURA et
al. 2002b). Thus, the genotypes of cultivars might be
linked to the phenotypes to obtain information useful
in breeding.
Databases of cultivar genotypes linked to QTLs
and other breeding information would make useful
breeding resources. An example was displayed in Fig.
3 as a prototype. To establish such a database, the integration of marker genotypes and breeding information
and association analysis of genetic resources will be
needed soon.
Fig. 2. Comparison of graphical genotypes of 2 cultivars, ' Kiyomi' and ' Tsunokaori' , displayed with QTLs
(arrowheads) for fruit characteristics (R, rind thickness; S, seed number; L, locule number; A, acid
content; C, carotenoid content). The genotypes of each locus of 2 cultivars were shown in the figure.
Fig. 3. An example of integration of genome information on a linkage map for breeding design. QTLs on
linkage group 4 for fruit characteristics are associated with the genotypes of breeding source cultivars.
29
5) DNA marker-mediating gene introgression from
a wide cross
CTV resistance is also an important breeding objective ( YOSHIDA 1993). Resistance is controlled by a
single dominant gene derived from Poncirus trifoliata
and mapped on linkage group 2. However, intergeneric hybrids, such as those between Citrus and Poncirus, have many unfavorable traits such as fruit
bitterness, high acidity, poor flavor, deciduous growth
habit, and long spines. As a result, backcrossing is
needed to introgress more citrus alleles. Therefore, a
breeding program to produce CTV-resistant plants
with edible fruit requires many years and several generations. But graphical typing of seedling progeny derived from Poncirus could be used to confirm the
degree of replaced alleles. The degree of replacement
in the BC2 generation was estimated as 35% to 73%
among 124 marker loci scattered throughout the genome (OMURA et al. 2005). The graphical genotype
of linkage group 2 also showed that 7 progeny among
94 individuals contained recombination within 10 cM
of the CTV resistance locus (Fig. 4). These recombi-
nants could be candidate parents for the next generation to produce introgression lines.
Summary
Citrus genome analysis in Japan was initiated to
obtain basic information to improve fruit characteristics through systematic breeding. It has focused on
EST cataloging. In addition to the use of ESTs to investigate fruit physiology, EST sequences were also
used to design DNA markers and to construct linkage
maps. CAPS markers were derived as tools for selection in practical breeding programs, for cultivar genotyping, and for linking markers on the linkage map to
physical maps of BAC clones. The recent status of
DNA markers and future work in Citrus genome
analysis and breeding are described.
References
1. ENDO , T., SHIMADA , T., FUJII , H., KOBAYASHI , Y.,
, T., and OMURA ., M. (2005) Ectopic expression
Fig. 4. Graphical representation of the CTV resistance gene region in linkage group 2 in BC2
progeny derived from Poncirus trifoliata. The susceptibility (S) or resistance (R) of lines
recombinant near the Ctv locus and of the parents is displayed. Replacement (%) shows
the percentage of loci that Poncitrus allele was replaced with Citrus allele by back-cross
among 124 loci placed in all linkage groups..
30
Mitsuo OMURA
of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata
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T., and OMURA , M. (2003) Evaluation of a CAPS
method based on ESTs in Citrus. Proc. Intl. Soc. Citricult. IX Congr. 2000:116-117.
15. YAMAMOTO , M., MATSUMOTO , R., and YAMADA , Y.
(1995) Relationship between sterility and seedlessness
in citrus. J. Jpn. Soc. Hort. Sci. 64:23-29.
16. YAMAMOTO , M., MATSUMOTO , R., OKUDAI , N., and
YAMADA, Y. (1997) Aborted anthers of Citrus result
from gene-cytoplasmic male sterility. Sci. Hort. 70:914.
17. YAMAMOTO , A., KATO , K., SUGIYAMA , A., SHIMADA , T., ENDO , T., FUJII , H., SHIMIZU , T., and
OMURA, M. (2005) Development of DNA markers
based on genome DNA sequence of Miyagawa waseí
BAC clones. J. Jpn. Soc. Hort. Sci. 72 (Suppl. 2): 282.
18. YOSHIDA , T. (1993) Inheritance of immunity to citrus
tristeza virus of trifoliate orange in some citrus intergeneric hybrids. Bull. Fruit Tree Res. Stn. 25:33-43.
19. YOSHIDA , T., NESUMI , H., YOSHIOKA , T., ITO , Y.,
UENO , I., and YAMADA , Y. (2005) Kankitsu Chukanbohon Nou 5 Gou (Citrus Parental Line Norin No. 5) is
useful for breeding seedless and early maturing cultivars. Bull. Nat. Inst. Fruit Tree Sci. 4:47-52.
31
カンキツにおけるゲノム解析と育種
大村三男
静岡大学
カンキツのゲノム解析は，現在，アメリカ，ス
ペイン，ブラジルなど世界各地で進められている
が，我が国では，１９９３年から主として果実形質の
改良の基礎的情報を得る目的で開始された。主と
図を構築するために利用された。ここで作成され
たCAPSマーカーは，無核性や耐病性などの育種
における重要形質の早期選抜マーカーとしてや，
品種のタイピングの利用，さらには，連鎖地図と
してウンシュウミカンのEST解析を行い，２００５年
までに３
３，
１０８を解析した。ESTは，果実特性に関
与の可能性のある遺伝子候補として分子生物学
的，生化学的研究に利用されるばかりでなく，そ
の配列に基づいたDNAマーカーを作成し，連鎖地
BACクローンによる物理地図を連結するマーカー
としても期待されている。DNAマーカー作成の
現状と育種へのDNAマーカーの適用の試みにつ
いて紹介した。
32
Mitsuo OMURA
質疑応答
中川：ウンシュウミカンのマップの中に多胚の遺
伝子座がマップされていましたが，あれはいわ
ゆる不定胚形成のアポミクシスの遺伝子座みた
いなものと同じと考えていいのでしょうか。
大村：そういうことです。カンキツの珠心胚形成
の能力は一つの主働遺伝子によって決まってい
ます。胚数に関与する遺伝子についてはまた別
のローカス（locus）があると言われていますが，
先ほど第一連鎖群上に示したのは，多胚を形成
するかしないかに関わる主働遺伝子座になりま
す。
NIAS, Japan
33
FUNCTIONAL GENOMICS BASED ON THE INTEGRATION OF
METABOLOMICS WITH TRANSCRIPTOMICS
Masami Yokota HIRAI1 and Kazuki SAITO1,2
1
RIKEN Plant Science Center, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences,
2
Chiba University, Inage-ku, Chiba 263-8522, Japan
2
Introduction
Plants produce a wide diversity of compounds
that we use for foods, medicines, flavors, and industrial materials. To improve the productivity of plants
by modifying the genes involved in the synthesis of
useful compounds or by strictly controlling plant
growth, it is essential to understand the plant' s metabolic processes and their regulatory mechanisms as a
whole.
Because plants are sessile, they have evolved a
metabolic system which is robust against changes in
environmental conditions. In response to changes in
external circumstances, metabolite levels are adjusted
by modulation of gene expression, protein modification, and enzymatic activity, leading to a new state of
metabolic equilibrium. Such manifold regulations
make it hard to understand plant metabolism as a
whole solely by ' traditional' methods such as molecular biology, biochemistry, and forward and reverse genetics. In recent years, however, novel technologies
for comprehensive analysis of the transcripts, proteins, and metabolites have opened the door for the
elucidation of metabolic systems as a whole.
Our final goal is to elucidate overall plant metabolism as an integrated system. For this purpose, all
genes and metabolites in plant cells, as the components of the system, should be identified. In the model
plant Arabidopsis thaliana, approximately 26 000
genes are predicted on the basis of nucleotide sequence information; however, only half of these genes
have had a function assigned on the basis of sequence
similarity to known genes, and the functions of only
about 11% have been confirmed experimentally.
Worse, no catalog of metabolites in the cell is yet
available. Hence, one of our immediate aims is to
identify the functions of unknown genes and to identify the full range of metabolites present in the cell. At
the same time, we intend to clarify the networks of
genes and metabolites, and to obtain an image of overall metabolism with the help of bioinformatics.
To attain this goal, we have adopted a strategy of
integration of metabolomics and transcriptomics. By
comprehensive analysis of the metabolome and transcriptome, and using multivariate analyses, we can
speculate on the networks between pathways, genes,
and metabolites. Such network analysis enables us to
identify the functions of unknown genes. By this strategy, we have successfully identified the functions of
the genes involved in sulfur metabolism and in flavonoid accumulation (Hirai et al. 2005; Tohge et al.
2005). In this article we introduce a functional genomics study of sulfur metabolism.
Materials and Methods
Wild-type Arabidopsis plants were grown on
sulfur-sufficient agar-solidified control medium for 3
weeks and then transferred to sulfur-deprived or control media. Rosette leaves and roots were harvested at
3, 6, 12, 24, 48, or 168 h after transfer for metabolome and transcriptome analyses.
34
Masami Yokota HIRAI and Kazuki SAITO
Metabolome analysis was carried out by combining non-targeted and targeted analyses. Non-targeted
metabolome analysis was conducted by Fourier-transform ion-cyclotron-resonance mass spectrometry (FTMS) according to the method of Tohge et al. (2005).
In brief, metabolites were extracted in triplicate from
each sample. Extracts were analyzed by two ionization methods, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), in
positive and negative ion modes. The mass spectra
from each analysis were integrated after calibration,
creating a peak list that contained the accurate mass
and absolute intensity of each peak. To compare and
summarize data across different ionization modes and
polarities (polar and non-polar), we converted all detected mass peaks to their corresponding neutral
masses assuming hydrogen adduct formation. Approximately 2000 to 3000 mass peaks were observed
in a single sample. For targeted metabolite analyses,
anions, organic acids, and sugars were measured by
capillary electrophoresis, and amino acids by highperformance liquid chromatography (HPLC) (Hirai et
al. 2003).
Transcriptome analyses were conducted with a
DNA microarray which carried 21 500 Arabidopsis
genes (Agilent Arabidopsis 2 oligoarray).
Results and Discussion
Sulfur is an essential macronutrient required for
plant growth. It is found in amino acids, oligopeptides
such as glutathione (GSH) and phytochelatins, vitamins and cofactors, sulfolipids, and a variety of secondary products (Saito 2004). Sulfur enters a plant
primarily through the roots as inorganic sulfate. After
activation to adenosine 5í-phosphosulfate (APS), sulfate is mainly assimilated into sulfide, and then coupled with O-acetylserine (OAS) to yield cysteine (Fig.
1). The thiol group of cysteine in proteins plays a role
in maintaining protein structure by forming disulfide
bonds. GSH plays a role in the various redox reactions in the cell, and in the detoxification of xenobiotics, mediated by GSH S-transferase (GST). Besides
the major pathway to cysteine, APS is converted into
3í-phosphoadenosine 5í-phosphosulfate (PAPS), which
is used for the sulfation of organic compounds such as
Fig. 1 Sulfur assimilation pathway and glucosinolate metabolism. APS, adenosine 5í-phosphosulfate; PAPS, 3í-phosphoadenosine 5í-phosphosulfate; Sultr, sulfate transporter; APS, ATP sulfurylase; APR, APS reductase; APK,
APS kinase; SIR, sulfite reductase; Serat, serine acetyltransferase; Bsas, O-acetylserine(thiol)-lyase (a member
of the beta-substituted alanine synthase family); CGS, cystathionine gamma synthase; CBL, cystathionine beta
lyase; MetS, methionine synthase; GSH1, gamma-glutamylcysteine synthetase; GSH2, glutathione synthetase;
MAM, methylthioalkylmalate synthase; CYP, cytochrome P450; SUR1, a C-S lyase encoded by the
SUPERROOT1 gene; S-GT, S-glucosyltransferase; AtSOT, sulfotransferase.
FUNCTIONAL GENOMICS BASED ON INTEGRATIVE METABOLOMICS
glucosinolates (GLSs) and flavonoids (Fig. 1). Sulfurcontaining secondary metabolites such as GLSs in the
Cruciferae are defense compounds against herbivores
and pathogens. Because of such important roles of sulfur in plant cells, sulfur deficiency (-S) causes stunted
growth and chlorosis of plants, and plants finally die
of sulfur starvation. However, if the degree of sulfur
deficiency is not too severe, plants can grow apparently normally. This means that the plant has a metabolic system that is robust against S and can manage
on a limited sulfur supply.
To elucidate how plants can manage on a limited
sulfur supply and grow normally, we conducted metabolomic and transcriptomic analyses. Arabidopsis
plants were grown on S-sufficient agar-solidified control medium for 3 weeks, transferred to sulfur-deprived or control media, and harvested at 3, 6, 12, 24,
48, or 168 h after transfer. Sulfur-starved plants did
not show apparent morphological changes until at
least 1 week (168 hours) after transfer. Rosette leaves
and roots were subjected to metabolome and transcriptome analyses. After appropriate normalization of metabolome and transcriptome data, we calculated the
log ratio of metabolite (mass peak) level and transcript level in the treated sample to the corresponding
levels in the control samples, and processed the results by batch-learning self-organizing map (BLSOM) (Kanaya et al. 2001; Abe et al. 2003). BLSOM is an improved form of the original SOM (Kohonen 1990; Kohonen et al. 1996) in that the initial
weight vectors are set by principal-components analysis and the learning process is designed to be independent of the order of input of vectors, and hence the
result is reproducible.
1) Classification of Metabolites and Genes According to the Time-dependent Pattern of Change
To clarify gene-to-gene and metabolite-to-gene
networks, we integrated metabolome and transcriptome data into a single matrix and analyzed them by
means of BL-SOM. To classify the metabolites and
genes on the basis of the time-dependent pattern of
change in response to -S, we selected the metabolites
35
and genes that exhibited an apparent change in accumulation level over 168 h after transfer to -S. For each
set of ca. 1000 metabolites and ca. 10 000 genes selected, the sum of the squares of the six log ratio values at the six time points was set as equal to one so
that relative log ratio values could be calculated. Ca.
1000 metabolites (or mass peaks) and ca. 10 000
genes were classified by BL-SOM into 40 29 cells
(leaves; Figs. 2A, B) and 40 24 cells (roots; data not
shown) on the map on the basis of the time-dependent
pattern of the change in response to -S. Groups of metabolites and genes exhibiting similar accumulation
patterns were clustered in the same or neighboring
cells. Six GLS molecular species in leaves were clustered (Fig. 2A). GLSs are synthesized from several
amino acids such as chain-elongated Met and Trp
through a number of reactions, and are degraded into
isothiocyanates (ITCs) (Fig. 1). The GLS contents
and the expression level of the genes encoding GLS
biosynthesis and degradation enzymes changed in response to sulfur and nitrogen nutrition (Hirai et al.
2004). ITCs were also detected by FT-MS and clustered by BL-SOM (Fig. 2A). The pattern of accumulation of ITCs was a mirror image of that of GLSs (Fig.
2C). These results suggest that GLS metabolism is coordinately regulated in leaves. Most of the GLS biosynthesis genes in Arabidopsis have been identified
(MAMs, CYP79s, CYP83s, and SUR1 in Fig. 1).
These genes were clustered into the same region on
the map (Fig. 2A), supporting the idea of coordinated
regulation of GLS metabolism.
2) Functional Identification of Novel Glucosinolate
Biosynthesis Genes
In GLS biosynthesis, desulfoGLSs are sulfated,
but no gene responsible for the sulfation had been
identified. By BL-SOM three out of 18 putative sulfotransferase genes of Arabidopsis (AtSOT16, 17, and
18) were clustered with known GLS biosynthesis
genes, strongly suggesting their involvement in GLS
biosynthesis (Figs. 2A, D). In vitro enzymatic assay
using respective gene products proved that these three
genes encode PAPS:desulfoGLS sulfotransferases (Hi-
36
Fig. 2. BL-SOM analyses of time-course metabolome and transcriptome data from leaf samples. Approximately 1000
metabolites and 10000 genes were classified by BL-SOM according to the time-dependent pattern of changes
in accumulation and expression.
(A) A self-organizing map showing clustering of GLSs, ITCs, and GLS biosynthesis genes. Each cell was colored
according to the relative log ratio values of the metabolites and genes therein at 3 h after transfer.
(B) 3D view of self-organizing map.
(C) Changes in the contents of GLSs (green lines) and ITCs (blue lines) in leaves. The ordinate scale indicates the
relative log ratio value.
(D) Changes in expression of GLS biosynthesis genes. DIOX1, a gene involved in side-chain modification of
GLSs. The ordinate scale indicates the relative log ratio value. The following abbreviations indicate the side
chain groups of glucosinolates and isothiocyanates: 3-msp, 3-methylsulfinylpropyl; 4-mtb, 4-methylthiolbutyl;
7-msh, 7-methylsulfinylheptyl; 8-mso, 8-methylsulfinyloctyl; i-3ym, indol-3-ylmethyl; 4mi-3ym, 4methoxyindol-3-ylmethyl; 4-msb, 4-methylsulfinylbutyl; 5-msp, 5-methylsulfinypentyl.
rai et al. 2005). In the same way, we could putatively
identify the genes encoding C-S lyases, S-glucosyltransferase, and GSTs involved in GLS biosynthesis,
some of which were identified recently by other
groups (Douglas Grubb et al. 2004).
Summary
We integrated metabolomics and transcriptomics
to predict gene function, especially in secondary metabolism. The regulation of secondary metabolites at
the transcriptional level may be dominant over regulation at the translational and enzymatic activity levels,
and hence the transcript profile may directly determine the metabolite profile. We believe that almost all
genes involved in secondary metabolism can be identified by the approach presented here. This type of functional genomics can be applied to novel biosynthetic
pathways in non-model plants, crops, and medicinal
plants by using transcriptome analysis such as cDNAAFLP and cDNA subtraction as substitutions for
DNA arrays.
Acknowledgements. We are grateful to our co-workers, who are mentioned in our recent publications on
metabolomics and transcriptomics. Part of our study
was supported by Core Research for Evolutional Science and Technology of Japan Science and Technology Agency, and by Grants-in-Aid from the Ministry
of Education, Science, Culture, Sports and Technology, Japan.
References
1.
2.
Abe T, Kanaya S, Kinouchi M, Ichiba Y, Kozuki T, Ikemura T (2003) Informatics for unveiling hidden genome signature. Genome Res 13:693-702
Douglas Grubb C, Zipp BJ, Ludwig-Muller J, Masuno
MN, Molinski TF, Abel S (2004) Arabidopsis glucosyl-
3.
4.
5.
transferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis. Plant J 40:893-908
Hirai MY, Fujiwara T, Awazuhara M, Kimura T, Noji
M, Saito K (2003) Global expression profiling of
sulfur-starved Arabidopsis by DNA macroarray reveals
the role of O-acetyl-L-serine as a general regulator of
gene expression in response to sulfur nutrition. Plant J
33:651-663
Hirai MY, Yano M, Goodenowe DB, Kanaya S,
Kimura T, Awazuhara M, Arita M, Fujiwara T, Saito K
(2004) Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis. Proc Natl Acad Sci USA
101:10205-10210
Hirai MY, Klein M, Fujikawa Y, Yano M, Goodenowe
DB, Yamazaki Y, Kanaya S, Nakamura Y, Kitayama
M, Suzuki H, Sakurai N, Shibata D, Tokuhisa J, Reichelt M, Gershenzon J, Papenbrock J, Saito K (2005)
Elucidation of gene-to-gene and metabolite-to-gene networks in Arabidopsis by integration of metabolomics
and transcriptomics. J Biol Chem 280:25590-25595
6.
37
Kanaya S, Kinouchi M, Abe T, Kudo Y, Yamada Y,
Nishi T, Mori H, Ikemura T (2001) Analysis of codon
usage diversity of bacterial genes with a self-organizing map (SOM): characterization of horizontally transferred genes with emphasis on the E. coli O157
genome. Gene 276:89-99
7. Kohonen T (1990) The self-organizing map. Proc IEEE
78:1464-1480
8. Kohonen T, Oja E, Simula O, Visa A, Kangas J (1996)
Engineering applications of the self-organizing map.
Proc IEEE 84:1358-1384
9. Saito K (2004) Sulfur assimilatory metabolism. The
long and smelling road. Plant Physiol 136:2443-2450
10. Tohge T, Nishiyama Y, Hirai MY, Yano M, Nakajima J,
Awazuhara M, Inoue E, Takahashi H, Goodenowe DB,
Kitayama M, Noji M, Yamazaki M, Saito K (2005)
Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants overexpressing an MYB transcription factor. Plant J 42:218325
38
メタボロミクスとトランスクリプトミクスの統合による植物ゲノム機能科学
平井優美１）・斉藤和季１，２）
１）
理化学研究所植物科学研究センターメタボローム基盤研究グループ
〒２
３
０−０
０
４
５横浜市鶴見区末広町１−７−２
２
２）
千葉大学大学院薬学研究院
〒２
６
３−８
５
２
２千葉市稲毛区弥生町１−３
３
食糧や木材，医薬品材料としての植物の有用性
はその複雑な代謝産物にある。植物の有用物質生
産能力を代謝工学等により向上させるためには，
関連する代謝系全体の制御機構の理解が極めて重
要である。我々は，全代謝産物（メタボローム）
と全転写産物（トランスクリプトーム）を解析し
て遺伝子発現と代謝物蓄積との間のネットワーク
すなわち代謝制御機構の全体像をシステムとして
明らかにすることを目指している。本研究では，
シロイヌナズナのメタボロームおよびトランスク
リプトームデータを統合解析することにより，特
定の代謝経路における複数の未知遺伝子の機能を
包括的に予測する方法を確立した。
野生型シロイヌナズナを硫黄栄養十分のコント
ロール培地を用いて約３週間栽培したのち，コン
トロール培地もしくは硫黄欠乏培地に移植して最
長１週間栽培した。これらの植物の葉と根を移植
後３，
６，１
２，２
４，４
８，１
６
８時間後にサンプリング
し，メタボロームおよびトランスクリプトーム分
析に供した。非ターゲットメタボローム分析とし
てフーリエ変換イオンサイクロトロン共鳴質量分
析（F
TM
S）を，ターゲット分析としてHP
LCおよ
びキャピラリー電気泳動を行って約２，
０
０
０代謝物
（マスピーク）のデータを得た。トランスクリプ
トーム解析は市販のオリゴD
NAマイクロアレイ
（A
gi
l
e
nt A
rab
id
ops
is2 Oli
go mic
ro
）を用いて，
ar
ray
搭載された２
１，
５
０
０遺伝子の発現解析を行った。
得られたデータを適切な方法により標準化・前
処理したのち，大きな変動を示さなかった代謝物
および遺伝子のデータを除去して，残った約
１，
０
０
０代謝物と約１０，
０
０
０遺伝子のデータを単一の
マトリクス（データテーブル）に統合し一括学習
型自己組織化マッピング法（B
L-S
OM ）により解
析した。B
L-S
OM は結果の再現性および精度に優
れ，同じ蓄積パターンや発現パターンを示す代謝
物や遺伝子はマップ上の同一あるいは近傍のセル
の中に分類される。どのような代謝物および遺伝
子が一緒にクラスタリングされたかを調べること
で，遺伝子機能を包括的に予測することができ
た。例として，アブラナ科植物の病害虫に対する
防御物質として知られているグルコシノレート
（G
LS）類が互いに近傍のセルにクラスタリングさ
れていたことからG
LS合成が同調的に制御されて
いることが示唆された。M
A
M やC
Y
Pなどの既知
のG
LS生合成遺伝子群も互いに近傍のセルに分類
されており，G
LS生合成系が生合成酵素遺伝子の
転写産物蓄積のレベルで制御されていることが示
された。これらと同調的に発現する機能未知の遺
伝子はG
LS合成に関与することが予測された。そ
れらのうち３つのp
uta
t
v
i
es
ulf
o
tr
ansf
e
遺伝子に
ras
e
ついて組換えタンパク質を作製して酵素活性を調
べたところ，３つともd
esu
lf
o
GLS su
l
fot
ra
n
sf
e
活
rase
性を示し，予測の正しいことが証明された。他に
もG
LS生合成酵素の候補遺伝子を複数予測できた。
本方法は，シロイヌナズナ以外の有用植物にお
ける遺伝子機能同定にも有効であろう。
質疑応答
大杉：大変興味深く聞かせていただきました。誤
差といいますか，サンプリングするときに，シ
ロイヌナズナを今おっしゃられたような条件で
栽培するときには，結構揃うのではないかと思
うのですが，例えば外で栽培したものを材料に
使うとか，なかなか揃わない場合もあると思う
のです。こういうメタボロミクスの実験をやる
場合に，どれくらいのサンプル数を使うのか，
その辺が非常に興味があるのですが，ちょっと
教えていただけますか。
平井：おっしゃるとおりで，メタボロミクスの最
大のポイントの一つと申しておりますが，今ナ
ズナを実験室の中で，しかも寒天培地，無菌栽
培というように，かなりコントロールされた環
境で育てています。それでもやはり個体別に測
りますと，かなり違うのです。そこは如何とも
し難いというか，ばらつきをなくす最大限の努
力をしているわけですが，大体GC-MS（GCマ
ス）などで分析している人に言わせますと，解
析によるのですが，５検体程度で十分な場合も
あるし，違いを引き出すような解析をしようと
39
思うと，やはり３０個体ぐらいの分析が必要であ
ると言っております。
草場：マスで検出した２，
０００位の代謝物というこ
とで，メジャーな代謝物ということになります
が，そのターゲットした解析なども含めれば，
どのくらいの数の代謝物について，この手法が
適用できるのでしょうか。
平井：FT/MS（FTマス）の最大の欠点は，化合物
の同定が非常に難しいのです。今２，
０００と申し
ましたが，名前が実際につくのは，実はせいぜ
い１割あればいいぐらいかと思います。それは
これからの課題なのですが，案外アミノ酸など
は経験上あまり出てきていないことが分かりま
した。だから，アミノ酸はターゲット分析が必
要だったわけですが，その一方で，グルコシノ
レートのような，硫黄を含んでいるものでは精
密マスの値が，特殊で組成式が決めやすいので
す。結局（FT/MSでは）C，H，O，Nといった
ものの分子量の算数的な組み合わせで，組成式
が決まります。その場合，一般的な一次代謝に
かかわるような代謝物というのは，実ははっき
りは見えてこないです。
NIAS, Japan
41
ADVANCED UTILIZATION OF BIOLOGICAL INFORMATION
Yukiko YAMAZAKI
Genetic Resource Informatics Laboratory, Center for Genetic Resource Information,
National Institute of Genetics
Genetic Resource Information Center
The National Institute of Genetics has developed, preserved and distributed genetic resources to
researchers requiring these materials for a considerable period. The need for such a service arose because
genetics has historically always depended upon variations in phenotype as a source of genetic resources.
These resources are still of importance, even in the genomic science era. Large-scale functional genomics
has increased the demand for mutants because the
number of mutants available for any organism holds
the key to the success of functional gene analysis of
that organism. This necessity is particularly apparent
if one considers that the number of different mutants
required for genetic studies of an organism must exceed the total number of genes of that organism. A
wide variety of bioresources are thus required for experimental studies for research on polygenic phenotypes and complicated genes such as the QTL genes.
The National BioResource Project (http://www.
nbrp.jp/) funded by the Ministry of Education, Culture, Sports, Science and Technology in Japan was established in 2002 to meet this demand. In the project,
twenty-four resource centers were selected to collect,
preserve and distribute the resources held for each organism. Each center is also required to promote the effective utilization of genomic resources, such as DNA
clones and mutants produced by large-scale mutagenesis. The project is also noteworthy from the point of
view of funding.
The Center for Genetic Resource Information
was established in 1997 to serve as a source of biore-
source information. The center is also involved in the
National BioResource Project and has been contributing to the project in cooperation with other resource
centers.
Genetic Resource Database
So far we have constructed thirty Genetic Resource Databases for nineteen different organisms and
have made them available to the public through the
internet (http://www.shigen.nig.ac.jp/). Activities of
the National BioResource Project and URL collections of worldwide genetic resource-related sites are
available at http://www.nbrp.jp/ and
http://shigen.nig.ac.jp/wgr/http://shigen.nig.ac.jp/wgr/,
respectively. The Genetic Resource Databases also
contain a variety of resources including DNA clones,
wild strains, experimental strains, spontaneous mutants, ENU- and/or MNU-induced mutants, radiationinduced mutants, recombinant inbred lines, transgenic
Fig.1 Structure of bioresource data
42
Yukiko YAMAZAKI
strains, enhancer trap lines and introgression lines.
Figure 1 shows the various types of data that are available from these databases. The vertical axis (Y) refers to the various levels for which the given resource
applies, such as individual body, organ, tissue, cell, genome, chromosome, genes and DNA. All of the information pertaining to the various species held can be
located somewhere on this 3D chart. The horizontal
axis (X) refers to the variety of species and the depth
of the 3D object (Z) refers to a polymorphism of each
species. The time scale, such as gene expression or developmental stage, is given by the depth of each cube.
A model strain for each species, usually one that has
been studied extensively, is located at the front of the
space. The other strains listed behind the model are
described as how they differ from the model. While
the amount of data available at present is limited, we
expect that the amount of data available for an increasing range of species will increase in the future. At this
symposium, I would like to introduce an approach
that can be adopted to maximize the potential benefits
of the data.
DNA data can be compared easily because communication thereof can be done in a common language. In addition, several key features are shared
among the International DNA Databanks. Descriptions of gene functions, however, vary depending
upon species because each species has a different scientific history. Enzyme commission numbers (EC) are
one solution, but these are insufficient for describing
all gene functions. To solve the problem, the respected Drosophilan geneticist, Dr. Ashburner, developed FlyBase and first introduced the concept of
Ontology into the biological database world. Gene ontology (GO) is widely known as most of the model-organism databases have cooperated with each other in
constructing gene ontologies. Currently, GO is indispensable for the functional classification of genes.
Ontology, a powerful tool towards advanced utilization of Biological Information
The word ontology means structured concept
In the context of biological ontologies, we use a
unique ID which is assigned to a certain concept and
is shared among various species, and is also independent of the nomenclature that has been used to date.
Consequently, different project members can share
tasks for creating a common structure of concepts by
simply adding a new concept defined with its parent
concept if it is necessary. This simple idea has allowed ontology to be applied a variety of biological
fields, including cellular structure, developmental cytology, phenotypes and anatomy.
Plant Ontology (PO) consists of two ontology
types, plant growth/developmental stages and plant
structural ontology. In 2005, the Plant Ontology Consortium (POC) released the first PO for arabidopsis,
soybean, maize and rice (http://www.plantontology.
org/). Oryzabase (http://www.shigen.nig.ac.jp/rice/
oryzabase/), a Rice Science Database we developed,
contains data on the various aspects associated with
rice development stages within each organ. This featured information is similar in structure to that of PO.
PATO stands for ìPhenotype And Trait Ontologyî,
and the Zebrafish and the Medaka database groups are
currently intensively constructing this ontology to describe the phenotypes of their mutant collections.
The construction of these ontologies is complicated by the need to manually extract all of the relevant information from journal articles. However,
ontology promises an exciting future, because once
we get reasonably consistent ontologies within various fields, then we can apply this information to a
greater number of studies and make data more useful
and content-rich than was previously possible.
ADVANCED UTILIZATION OF BIOLOGICAL INFORMATION
43
バイオ情報の高次利用に向けて
山崎由紀子
国立遺伝学研究所･生物遺伝資源情報総合センター
〒４１１−８５４０静岡県三島市谷田１１１１
国立遺伝学研究所は研究材料としての生物遺伝
資源（バイオリソース）を開発，保存，提供する
系統保存事業を長い間行ってきました。遺伝学の
研究には多様な形質をもつ多数の系統が必要でし
たから当然のことですが，分子生物学やゲノム科
学の時代においても，バイオリソースが不要にな
ることはありませんでした。むしろポストゲノム
時代に入ってからは，大量の遺伝子解析を可能と
する強力な材料として，以前にも増して変異体な
どの需要が増えてきています。
このような時代のニーズにこたえて，２００２年に
はナショナルバイオリソースプロジェクト（National BioResource Project: NBRP）（http://www.
nbrp.jp/）が始まりました。生物種毎にセンター化
を図り，バイオリソースの収集，保存，提供体制
を整備することを目的とした事業です。生物遺伝
資源情報総合センターはバイオリソースの情報セ
ンターとして１９９７年に設立されましたが，NBRP
でも「情報」の中核機関として情報整備を進めて
います。
当センターはこれまでに，微生物，植物，動物
を含む１９種類の生物種をカバーする遺伝資源デー
タベースを構築して公開しています（http://www.
shigen.nig.ac.jp/）。データベースに蓄積される情
報は，多様な生物種，同一種内の多型，そして個
体，細胞，染色体，DNAなど多次元の情報です。これらの大量かつ多次元の情報を最大限活用する
ための試みの１つとして，今日はバイオオントロ
ジーを紹介します。
DNA情報はATGCの共通言語があることによっ
て，すべての生物種のデータを対象とした相同性
解析が可能です。
一方，遺伝子の機能に関する
記述を共通化する言語はありません。そこで，種
横断的な遺伝子機能の比較を可能とするシステム
として，Gene Ontology （GO：遺伝子オントロ
ジー）という考え方が登場しました。
「オントロジー」は日本語で「概念の構造化」
あるいは「構造化した概念」という意味です。「表
記は異なるけれど実は同じ意味（概念）を表す」
ものを同一の番号（ID）で管理しようという考え
方です。バイオリソースデータベースと深い関連
があるオントロジーにPlant Ontology（PO http://
www.plantontology.org/）があります。これは植物
の構造（形態）と発生（生長）に関する概念構造
です。
もう１つ形質に関するオントロジーとし
てPATO（Phenotype And Trait Ontology）がありま
す。POは多くのモデル地植物のデータベースが
共同で構築をはじめています。PATOはゼブラ
フィッシュやメダカのグループが取り組んでいます。
オントロジーの構築は論文から情報を抽出する
地味で時間と労力を要する作業を伴います。しか
し一見絶望的な作業も，試行錯誤を繰り返すうち
に合理的な構造を見出し，いつの間にか普及して
いきます。同じオントロジーを共有する生物種が
増えると，オントロジーはより大きな概念を取り
扱えるようになり，将来すべての生物種の情報を
オントロジーを用いて構造化することができるか
もしれないという希望が見えてきます。
44
Yukiko YAMAZAKI
質疑応答
奥本：これは一つ一つのデータをだれかが打ち込
まなければいけないのですよね。その言葉を何
か自分で見つけて来られるのでしょうか。
山崎：はい。私は１月にゼブラフィッシュのグ
ループのところに行ったのですが，それをみん
なでやっているのです。１
７人位いるのですが，
本当に一人一人，毎日朝から晩まで論文を読ん
で，もちろんプラットフォームは作ってもらっ
ているので，割と操作性はいいのですが，やっ
ていることは論文を読んで，特にミュータント
の論文をピックアップして入れているのです。
けれども，そうやって３，
０
０
０とか，たまっていく
わけですよね。でき上がってしまうと，本当に
押しも押されぬという形になってしまうので圧
倒されました。そこでやっている方はFl
y Ba
s
e
から来たとか，イーストのデータベースで仕事
をしていたけれども今度はゼブラフィッシュに
来たとか，
そういう交流もあるみたいなのです。
奥本：お互いに打ち込んだデータをチェックしあ
うということもやるのですか。
山崎：というか，プラットフォームが一緒なの
で，見ることはできるのです。P
ATOの場合は
非常にまだまだ難しい問題があって，一つの論
文を読んでも，入れ方が人によって違う。読み
が深い人と読みが浅い人，例えば何とかがおか
しい，アブノーマル（a
bno
rm
a
l
）と入れる人と，
何とかが短いと入れる人と，あるいはサイズが
トランケート（t
run
ca
t
）していると入れる人で
e
は，ものの見方が全然違うということで，そう
いうことも問題にはなっていましたが，ひたす
らやっていました。
NIAS, Japan
45
NATURAL VARIATIONATION AND THE STUDY FOR ENHANCING
GENETIC DIVERSITY IN RICE
Shuichi FUKUOKA , Kaworu EBANA , Yusaku UGA , Makoto KAWASE
Genebank, National Institute of Agrobiological Science (NIAS)
Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602
Introduction
Asian cultivated rice (Oryza sativa L.) has great
morphological and physiological diversity. The beneficial traits found in germplasms have been introduced
into other rice through cross breeding. Since agronomic traits are controlled by multiple loci, genetic
improvement is time consuming, especially when introducing traits from exotic germplasms. Non-random
distribution of the benefitial traits in the gene pool
and the reproductive barierrs observed in the progenies of the cross between genetically differentiated
germplasms is also a factor associated with the efficiency of breeding. Therefore, information on the genetic controlling of the traits and structure of the gene
pool is of interest to the rice breeders who need to determine the cross- conbinations and establishing efficient precedures for germplasm enhancement.
Rice genome information can help improve
strategies for enhancing rice germplasm. A high-density genetic linkage map (KURATA et al. 1994; HARUSIMA et al. 1998) is a powerful tool for targeting
genes for agronomic traits and their selection (YANO
and SASAKI 1997). DNA markers are also useful for
depicting the structure of the gene pool and selecting
a subset of the germplasm panel to maximaze the use
of genetic diversity in cultivated rice (KOJIMA et al.
2005). Complete genome sequence information
(IRGSP 2005), a gene annotation database (ITO et al.
2005), full-length cDNA libraries (KIKUCHI et al.
2003) of a Japonica rice variety Nipponbare were developed as tools for identifying the structure and func-
tion of the genes controlling traits. New technologies
based on rice genome information have been accelerating genetic studies for practical breeding, establishing the connection of genetic studies with other basic
sciences.
The present paper focuses on a study of the genetic dissection of field resistance to rice blast, as an
example study of an important agronomic trait under
complicated genetic control, as well as a study revealing the genetic structure of cultivated rice. These studies present strategies for rice germplasm enhancement
incorporated with rice genome information.
Genetic dissection of field resistance to blast
Rice blast is a destructive disease of rice worldwide, and strengthening resistance to blast is an important breeding objective. Two types of resistance to
blast, complete resistance and field (partial) resistance
have been described in rice (EZUKA 1972). Complete
resistance induces a hypersensitive reaction and is
characterized by a resistant infection type. More than
20 loci for complete blast resistance have been identified (MC COUCH et al., 1994), and some were cloned
in rice (WANG et al. 1999; BRYAN et al. 2000). Despite their significant effect against rice blast, complete resistance genes were overcome by compatible
races of the pathogen several years after the varieties
with this type of resistance were released. Field resistance is usually incomplete and characterized by a susceptible infection type. Field resistance limits the
proportion of diseased leaf area and allows effective
46
control of the parasite under natural conditions. This
type of resistance is considered to be durable when exposed to new races of the pathogen. Japanese upland
rice has been used as a source of the genes conferring
field resistance to rice blast in a Japanese breeding
program that w as initiated in the 1920's. However,
field resistance in upland rice has not contributed
much to the inprovement of Japanese rice varieties
(INOUE et al. 1983), possibly due to the close linkage
between the genes for field resistance and certain undesirable characteristics. Some genetic studies reported that many genes with additive effects are
responsible for the expression of field resistance (HIGASHI and KUSHIBUCHI 1978, HIGASHI and SAITO
1985). These results suggest that identification of the
number of genes and their chromosomal locations are
important for understanding field resitance and for enhancing its use.
1) Mapping of QTL for field resistanceto rice blast
QTL analysis was carried out using 146 F4 lines
of a cross between theJapanese irrigated rice cultivar
Nipponbare and the upland cultivar Owarihatamochi
to determine the chromosomal regions involved in
field resistance to blast in Japanese upland rice (FUKUOKA and OKUNO 2001). Nipponbare and Owarihatamochi have no gene for complete resistance to
rice blast, based on the test using Japanese differential
blast races. The frequency distribution of the field resistance score based on the diseased leaf area (DLA)
in the F4 lines was continuous and ranged from 1
(highly resistant) to 10 (highly susceptible). The
scores for Owarihatamochi and Nipponbare were 2.8
and 8.0, respectively. We used 116 DNA marker loci
for QTL mapping and detected four QTLs located on
three chromosomes (Fig. 1). The resistant alleles on
chromosomes 4 and 12 come from Owarihatamochi,
while that on chromosome 9 is derived from Nipponbare. The two QTLs on chromosome 4, close to
marker loci G271 (qBR4-1) and G177 (qBR4-2), explained 45.7% and 29.4% of the phenotypic variation,
respectively, while the QTLs on chromosome 9
(qBR9-1) and 12 (qBR12-1) explained 7.9% and
13.7% of the total phenotypic variation. All together,
the four QTLs explained 66.3% of the total phenotypic variation.
Fig. 1 Chromosomal location of the QTLs for rice blast field resistance in F4 lines from the cross between irrigated rice Nipponbare and upland rice Owarihatamochi. ( adapted from FUKUOKA and OKUNO , 2001)
ENHANCEMENT OF GENETIC DIVERSITY IN RICE
2) Marker-assisted selection of field resistance in
backcrossed lines
To evaluate the effect of each QTL, advanced
backcrossed lines with putative QTLs of Owarihatamochi were developed by marker assisted selection using a highly susceptible lowland rice variety,
Aichiasahi, as the recurrent parent. During backcross
and selection, the proportion of upland rice chromosomes in these plants decreased to less than 6% based
on the genotypes of DNA markers. We selected three
BC2F1 plants that contained just one out of three blast
resistance alleles from Owarihatamochi. The field resistance in the 98, 44 and 46 BC2F3 lines for qBR4-1,
qBR4-2 and qBR12-1, respectively was assessed and
compared to the levels of field resistance among the
lines with Owarihatamochi-homozygous alleles, heterozygous alleles and Aichiasahi-homozygous alleles.
Genotypes at each QTL were estimated based on the
genotype of DNA markers around the QTLs. The average DLA score of the lines with the Owarihatamochiallele was significantly higher than those with the
Aichiasahi-allele at all three QTLs (Table 1). The effect was largest at the qBR4-1 and smallest at the
qBR12-1, in good accordance with the result of the
QTL analysis.
3) Map-based cloning of a field resistance gene
pi21
Genetic analysis for field resistance was conducted in 82 BC1F3 lines in which qBR4-1, a QTL
with the largest effect was segregated. A resistance
gene for this QTL was mapped as a single recessive
gene pi21 between the RFLP marker loci G271 and
Table 1. Average score for diseased leaf area of the lines
with different genotypes at QTLs
QTL
O-homozygous Heterozygous
47
G317 at a distance of 5.0 cM and 8.5 cM, respectively
(F and O 2001). An intensive DNA
marker survey using the end-fragment DNA sequence
of the P1 artificial chromosome (PAC) clones covering the pi21 region identified eleven DNA markers
around the pi21 locus. We used two mapping populations consisting of a total of 3717 plants for fine genetic mapping of pi21. The analysis delimited the
1.7kb-genomic region where the segregation of DNA
markers completely associated with the resistant/susceptible phenotypic difference. The search of
the gene annotation database and Nipponbare cDNA
clone identified that this region is within the coding region, and that the sequence variations between susceptible varieties Aichiasahi and Nipponbare, and
resistance variety Owarihatamochi, cause length-polymorphisms at the amino acid level. Deletion of the
Pi21 protein in Owarihatamoti was considered to result in an improvement in of field resistance. The molecular function of the candidate gene for pi21 is
unclear, and its DNA sequences have no similarity
with previously reported disease resistance genes. The
functional analysis of the gene is under progress.
4) Conclusions
DNA marker-based genetic dissection of a beneficial trait, field resistance to blast, efficiently identified chromosomal regions for the trait that enhances
the use of natural variation in the gene pool. Further
analysis using backcrossed lines enabled us to confirm the effect of each QTL for the trait, and the lines
are useful as a material for pre-breeding as well as of
one for basic research. Identification of the gene for
the trait will help enable precise genotyping of the
gene, and will contribute to further understanding of
the mechanism of the expression of field resistance.
A-homozygous
qBR4-1 3.1±0.46 (10) 5.7±0.77 (55) 6.9±0.75 (100)
qBR4-2 6.7±0.66 (67) 7.3±0.51 (83) 8.2±0.29 (100)
qBR12-1 6.4±0.29 (80) 6.8±0.64 (93) 7.0±0.57 (100)
O: Owarihatamochi, A: Aichiasahi .
Percentages of the diseased leaf area compared with A-homozygous lines are indicated in parentheses.
The analysis of the genetic structure in Asian cultivated rice
The intraspecific variation of Asian cultivated rice
has been investigated by many researchers. Distinct
differentiation within this species was first reported
48
by KATO et al. (1928), and has been supported by
many reports using various kind of methodologies,
such as morphological, and physiological characteristics, isozyme and DNA markers (ex. OKA 1953;
GLASZMANN 1987; ZHANG et al. 1992). When reviewing these studies in detail, there were some discrepancies in the results among methodologies,
suggesting that the key traits or factors for each methodology represent different aspects of genetic variation in Asian rice, and also suggesting the genetic
differentiation within each of the two major variety
groups. Since adaptation to diverse environments and
to different cultural practices can be factors in the genetic differentiation in rice, understanding the genetic
structure is useful for identifying varietal groups and
the chromosomal regions relevant to varietal differentiation. Such information will be helpful for screening
potential germplasms from the huge number of collections, and for determining their use. DNA markers
identified on the rice genome provide an efficient
means for detecting genetic variation and the distribution of this variation among the rice germplasm. The
markers that cover the rice genome are also useful for
detecting genetic differentiation and the genetic variation associated with characteristics for adapting to different environments and cultural practices.
Fig 2. Principal coordinate plots of 332 accessions of rice
based on DNA polymorphism at 179 RFLP loci. (
adapted from KOJIMA et al, 2005)
Fig 3. Characterization of the accession groups detected by
the principal coordinate analysis (adapted from KOJIMA et al, 2005)
1) Genetic structure of Asian cultivated rice based
on RFLP
A total of 332 accessions originating from 23
countries, and including 281 landraces and 51 modern
varieties were selected from the accessions maintained at the NIAS Genebank and used for the analysis using 179 RFLP marker loci on the high-density
genetic linkage map of rice (KURATA et al. 1994; HA-
and each chromosomal region showed mosaic structures with a combination of Indica- and Japonica-specific alleles. Hybridization and separation by the
reproductive barrier among the rice accessions may
account for the complicated mosaic structure of the
chromosomes.
et al.1998). Based on principal coordinate
analysis of the RFLP data, the accessions were classified into three major groups (Fig.2)(KOJIMA et al. in
press). Based on the proportion of alleles shared with
the Indica and Japonica reference varieties, one group
was assigned to Japonica, and the other two groups to
Indica. However, each accession harbored both Indicaand Japonica- specific alleles to some extent (Fig. 3),
2) Core collection as a rice diversity research set
The core collection of rice was chosen as a rice
diversity research set based on a genome-wide RFLP
survey of 332 accessions of Asian cultivated rice (KOJIMA et al. 2005). The RFLP data on the 332 accessions were subjected to cluster analysis and 67 groups
were recognized at a similarity index of 0.915. A single accession from each of the 67 groups was se-
RUSHIMA
lected. These 67 accessions retained 91% of the
alleles detected in the original 332 accessions, and
covered the variation of the initial set of accessions in
terms of several agro-morphological traits. The 69 accessions, including varieties from 19 countries and the
reference varieties, Nipponbare and Kasalath, were selected as a rice diversity research set. This collection,
which is presently well characterized at the molecular
level will be used for detailed genetic studies and rice
improvement. This set is distributed by the NIAS Genebank (contact address is
[email protected]). Accumulated data on the
various traits and on DNA polymorphism in the collection will be provided as feedback to users, which
should enhance the efficient use of rice genetic resources.
3) Association study in rice
Association analysis, or linkage disequilibrium
(LD) mapping, has been extensively used in animals
to dissect quantitative traits. This approach has recently been extended to plants such as maize and
Arabidopsis (FLINT- GARCIA et al. 2003). Association
analysis is potentially advantageous in the resolution,
49
speed and allelic range compared with F2-based QTL
mapping. LD, caused by the linkage and population
structures resulting from the history of the population,
plays a central role in association analysis and the extent of LD determines the applicability of association
study. The knowledge of LD in rice is limited, and
therefore it is important to determine the extent of LD
to perform an association analysis in rice. We are collecting polymorphism information to study is genomewide LD in rice.
4) Detection of the chromosomal regions associated
with cultural type differentiation among germplasms in northern Vietnam
A total of 129 rice landraces from northern Vietnam, 89 accessions of upland rice and 40 of lowland
rice, were analyzed by using 169 RFLP marker loci
(FUKUOKA et al. 1993). Based on principle coordinate analysis of the RFLP data, Vietnamese landraces
were classified into three groups; one of Indica, the
other two of Japonica. The two groups in the Japonica
rice accession were respectively corresponded with
upland and lowland rice, implying the genetic differentiation in the Japonica can be the result of adapta-
Fig.4. Chromosomal locations of RFLP marker loci and their association with genetic differentiation among
Vietnamese landraces. Marker loci diagnostic to upland-lowland cultural type differentiation in Japonica rice were indicated in boxes. (adapted from FUKUOKA et al, 2003)
50
tion to different cultural practices. In Indica
accessions, out of 115 polymorphic marker loci, no
marker loci were associated with cultural types differentiation. In Japonica rice, eight marker loci out of 81
polymorphic loci were associated with cultural type
differentiation (Fig. 4). Some of the loci associated
with upland-lowland differentiation were close to the
QTLs associated with root morphology and seedlingvigor (YADAV et al. 1997; ZHANG et al. 2001; C et
al. 2002). Such QTLs may possibly be related to the
adaptation of rice to upland condition.
5) Conclusions
Asian cultivated rice is clearly differentiated into
two major groups, as detected based on the RFLP survey. The genetic structure within these two groups is
complicated, possibly due to the result of a natural hybridization event in the past. The rice core collection
was chosen as a rice diversity research set based on
genome-wide RFLP data to represent the genetic diversity of this species in a small number of accessions. This collection is useful for further intensive
polymorphism surveying and characterization of rice
germplasm. An association study is a potential approach for dissecting natural variation in a species,
and needs further evaluation to determine the optimal
precedure in rice
5.
6.
7.
8.
9.
10.
11.
12.
13.
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52
イネの自然変異と多様性研究
福岡修一，江花薫子，宇賀優作，河瀬真琴
独立行政法人農業生物資源研究所ジーンバンク
茨城県つくば市観音台２−１−２
アジア栽培イネは様々な環境で栽培され，多様
な変異を保持している。しかしながら，それらの
形質変異の多くは遺伝様式が複雑であるため，効
率的に利用することは困難であった。イネゲノム
研究が生み出した塩基配列情報やDNAマーカー
などの研究資源は，遺伝的多様性，とりわけ形質
変異の解明に役立ち，多様性研究を大きく発展さ
せる。
いもち病圃場抵抗性は，いもち病菌レース変動
に対して安定した効果を示す有用な形質である。
陸稲品種の優れた圃場抵抗性は古くより知られる
が，複雑な遺伝様式を持つため，その育種的利用
は進まなかった。DNAマーカーを用いた遺伝解
析によって，陸稲品種オワリハタモチの圃場抵抗
性の主要なQTLが３カ所の染色体領域にあること
を明らかにし，最も作用力の大きいQTL，pi21の
座乗位置を決定した。さらに，戻し交雑集団を用
いた解析によって，圃場抵抗性に関わる新たな
QTLを明らかにした。また，pi21のマップベース
クローニングを試み，遺伝子予測やcDNAクロー
ンのデータベースを用いて候補遺伝子を特定し，
圃場抵抗性に関わる変異を遺伝子レベルで明らか
にした。既報の真性抵抗性遺伝子とは異なる構造
を持つpi21遺伝子の機能解析によって圃場抵抗性
の機構解明か可能となる。
このように，
ゲノム情報
を利用した有用形質の遺伝学的解析によって，自
然変異を解明し効率的な利用技術を開発に役立つ。
自然変異の育種利用をはかる上で，集団構造の
解明は重要な研究課題である。アジア栽培イネに
おける日本型イネ・インド型イネという亜種レベ
ルの分化は，多数の遺伝子座の変異に基づいてお
り，個々の要因を特定することは困難である。ゲ
ノム上に散在するDNAマーカーを用いて品種間
変異を調べると，イネの遺伝的多様性は日本型・
インド型という基本骨格の上に，品種群内外から
の遺伝子移入と新たに生じた変異が積み重なり構
成されることがわかった。日本型・インド型イネ
以外にも，水陸稲等の栽培様式や感光性等による
生態型の分化があり，ベトナム北西部の在来品種
のDNA変異を調査したところ，水稲・陸稲の栽培
様式と相関のある染色体領域を検出できた。この
ような領域の中には，根の形態に関与するQTLが
報告されており，品種間変異の解析によって品種
群や生態型に特徴的な変異の検索が可能である。
そこで，原産地情報およびDNA多型情報に基づ
き，アジア栽培イネ品種の変異を効率的に調査で
きる品種セット（コアコレクション）を選定した。
今後，コアコレクションに対してDNA多型情報と
形質変異情報の集積を行うことで，他殖性生物で
利用されている連関（association）解析や連鎖不平
衡（LD）解析を自殖性作物のイネに適用するため
の基礎的な情報を得ることができる。これによっ
て，遺伝子の進化的意義を解明，選択圧となる環
境要因の特定など，多様性解明に役立つ情報の発
掘が期待できる。
質疑応答
天野：圃場抵抗性，pi21というのは，欠失型と考
えてよろしいのですか。それならば，交配導入
するよりも，突然変異でガンマ線ででもたたい
てやったら，その本体が取れてくるのではない
かという気がしたのですが。それと，ハンガ
リーなどの外国でも真性抵抗性ではなく，圃場
抵抗性ではないかと思われる誘発例が結構出て
います。そのあたりのことを考えて圃場抵抗性
を期待するのだったら，突然変異がいい手段で
はないかという気がしたのですが，いかがで
53
しょうか。
福岡修一：確かに「オワリハタモチ」の対立遺伝
子は，抵抗性に関しては機能を失っておりまし
て，つまり「日本晴」の機能型の対立遺伝子が
壊れることによって，抵抗性が発現していると
考えています。ただ，まだはっきりしていない
のは，このpi21という遺伝子が圃場抵抗性だけ
に機能しているかどうかということと，あと，
壊れ方がどのような壊れ方でもいいかどうかと
いうことは，まだはっきりとしたデータは得て
いません。
NIAS, Japan
55
REVERSE GENETICS FOR FUNCTIONAL GENOMICS OF RICE
Akio MIYAO
Kannondai, Tsukuba, 305-8602
Introduction
The complete rice genome sequence was completed by December 2004 through the efforts of the International Rice Genome Sequencing Consortium
(IRGSP 2005). Access to the complete sequence has
created a paradigm shift for rice breeding from
phenotype-based or map-based breeding to genebased breeding. Both identification and characterization of agronomically important genes are necessary
for gene-based breeding. Currently, annotations of
rice genome sequences are proceeding under the collaboration of geneticists, molecular biologists and annotators throughout the world. However, most
annotated genes are still identified as a predicted gene
by some sort of similarity search or functional prediction program. For example, it is easy to identify a
gene that encodes a protein kinase, but difficult to deduce the target or the pathway involving the gene
product. Genes with no similarity to any other gene
with a known function remain to be characterized by
the traditional functional analyses of genetics and biochemistry.
Gene knockout mutagenesis is one of the suitable methods for the functional analysis of genes. TDNA (SALLAUD et al. 2003; JEONG et al. 2002; JEON
et al. 2000), Ds transposon (KOLESNIK et al. 2004;
GRECO et al. 2003; UPADHYAYA et al. 2002; CHIN et
al. 1999) and Tos17 retrotransposon (MIYAO et al.
2003; HIROCHIKA 2001) are used for rice gene disruption. RNAi technology has also been adopted for the
reduction of specific gene functions (MIKI et al.
2005). These technologies, like more traditional muta-
genesis, enable gene functions to be assigned from a
correlation between disrupted genes and aberrant phenotypes. In this paper, gene disruption lines of rice using retrotransposon Tos17 and the application of
functional analysis of rice genes are described.
Large-scale production of gene knockout lines using retrotransposon Tos17
Thirty-five percent of the rice genome has been
annotated with the aid of transposable elements. Most
transposable elements are inactivated upon insertion
into rice cells. Unregulated transposition may cause
the widespread disruption of genes, which would be a
serious disadvantage for living cells. Some transposons are activated under certain stress conditions. The
endogenous retrotransposon Tos17 is activated in cultured cells of Japonica cultivar Nipponbare (HIROCHIKA et al. 1996). Messenger RNA of Tos17 is
reverse transcribed to form a new copy of Tos17,
which is inserted into another location of the genome.
Tos17 transposition is immediately inactivated in
plants regenerated from cell culture and newly inserted copies of Tos17 segregate in the next generation. There are two copies of Tos17 in the wildtype
Nipponbare genome, and in cultured cells, one or two
copies of Tos17 are transposed per month. To obtain
efficient gene disruption lines, cells were cultured for
five months. We produced 50,000 insertion lines of
Nipponbare. If there is an average of 10 transpositions
in each line, there would be a total of 500,000 insertions. (MIYAO et al. 2003; HIROCHIKA et al. 2004)
Because Tos17 is an endogenous transposon, in-
56
Akio MIYAO
sertion lines are not defined as genetically modified
organisms (GMO) as T-DNA or Ac/Ds inserted rice
would be. Thus, large scale field experiments can be
conducted without the stringent regulations required
when working with GMO plants. This is a significant
advantage over other gene knockout lines.
Phenotypes of insertion lines
Because the genotype of Tos17 in regenerated
plants is heterozygous, the genotypes of seeds (M2
generation) from regenerated plants are segregated.
The phenotypes of 10 to 25 plants from each line
were observed in the field. The phenotyping effort is a
3 year collaborative project of 7 laboratories. To prevent confusion that could be caused by the used of different expressions for the same phenotype, fifty-one
common terms were adopted to describe phenotypes.
Details of phenotypes can be further explained with
additional comments and photographs. Phenotype
data is accessible via a web site (http://tos.nias.affrc.
go.jp).
Fifty percent of the regenerated lines show at
least one variation from the wild-type phenotype. Frequently observed phenotypes include sterile, dwarf
and pigment deficiency. The frequency of other minor
phenotypes, e.g., brittle, fine leaf, virescent, ranges
from 0.1 to 6 percent. Several hundred lines which
showed fine phenotypic segregation have been analyzed by DNA blot hybridization. The ratio of co-segregation between phenotype and genotype ranges
from 5 to 7 percent, with the remaining mutations not
attributable to Tos17. There are other mechanisms of
mutation in cultured cells of rice. Finding the source
of mutation in cultured cells is a remaining research
goal. Once the source is determined, gene disruption
lines will be more useful for functional genomics.
Large scale analysis of insertion sites of Tos17
Flanking regions of the Tos17 insertion were isolated by Thermal asymmetric interlaced (TAIL) and
suppression PCR methods using the PCR primer at
the 3í-end of Tos17 (MIYAO et al. 1998; LIU and
WHITTIER 1995; SIEBERT et al. 1995). For TAIL
PCR, flanking regions of the Tos17 insertion were amplified with random primers and the Tos17 3í-end
primer. For suppression PCR, blunt-end restriction enzyme digested genomic DNA is ligated with an adaptor fragment which is modified with NH3 at the 3í end
of the bottom strand, and the flanking regions were
amplified with Tos17 3í-end primer and adaptor
primer. Amplified fragments were separated by agarose gel electrophoresis (Fig. 1), extracted from gel
pieces, and sequenced by the BigDye terminator
method.
Tos17 insertion points in the rice genome were
identified by a BLASTN search of flanking sequences
against the rice genome. We have obtained more than
50,000 flanking sequences and identified about
21,000 insertion loci. The disparity in number between flanking sequences and identified loci is due to
the redundancy of flanking sequences. The distribution of insertion points on the rice genome is not ran-
Fig. 1 Amplification products generated by TAIL-PCR
(A) and suppression PCR (B). (A) Lane M contains DNA size markers (HindIII digests of genomic DNA and HincII digests of phiX174
DNA). Lanes 2 and 3 are products of the 2nd and
the 3rd TAIL-PCR, respectively. Products from
the 3rd reaction indicated by arrows are smaller
than those from the 2nd reaction because the
nested primer was used in the 3rd reaction. (B)
Lanes DraI, EcoRV, and ScaI are products of suppression PCR using the corresponding enzymes.
dom. The insertion frequency of Tos17 within coding
regions is 3-fold higher than in non-coding regions.
There are many hë ot spotsí with high insertion frequency, as has been observed in other transposonbased mutagenesis systems in both prokaryotes and
eukaryotes. The consensus target sequence of Tos17 is
ANGTT-(Target Site Duplication)-AACNT (Fig. 2).
Tos17 target site recognition allows some variation
from the consensus sequence. There is no target site
specificity for hot spots at the nucleotide sequence
level. However, there is a correlation between the location of protein kinase and disease resistance genes
and Tos17 hot spots. It is interesting that the insertion
preference of Tos17 correlates with the localization of
protein kinase and disease resistance genes, which are
clustered and frequently telomeric rather than pericentromeric. This apparent non-random distribution may
reflect some selection force for localization of these
genes in the process of development of the rice genome over time. The correlation between these gene
clusters and Tos17 insertion hot spots indicates that
there are some structural differences in these regions
(MIYAO et al. 2003).
Fig. 2 Base preferences of Tos17 Insertion Sites. Average base preferences at each position were obtained from 20,458 independent flanking
sequences. The target site duplication (TSD) sequence extends from positions 1 to 5. Numbers
with minus and plus signs are base numbers upstream and downstream, respectively, from the
TSD. The percentage of A (green), C (blue), G
(black) and T (red) at each position was plotted.
57
Web-based relational database for reverse genetics
Phenotypic and flanking sequence data are
stored in a relational database. In 1996, which marked
the beginning of the mutant panel construction project, there was no free UNIX- based system available
in Japan except for FreeBSD (http://www.freebsd.org).
Linux was not stable for the Japanese computing environment. For the same reason, we choose the PostgreSQL (http://www.postgresql.org) relational
database management system. FreeBSD is a highly reliable system that has had no serious problem during
its nine years in operation, making it an ideal platform
for internet access to the database.
The number of phenotype descriptions stored in
the relational database exceeds 120,000 lines, and
there are more than 59,000 photographic files. Sequence data from the Tos17 flanking regions are also
stored in the database. All of the recorded flanking sequences have been searched by BLASTN against the
rice genome pseudo-molecule constructed by IRGSP.
Full-length cDNA sequences have also been searched
by BLASTN, and locations on exons of the pseudomolecule were confirmed with the Sim4 program. Insertion map locations and cDNAs on the pseudo-molecule are plotted to png style image files on demand by
a perl script with GD module (Fig. 3; http://tos.nias.
affrc.go.jp/cgi-bin/tos17/ricegenome.cgi). Full-length
cDNA is evenly distributed throughout all chromosome regions, except for pericentromeric regions,
where there is a slightly lower density. Tos17 insertions are also distributed throughout all of the chromosomes. However, there is a greater distribution bias
for Tos17 than for full-length cDNA. The location of
Tos17 insertions seems to coincide with euchromatic
regions. For example, the heterochromatic regions described in a report of chromosome 4 have very few
Tos17 insertions (FENG et al. 2002).
Guest users are able to search the disruption mutant library using BLASTN on our web site. If the
query sequence aligns with one of the flanking sequences, the user can choose the mutant line and list
phenotypic descriptions with a photograph. Further-
58
Akio MIYAO
Fig. 3 Tos17 insertion and cDNA location map on 12 rice chromosomes. Insertion of Tos17 is shown
with a vertical salmon bar. Location of cDNA is shown with a vertical blue bar. One pixel represents 100kb.
more, a chromosomal location map of flanking sequences can be displayed with the option of zooming
in on an interactive chromosome map by clicking on a
pixel of cDNA or Tos17. A close-up of the corresponding region (200 kb) is drawn to another interactive map. The user can access the name of the cDNA
and insertion point of Tos17 from the map. Phenotypic data can be listed from the location map data of
Tos17. If the same phenotype is shown between allelic
mutants of the same gene, the function of the gene
may affect the phenotype. To confirm its function,
complementation of the disrupted gene with the transformed wild-type gene is required.
The mutant panel database is a powerful tool that
can be used to determine gene functions. All of the
mutant panel data are accessible from our web site
(http://tos.nias.affrc.go.jp) and seeds corresponding to
mutant lines can be obtained from the Rice Genome
Resource Center (A
et al. 2003; http://www.
rgrc.dna.affrc.go.jp).
Summary
We have produced more than 50,000 gene-disruption lines using an endogenous retrotransposon of
rice, Tos17. Phenotypes of each mutant line have been
observed and recorded in the field. Furthermore, more
than 10% of mutant line disruption points have been
mapped on the rice genome by flanking sequence
analysis. All data are stored and managed with a webbased relational database (http://tos.nias.affrc.go.jp).
The newest insertion point map on twelve chromosomes of rice can be created on demand with CGI program software. The Tos17 gene disruption mutants
can be searched using our database system. When the
target mutant is detected by BLAST search, phenotypic data of the mutant with a photo image can be obtained. Mutant seeds are available from the Rice
Genome Resource Center (http://www.rgrc.dna.affrc.
go.jp). This system will be useful not only for the
functional genomics of rice but also in gene-based
breeding.
Acknowledgements
This work was supported by grants from the Ministry of Agriculture, Forestry, and Fisheries of Japan,
the Enhancement of Center-of-Excellence, Special Coordination Funds for Promoting Science and Technology in Japan, and the Program for the Promotion of
Basic Research Activities for Innovative Biosciences.
I thank Dr. Hirohiko Hirochika his direction of my
study and for giving me the opportunity to lecture at
the Gamma Field Symposia. Phenotypic data were the
results of Mutant Panel Project participantsí efforts.
Flanking sequence data were the results of technical
staff efforts, especially Miss Yumiko Yamashita in the
STAFF institute.
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J. -J., PARK , S., LEE , H. S., AN , K., AN , G. (2002). TDNA insertional mutagenesis for activation tagging in
rice. Plant Physiol. 130: 1636-1644.
KOLESNIK , T., SZEVERENYI, I., BACHIMANN , D., KUMAR , C. S., JIANG , S., RAMAMOORTHY , R., CAI , M.,
MA , Z. G., SUNDARESAN , V., RAMACHANDRAN , S.
(2004). Establishing an efficient Ac/Ds tagging system
in rice: large-scale analysis of Ds flanking sequences.
Plant J. 37: 301-314.
LIU , Y., and WHITTIER , R.F. (1995). Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC
clones for chromosome walking. Genomics 25: 674681.
MIKI , D., ITOH , R., and SHIMAMOTO , K. (2005). RNA
silencing of single and multiple members in a gene
family of rice. Plant Physiol. 138(4):1903-1913.
MIYAO , A., YAMAZAKI , M., and HIROCHIKA , H.
(1998). Systematic screening of mutants of rice by sequencing retrotransposon-insertion sites. Plant Biotechnology, 15(4):253-256.
MIYAO , A., TANAKA , K., MURATA , K., SAWAKI , H.,
60
Akio MIYAO
TAKEDA , S., ABE , K., SHINOZUKA , Y, ONOSATO , K.,
and HIROCHIKA , H. (2003). Target site specificity of
the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in
retrotransposon-rich regions of the genome, Plant Cell,
15(8):1771-1780.
16. SALLAUD , C., MEYNARD , D., BOXTEL , J., GAY , C.,
BES , M., BRIZARD , J. P., LARMANDE , P., ORTEGA , D.,
RAYNAL , M., PORTEFAIX , M., OUWERKERK , P. B.,
RUEB , S., DELSENY , M., GUIDERDONI , E. (2003).
Highly efficient production and characterization of TDNA plants for rice (Oryza sativa L.) functional ge-
nomics. Theor. Appl. Genet. 106: 1396-1408.
17. SIEBERT , P. D., CHENCHIK , A., KELLOG , D. E., LUKYANOV , K. A., and LUKYANOV , S. A., (1995). An improved PCR method for walking in uncloned genomic
DNA. Nucleic Acids Res. 23: 1087-1088.
18. UPADHYAYA , N. M., ZHOU , X. -R., ZHU , Q. -H.,
RAMM , K., WU, L., EAMENS , A., SIVAKUMAR , R.,
KATO , T., YUN, D. -W., SANTHOSHKUMAR , C.,
NARAYANAN , K. K., PEACOCK , J. W., DENNIS , E. S.
(2002). An iAc/Ds gene and enhancer trapping system
for insertional mutagenesis in rice. Funcl. Plant Biol.
29: 547-559.
61
逆遺伝学的手法によるイネの遺伝子機能解析
宮尾安藝雄
農業生物資源研究所分子遺伝研究グループ遺伝子機能研究チーム
〒３０５−８６０２茨城県つくば市観音台２−１−２
イネの全ゲノム塩基配列が解読され，コードさ
れている遺伝子のゲノム上の位置を把握できるよ
うになった。すでにその機能が明らかになってい
隣接塩基配列を検索することにより，任意の遺
伝子の破壊系統の検索を行うシステムを構築した
（http://tos.nias.affrc.go.jp/）。また，検索された系統
る遺伝子も存在するが，残された多くの機能未知
の遺伝子の中には，育種を行っていく上で重要な
遺伝子が存在すると考えられ，これらの遺伝子の
機能解明を行うことが課題となっている。遺伝子
の機能を解析する方法の一つに，遺伝子破壊法が
あげられる。これまでに，日本晴に内在性のレト
ロトランスポゾンTos17を用いた遺伝子破壊系統
を約５万系統作出した。作出した系統の表現型を
圃場で観察し，画像と共にデータベース化を行っ
た。また，転移したTos17の隣接塩基配列を解析
して，系統ごとのTos17のゲノム上の挿入位置を
データベース化した。
リストより，それぞれの系統の表現型を調べるこ
とができる。これらの整理された破壊された遺伝
子と表現型の関係より，多くの遺伝子の機能解析
が可能となった。
さらに，最新の染色体別の全塩基配列データに
対して，Tos17の挿入位置を示したマップを作成
し，任意の位置のTos17の挿入状況を知ることが
できる。発現遺伝子の位置情報と合わせて解析を
進めることにより，ゲノム全体にわたる遺伝子機
能地図が出来ると考えている。このような遺伝子
機能情報から育種を行う‘Gene-based’育種が行
える日も近いと考えている。
NIAS, Japan
63
DEVELOPMENT AND UTILIZATION OF GENOME INFORMATION IN
VEGETABLE CROPS
Hiroyuki FUKUOKA
Laboratory of Breeding Technology, National Institute of Vegetable and Tea Science, National Agriculture and Biooriented Research Organization, Kusawa 360, Ano, Tsu, Mie 514-2392, Japan
Introduction
The number of vegetable crops sold on the Japanese market tops out at around 150 items belonging to
33 taxonomic families. Of these, the Ministry of Agriculture, Forestry and Fisheries has designated 14
items (cabbage, Chinese cabbage, radish, spinach, tomato, eggplant, potato, bell pepper, cucumber, carrot,
lettuce, onion, bunching onion and taro) as the vegetables which are particularly indispensable for consumers, making the stability of their supply a matter of
public policy. Even these 14 items cover a wide range
of higher plant taxa, and are classified into 11 genera
belonging to 8 families. This taxonomic diversity is a
defining characteristic of the vegetable crops that support the nationís healthy dietary culture. Therefore,
from an agronomic perspective, a strategy is required
for applied genomic research in vegetables that is different from the comprehensive basic science approach
used in model plants such as Arabidopsis and rice.
While full advantage should be taken of the enormous
amount of genetic information obtained from the
model plants, due to the practical limitations of currently available technology the development of genome information in vegetable crops cannot be
pursued in the same way. In order to sustain the diversity of vegetable production, supply and consumption,
the research and development of vegetable genome information should be directed to satisfy both priority
and versatility in terms of raising the level of research
progress in the vegetable crops as a group. In this paper, recent research activity and perspectives at
NIVTS ranging from basic genome analysis to more
applied technological development will be introduced.
Large scale development of DNA markers and construction of high resolution linkage maps: Eggplant as a model of a low DNA polymorphism
vegetable.
Eggplant is one of the most important and popular vegetables in Asia, the Middle East and Mediterranean regions. However, information about its genome
is rather limited compared to other solanaceous vegetables such as tomato (Tanksley et al. 1992, Van der
Hoeven et al. 2002) and pepper (Lefebvre et al.
2002), partly because it is relatively less important in
production and consumption in Europe and the US.
Although eggplant exhibits a wide variety of agricultural and biological traits, DNA polymorphism among
the cultivars is rather rare and therefore it is not easy
to develop a sufficient number of molecular genetic
markers for the construction of a genetic map (Nunome et al. 2003). This low polymorphism is relatively common to autogamous vegetable species such
as tomato, cucumber and melon, possibly because of
the selection of limited genetic sources for modern
high quality breeding that does not require consideration for the genetic diversity that is necessary to avoid
inbreeding depression (Alvarez et al. 2001).
We started large-scale isolation of microsatellites
in eggplant to develop an eggplant genetic map which
consists of landmarks that are of genome-wide distribution, co-dominantly inherited and highly polymor-
64
Hiroyuki FUKUOKA
phic among cultivars (Tautz 1989). With technical
improvements in procedures for enrichment of
microsatellite-containing genomic DNA fragments (T.
Nunome et al. in preparation), development of a largescale sequence processing computing pipeline (Fukuoka et al. 2005) and high-throughput genotyping
workflow management, more than 1,000 microsatellites have been identified and around 120 loci have
been mapped using an F2 population derived from
intra-specific crosses between eggplant cultivars. An
eggplant genetic map has already been reported by
Doganlar et al. (2002). An F2 population derived
from inter-specific crosses with wild relatives was
used as a mapping population. RFLP markers were
mainly used, which are not as convenient as microsatellites in terms of applied utility. The markers located
on our linkage map are useful for genetic analyses of
important characters in eggplant because PCR-based
microsatellite markers are much more versatile than
AFLP and RAPD markers. For example, applications
using microsatellite markers are not limited to the
population used for map construction. Genetic analyses of resistance to soil-borne diseases and other agronomically important traits using these genetic maps
are underway. The procedure for microsatellite
marker development in this study has proven to be efficient and compatible in other vegetables such as
bunching onion, melon and yam in other laboratories
and the use of eggplant as a model plant for vegetables with low DNA polymorphism has thus facilitated
marker resource accumulation in several different
vegetable crops.
Genetic marker development using gene coding
sequences has also recently begun. The advantage of
genetic markers based on gene sequences is that
orthologous regions can be identified by sequence
comparisons among related species. Around 2,000
unique sequences have been identified as candidate
genes for marker development by comparative sequence analysis among Arabidopsis, tomato and eggplant, which should make it possible to identify
corresponding chromosome regions (unpublished
data). Marker development and mapping based on sin-
gle nucleotide polymorphisms (SNPs) is now underway mainly by comparing the introns of selected
genes. This comparative genomic approach would be
helpful for development of molecular markers between microsatellte-based landmarks, and for the integration of genetic linkage maps constructed in various
solanaceous vegetables.
Arabidopsis genomic information in genetic
marker development and gene isolation in cruciferous vegetables.
Clubroot resistance is one of the most important
targets for cruciferous vegetable breeding. The disease, caused by the fungus Plasmodiophora brassicae, is soil transmissible and is difficult to control
with cultural and chemical methods. Breeding for resistance is thus the most effective and important control strategy. The results of extensive screening for
genetic resistance to clubroot in Chinese cabbage
(Brassica rapa) indicate that some turnip (also B.
rapa) lines have strong resistance to the disease. Several clubroot-resistant (CR) varieties of Chinese cabbage have been successfully bred and released using
the resistant genes derived from turnip. Recently it
has been reported, however, that some resistant varieties have already succumbed to super-virulent Plasmodiophora races.
In order to analyze the genetic background of
clubroot resistance and to construct an efficient breeding system based on marker-assisted selection, we
constructed a molecular marker-based linkage map,
and mapped the resistant genes in Chinese cabbage
mainly based on RFLP and microsatellite markers
(Suwabe et al. 2004). Two resistance genes (Crr1 and
Crr2) have been located in different linkage groups of
the B. rapa linkage map between two sets of microsatellite markers. Crr1 and Crr2 gave different resistance
reactions against different pathogen isolates and likely
have independent biological functions (Suwabe et al.
2003). A sequence analysis of adjacent microsatellite
markers revealed that the chromosomal regions
around Crr1 and Crr2 would correspond to the same
DEVELOPMENT AND UTILIZATION OF GENOME INFORMATION IN VEGETABLE CROPS
region in the Arabidopsis genome, suggesting that
these two genes are derived from the same origin
(Suwabe et al. 2005). Thanks to partial but highly conserved colinearity between the Brassica and Arabidopsis genomes, we succeeded in developing several
SNPs and Indel markers around the resistant genes using Arabidopsis genome sequence information. One
SNP marker in particular co-segregates with the Crr1
phenotype in 1920 F2 individuals, indicating that the
marker resides very closely to the resistance gene. A
chromosome walk to isolate Crr1 and Crr2 is in progress.
It has been suggested that there are clubroot resistance genes in addition to Crr1 and Crr2 (Hirai et al.
2004). In the near future it will be possible to develop
selection markers using all of the clubroot resistance
genes composing a gene family to construct a markerassisted breeding system for super-resistant cultivars.
Variety tagging using DNA marker technology.
Recent efforts by rural vegetable growers and local governments to preserve and expand market share,
and an increase in imported fresh agricultural products that can be fraudulently marketed as local produce have increased the need for variety and producer
identification technologies. DNA markers have the
potential to play an important role in the development
of accurate and reliable variety tags. The most prominent example of these emerging technologies is in
strawberry, where the identification method for almost
all cultivars produced and/or quoted in the Japanese
market has been implemented (Kunishisa et al. 2005).
Early application of this technology in strawberry is at
least in part due to its vegetative propagation as
clones, making genetic variation within a cultivar extremely rare. In contrast, within the allogamous vegetables such as bunching onion, the frequency of DNA
polymorphism within a variety is too high to determine a unique identifiable genotype, though uniformity of the phenotype required for an established
variety is not affected. Thus, we have proposed a
breeding method which makes DNA marker-based va-
65
riety identification possible for allogamous vegetable
crops (Tsukazaki et al. 2004), based on the idea that
the chromosomal region around the marker loci exhibiting intra-varietal polymorphisms would not contain
variety-determining genes. Thus, if individuals with
an arbitrary homozygous marker genotype are selected from a breeder stock farm , the character of the
variety is not likely to be changed. To demonstrate the
feasibility of this approach, bunching onion individuals that are homozygous at two or four microsatellite
marker loci were selected from a population of a local
variety and several agronomical traits in the progeny
population were examined. As compared to the original varietal population, no significant differences
were observed in the selected population, suggesting
that the procedure would make cultivar identification
possible for allogamous crops.
Conventional variety identification methodology
has been limited to existing varieties and the strategy
has been based on screening DNA polymorphisms
that are found by chance. The procedure we have proposed is based on a new concept wherein a marker
genotype is positively controlled for during the breeding procedure to tag the variety. This method can provide a powerful tool for differentiation of local
produce.
Perspective
Heretofore, development of genomic information
resources in each vegetable species has been mainly
oriented toward its significance to practical breeding.
Continuous and comprehensive research activities
which could be considered as iomics have not as yet
been a priority in Japan. Recently, as a model of solanaceous plants, which are situated in a phylogenically
peculiar cluster and involve a number of agronomically important crops, sequencing of the tomato genome has been started by an international consortium
(Mueller et al. 2005). It is worthy of special mention
that the Japanese research community has been organized and are included in various activities such as contribution to the sequencing of chromosome 8, full-
66
Hiroyuki FUKUOKA
length cDNA sequencing, DNA array distribution,
and collection of induced mutant lines. The same kind
of collaborating framework has been activated in cruciferous and cucurbitaceous vegetables. In light of the
success that cooperative programs have had with
other important crops, it is likely that this kind of activity will become common in a number of vegetable
crops.
References
ALIVAREZ, A.E., van de WIEL , C.C.M., SMULDERS ,
M.J.M., and VOSMAN , B. (2001) Use of microsatellites
to evaluate genetic diversity and species relationships
in the genus Lycopersicon. Theor. Appl. Genet.
103:1283-1292.
DOGANLAR , S., FRARY , A., DAUNAY , M.-C., LESTER ,
R.N., and TANKSLEY , S.D. (2002) A comparative genetic linkage map of eggplant (Solanum melongena)
and its implications for genome evolution in the Solanaceae. Genetics 161: 1697-1711
FUKUOKA , H., NUNOME , T., MINAMIYAMA , Y., KONO , I.,
NAMIKI , N., and KOJIMA , A. (2005). read2Marker: a
data processing tool for microsatellite marker development from a large data set. BioTechniques 39: 472-476.
HIRAI M., HARADA , T., KONO , N., TSUKADA , M.,
SUWABE , K., and MATSUMOTO , S. (2004) A novel locus for clubroot resistance in Brassica rapa and its linkage markers. Theor. Appl. Genet. 108: 639-643.
KUNISHIMA, M., FUKINO , N., and MATSUMOTO , S. (2005)
CAPS markers improved by cluster-specific amplification for identification of octoploid strawberry (Fragaria x ananassa Duch.) cultivars, ant their disomic
inheritance. Theor. Appl. Genet. 110: 1410-1418.
LEFEBVRE , V. PFLIEGER , S. THABUIS , A. CARANTA , C.,
BLATTES , A, CHAUVET , J.-C., DAUBEZE , A.-M, and
PALLOIX , A. (2002) Towards the saturation of the pepper linkage map by alignment of three intraspecific
maps including known-function genes. Genome 45:
839-854.
MUELLER , LA., TANSKLEY , S.D., GIOVANNONI , J.J., van
ECK , J., STACK , S., CHOL, D., KIM , B.D., CHEN , M.,
CHENG , Z., LI, C., LING , H., XUE , Y., SEYMOUR , G.,
BISHOP , G., BRYAN , G., SHARMA , R., KHURANA , J.,
TYAGI , A., CHATTOPADHYAY , D., SINGH , N.K.,
STIEKEMA , W., LINDHOUT , P. J
, T., LANKHORST ,
R.K., BOUZAYEN , M., SHIBATA , D., TABATA , S.,
GRANELL , A BOTELLA , M.A., GIOVANNI G., FRUSCIANTE , L., CAUSSE , M., and ZAMIR , D. (2005) The Tomato Sequencing Project, the first cornerstone of the
International Solanaceae Project (SOL). Comp. Func.
Genomics 6: 153-158.
NUNOME , T., SUWABE , K., OHYAMA , A., and FUKUOKA ,
H. (2003) Characterization of Trinucleotide microsatellites in eggplant. Breed. Sci. 53: 77-83.
SUWABE , K., TSUKAZAKI , H., IKETANI , H., HATAKEYAMA , K., FUJIMURA , M., NUNOME , T., FUKUOKA ,
H., MATSUMOTO S., and HIRAI , M. (2003) Identification of two loci for resistance to clubroot (Plasmodiophora brassicae Woronin) in Brassica rapa L. Theor.
Appl. Genet. 107: 997-1002.
SUWABE , K., IKETANI , H., NUNOME , T., OHYAMA , A., HIRAI , M., and FUKUOKA , H. (2004) Characteristics of
microsatellites in Brassica rapa genome and their potential utilization for comparative genomics in Cruciferae. Breed Sci 54: 85-90.
SUWABE , K., TSUKAZAKI , H., IKETANI , H., HATAKEYAMA , K., KONDO , M., FUJIMURA , M., NUNOME , T.,
FKUOKA , H., HIRAI , M., and MATSUMOTO , S.
(2005) SSR-based comparative genomics between
Brassica rapa and Arabidopsis thaliana: the genetic origin of clubroot resistance. Genetics (in press).
TANSKLEY , S.D., GANAL , M.W., PRINCE , J.P., De
VICENTE , M.C., BONIERBALE , M.W. BROWN , P., FULTON , T.M. Giovannoni, J.J., and Grandillo, S. (1992)
High density molecular linkage maps of the tomato and
potato genomes. Genetics 132:1141-1160.
TAUTZ , D. (1989) Hypervariability of simple sequences as a
general source for polymorphic DNA markers. Nucleic
Acids Res. 17: 6463-6471.
TSUKAZAKI , H., FUKUOKA , H., SONG , Y.S., YAMASHITA ,
K., and KOJIMA , A. (2004) Proposal of breeding
method prerequisite to variety identification in allogamous crops, e.g., bunching onion (Allium fistulosum).
In: 4th intíl. ISHS Symp. on edible Alliaceae. Chinese
Academy of Agricultural Sciences, Beijin. pp199.
VAN der HOEVEN , R., RONNING , C., GIOVANNIONI , J., Martin, G., and TANKSLEY , S. (2002). Deductions about
the number, organization, and evolution of genes in the
tomato genome based on analysis of a large expressed
sequence tag collection and selective genomic sequencing. Plant Cell 14: 1441-1456.
DEVELOPMENT AND UTILIZATION OF GENOME INFORMATION IN VEGETABLE CROPS
67
野菜におけるゲノム情報の開発と利用
福岡浩之
（独）農業・生物系特定産業技術研究機構野菜茶業研究所育種工学研究室
多品目を擁する野菜におけるゲノム研究では，
イネやシロイヌナズナなどで蓄積されたモデル植
物のゲノム研究の成果を受け，その成果やノウハ
ウをどう活用して研究を加速化し実用化へつなげ
てゆくか，野菜の特質である多様性を損なわない
研究開発をどう進めていくか，などを考えつつ，
効率的な研究開発を行うことが求められている。
そこで，私たちは，研究対象の重点化および多品目
に対応する汎用的な技術開発と基盤整備の双方を
両立させることを念頭に置き，ナスの全ゲノムを
カバーする詳細マーカー連鎖地図の作製とマーカー
開発，ハクサイ根こぶ病抵抗性に関する詳細遺伝
解析，DNAマーカー品種識別法の開発と実用化，
の３つの課題をモデルとして研究を進めている。
ナスはゲノム情報の蓄積が乏しく系統間の
DNA多型頻度も低い野菜の典型であるといえる。
そこで，マイクロサテライト（SSR）マーカーの
大量開発を行い，多型頻度の高い近縁種との交配
集団を用いずに構築されたものとしては初めてと
なるマーカー連鎖地図を構築した。また，ナス科
野菜であるトマト，ピーマンのゲノム情報との統
合を目的として，発現遺伝子配列を利用したマー
カー開発にも着手している。ハクサイでは，根こ
ぶ病抵抗性の遺伝を詳細に解析し，２つの独立な
連鎖群に座乗し異なるレースに対する抵抗性反応
に分化がみられる抵抗性遺伝子座Crr1およびCrr2
を見いだした。シロイヌナズナのゲノム情報を活
用して抵抗性遺伝子座近傍の連鎖地図の詳細化を
行い，抵抗性と非常に強く連鎖する一塩基多型
マーカーを開発した。現在，これを用いてCrr1お
よびCrr2遺伝子座のマップベースクローニングが
進行中である。DNAマーカーを用いた品種や産
地の判別技術開発にも取り組んできており，栄養
繁殖性のイチゴにおいて完成度の高い品種判別技
術をすでに確立し実用化している。その一方，ネ
ギなどの他殖性野菜では，品種の要件である表現
型の均一性は十分確保されているにもかかわらず，
品種内でのDNA多型頻度が著しく高く，品種を特
定するマーカー遺伝子型が一意に決定できないこ
とが最近明らかになってきた。そこで，育種過程
でいくつかのマーカー遺伝子座をそろえることに
よって品種識別を人為的に可能としようとする試
みを進めている。本手法は系統育成過程で積極的
にマーカー遺伝子型を制御して品種を標識すると
いう新しい考え方に基づいており，DNAマーカー
のリソース整備が進めば多くの品目に適用できる
と考えられる。
以上のように，野菜においても応用研究として
のDNAマーカーのリソース整備が進み，これを利
用した詳細な遺伝解析も可能となってきている。
その一方で，近年ではナス科，アブラナ科，ウリ
科などの野菜においてもモデル植物で行われたよ
うな国際コンソーシアムによる網羅的なゲノム情
報基盤整備が進められるようになってきている。
我が国の野菜の遺伝・育種研究勢力もこのような
動きに積極的に関与し，国際的プレゼンスを確保
していく必要があろう。
NIAS, Japan
69
USE OF -RAY-INDUCED MUTATIONS IN THE GENOME ERA IN RICE
Makoto K
Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan
Introduction
Ionizing radiation has been used for inducing mutations and improving crops since the discovery by
STADLER (1928) that X-rays could induce mutations
in barley. At the end of 2004, the whole genome sequence of rice was determined (I
R
GS
P
, 2005). What
can -ray-induced mutations contribute now that this
has been achieved? One answer could be the elucidation of the functions of the numerous genes revealed
by the complete sequence of the rice genome. This includes identification of mutants through reverse genetics and the isolation of genes containing mutations
through forward genetics using molecular markers
and sequence information. Another answer could be
mutation breeding using reverse genetics. But first we
must know what kinds of DNA lesions are caused by
-rays. In this article, I describe the production of
DNA lesions, and then discuss how -ray-induced mutations can contribute to the elucidation of gene function and to mutation breeding.
-rays
Characteristics of Ionizing radiation is typically classified into lowlinear-energy-transfer (LET) radiation, such as -rays
and X-rays, and high-LET radiation, such as a particles and heavy ion particles. Low-LET radiation gives
a relatively low energy per unit length of the particleís
path, and transfers energy evenly to the irradiated
area. High-LET radiation gives a relatively high energy per unit length of the particleís path and transfers
energy to very limited regions. This difference is
thought to cause different types of damage to chromosomal DNA (SACHS et al., 2000). We analyzed DNA
lesions caused by -rays as a representative of lowLET radiation.
The pollen-irradiation method
Sequence analyses of -ray-induced mutations in
plants have been widely carried out. These mutations
include deletions, translocations, and sometimes insertions (S
et al., 1992, B et al.,
1996, B
et al., 1997, Net al.,
2003). In these studies, however, mostly M2 plants or
their progeny were analyzed, which could lead to biased results, because mutations with defective transmissibility to progeny are excluded from this type of
analysis.
1) Principle of pollen irradiation
To analyze various mutations induced by ionizing radiation, including nontransmissible mutations,
we used the pollen-irradiation method (Sand
R, 1948). The experimental procedure was as
follows (Figure 1): (1) The pollen of wild-type plants
was irradiated. (2) The irradiated pollen was used to
pollinate plants homozygous for a recessive marker
mutation. (3) F1 plants were screened for the recessive
marker phenotype. Because the whole body of an F1
plant is derived from a single fertilized egg, this
method can avoid chimeras, which make the molecular analysis of mutations difficult.
Pollen longevity is important for this analysis.
70
Makoto KUSABA
one DNA broken end with another without specificity
(CRITCHLOW and JACKSON , 1998, BRITT 1999, GORBUNOVA and LEVY , 1999, LIEBER et al., 2003).
NHEJ is the major repair mechanism in plants. The induction of deletions by γ-irradiation is thought to
work as follows. NHEJ can be precise but is often accompanied by small deletions, and sometimes by very
large deletions (SACHS et al., 2000). When the outer
DNA fragments of the three fragments generated by
two DSBs in one chromosome are joined together, the
consequent omission of the middle fragment causes a
large deletion.
Fig. 1 The principle of the pollen-irradiation method.
Deletions and deleted genes are shown in gray.
For this and other reasons, Arabidopsis thaliana was
chosen in preference to rice. Ecotype Columbia (Col)
was used as the pollen donor for mutagenesis, and
ecotype Landsberg erecta (Ler) was used as the pollen recipient. This enables the identification of
radiation-induced mutations in pollen by using molecular markers that distinguish the Col and Ler genomes. As a recessive visible marker mutation, the
trichomeless mutation gl1-1 in the Ler background
was used.
2) Induction of DNA lesions
Of the 46 gl1 mutants induced with -rays, only
2 had point mutations (4- and 1-bp deletions). One
had a reciprocal translocation. All the remaining mutants carried large deletions, ranging from 80 kbp to
more than 6 Mbp; the latter corresponds to more than
5% of the whole genome. The average size of the deletions was 2 Mbp.
Nonhomologous end joining (NHEJ) is a doublestrand break (DSB) repair mechanism, which rejoins
3) Transmissibility of mutations
All the gl1 mutants with small deletions were
fully fertile, and the mutations were transmitted to
progeny normally. On the other hand, most mutants
carrying large deletions around the GL1 locus were
semi-sterile, suggesting a correlation between large
deletions and semi-sterility. These large deletions did
not appear to be transmitted even heterozygously. The
most likely explanation for these observations is that
the large deletions contained a gene or genes required
for the formation or viability of pollen and egg cells:
only egg cells and pollen that do not carry the large
deletions can undergo successful fertilization, explaining the semi-sterility and nontransmissibility of the deletions.
In a separate experiment, three mutants induced
by carbon ion particles (220 MeV) showed full fertility even though they carried large deletions around the
GL1 locus. (NAITO et al., 2005). These deletions were
transmitted only heterozygously. Because one-fourth
of the progeny seeds did not germinate, we hypothesize that fertilized eggs homozygous for the deletions
can develop into seeds but lack the ability to germinate. In addition, SHIKAZONO et al. (2003) isolated
gl1 alleles carrying a large deletion that were transmitted normally, suggesting that the deleted regions do
not contain the gene involved in transmission. Comparison of the deleted regions of these three classes of
mutants predicts a gene or genes essential for gamete
development or viability, and a gene or genes in-
USE OF -RAY-INDUCED MUTATIONS IN THE GENOME ERA IN RICE
volved in seed germination around the GL1 locus
(NAITO et al., 2005). These results imply that the
transmissibility of large deletions depends on what
gene the deletion involves. The lines of information
obtained in this experiment are thought to be common
to rice.
Transmissible DNA lesions caused by -rays in rice
The pollen irradiation study revealed that -rays
induce both large and small deletions, but mainly the
former. Most large mutations, which cause defects in
transmissibility, cannot be used for mutation breeding
and analysis of genes except in particular cases (for
example, in improving vegetatively propagated
crops). In this regard, it is important to know the characteristics of DNA lesions in transmissible mutations
induced byγ-rays.
We analyzed 9 genes withγ-ray-induced mutations whose wild-type sequences and mutant characters are already known, using sequencing of PCR
products or PCR-based estimation of deleted regions.
The DNA lesions we analyzed are classified into 2
classes. Six of the 9 mutations were point-like mutations (1- and 3-bp deletions), and 3 were large deletions (>10 kb) (Morita et al., unpublished results). No
intermediate-size deletion was obtained. This could
be informative for developing a method to isolate mutants by reverse genetics. Because these mutations
were selected according to phenotype, the mutations
not affecting phenotype were not included. Although
it is very important to know what kind of mutation
tends to be isolated in mutagenesis by γ-rays, reverse
genetics might be required to know the exact distribution of DNA lesions caused by γ-rays.
71
cantly to elucidating the functions of the predicted
genes (YAMADA et al., 2003). Other reverse genetics
approaches than insertional mutagenesis have been established. Targeting Induced Local Lesions IN Genomes (TILLING) detects nucleotide substitutions
and small deletions/insertions (McCallum et al., 2000;
Figure 2). In this method, a DNA fragment amplified
with a primer set for a particular gene by PCR is heatdenatured and then annealed. The annealed DNA fragment is treated with CEL I, a heteroduplex-cleavage
enzyme from celery. If the genome DNA templates
contain a mutant gene, part of the mutant DNA fragment will form a heteroduplex with the wild-type
DNA fragment. Electrophoresis will reveal three
bands: the uppermost band, which contains wild-type
and mutant homoduplexes, and the lower two bands,
which are derived from the heteroduplex cleaved by
CEL I. Detection of the lower two bands means that
the plants used for isolation of DNA contain mutants
of the gene. In rice, genome-wide reverse genetics using insertional mutagenesis has been carried out
mainly with the endogenous retrotransposon Tos17,
whose transposition is activated by tissue culture (H
Reverse genetics using -ray-induced mutants
In the post-genome era, the main objective is to
elucidate the function of genes predicted from the
whole genome sequence revealed by genome projects.
In A. thaliana, ample T-DNA-tagged lines and the TDNA-flanking sequences are contributing signifi-
Fig. 2 The principle of TILLING. Among the re-natured fragments, only heteroduplexes are digested
with CEL I. Resultant fragments are shown with
arrows.
72
Makoto KUSABA
ROCHIKA ,
2001). A large-scale flanking-sequence-tag
database is available. However, Tos17 transposition
shows insertion hot spots, and coverage of the whole
genome is not enough. T-DNA tagged lines are also
available in rice (HIROCHIKA et al., 2004), but the
population scale is not large enough at present. This is
partly because the rice plant is large, and much more
space in greenhouses is required for growing rice than
for growing Arabidopsis. Therefore, the use of -rayinduced mutants in reverse genetics screening could
contribute to the analysis of gene function in rice.
Gamma-rays induce small deletions as well as large
deletions, suggesting that TILLING can be applied to
g-ray-induced mutants. In fact, -ray-induced mutations were successfully identified by TILLING-based
screening (Sato et al., 2006).
Point mutations, typically induced by chemical
mutagens, and insertion mutations, typically induced
by transposons and T-DNA, are useful for obtaining
mutations in a particular gene. But functionally redundant genes frequently exist in plant genomes. The generation of double mutants by crossing of mutants for
each gene might elucidate the function of two genes
with redundancy. However, a number of redundant
genes exist as multiple repeats, and generating double
mutants by breeding is difficult owing to very high genetic linkage. T-DNA or EMS disrupts only one gene
per mutation, but a large deletion could be useful because it could delete several tandemly repeated genes
simultaneously. Gamma-rays could be useful for this.
A PCR-based system of detecting deletions has
been developed using fast-neutron-induced mutants
(L et al., 2001). In this method, a primer set is designed to amplify a particular gene. If the targeted
gene contains a deletion within the amplified region, a
smaller product than expected will be amplified (Figure 3). Because this method is more sensitive to TILLING, a larger DNA pool can be used for screening.
Use of large deletions for map-based cloning
Gamma-rays frequently induce large deletions.
As mentioned earlier, whether or not a large deletion
Fig. 3 The principle of the PCR-based deletion screening method. The case of Lgc1 is shown. Lgc1 is a
mutated GluB5 lacking a 3.5-kb 3í-region. The
primers amplify a 5.0-kb fragment in the wild
type and a 1.5-kb fragment in Lgc1. In this experiment, the 3.5-kb deletion can be detected
even in a 1/10 000 dilution.
is transmitted to progeny depends on what gene is deleted. This means that a large deletion in a region
where gene density is low, such as near a centromere,
tends to be transmitted to progeny. But map-based
cloning of a gene in a region where genetic recombination is suppressed, such as near a centromere, is
very difficult. A mutant allele carrying a large deletion could be useful for map-based cloning of such a
gene. A deletion larger than 100 kb could be found
easily. PCR-based markers that are not amplified are
thought to be deleted from the mutant genome. It is
not necessary to use many markers with very high
density. Identification of a deletion of several million
base pairs long could make it possible to significantly
limit the candidate region for the mutation gene. A
100-kb region will contain many genes even near a
centromere. But because the region contains many retroelements and repeats, candidates for the mutant
USE OFγ-RAY-INDUCED MUTATIONS IN THE GENOME ERA IN RICE
gene are few. In fact, we successfully isolated a mutant gene near a centromere by map-based cloning using a large-deletion allele and an allele with a point
mutation (Morita, Kusaba, and Nishimura, unpublished data).
Conclusion
Gamma-rays induce large and small deletions.
This characteristic could be useful for analyzing gene
function in the genome era. Most results described to
date were obtained using mutants induced by acute irradiation. It would be useful to develop a novel mutagenesis system regulating characteristics of DNA
lesions and mutation frequency through the use of
various irradiation conditions.
Acknowledgement
I thank Dr. Minoru Nishimura, Dr. Ryouhei
Morita, and Mr. Ken Naito for providing unpublished
data and helpful discussions.
This work was supported by a grant for nuclear
research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
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BUSCHGES R., HOLLRICHER K., PANSTRUGA R., SIMONS
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MCCALLUM , C.M., COMAI , L., GREENE , E.A., HENIKOFF ,
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TANAKA A. et al. (2005) Transmissible and nontransmissible mutations induced by irradiating Arabidopsis
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NAKAZAKI T., OKUMOTO Y., HORIBATA A., YAMAHIRA ,
S., TERAISHI M. et al. (2003) Mobilization of a transposon in the rice genome. Nature 421: 170-172.
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143-146.
SASAKI T., MATSUMOTO T., ANTONIO B.A., NAGAMURA
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SATO Y., SHIRASAWA K., TAKAHASHI Y., NISHIMURA M.,
NISHIO T. (2006) Mutant selection from progeny of
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SHIKAZONO , N., YOKOTA Y., SATOSHI S., SUZUKI G.,
WATANABE H. et al. (2003) Mutation rate and novel tt
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74
Makoto KUSABA
ポストゲノム時代におけるガンマ線誘発突然変異の利用
草場信
東京大学大学院農学生命科学研究科
〒１
１
３−８
６
５
７東京都文京区弥生１−１−１
ガンマ線により誘発される突然変異の特徴づけ
をするため，致死性の突然変異をも捉えることが
できる花粉照射法により突然変異体を単離し，解
析した。分子マーカーを用いた欠失サイズの推定
の結果，ガンマ線急照射によるDNA変異の多くは
平均で２Mbにも及ぶ巨大欠失であり，点様の突
然変異は１割以下でしかなかった。また多くの巨
大欠失は半数体致死であり，後代に全く遺伝しな
かった。実際に遺伝子機能の解析や育種に応用す
るには後代に遺伝する突然変異体を用いなければ
ならない。そこで放射線育種場で維持されている
イネ突然変異体あるいは新規に得られた突然変異
体のうち原因遺伝子が既に判明しているものに関
して，それぞれの突然変異体のDNA変異を調査し
た。その結果，ガンマ線急照射では１−数bp程度
の小さい欠失と１０kbを超えた大きな欠失に二分さ
れる傾向があることが判明した。
このようにγ線誘発突然変異には大きな欠失と
小さな欠失が存在することが明らかとなった。こ
の特徴を考慮し，前者に関してはTILLING法を，
後者に関してはPCRを用いた欠失の検出法により
ガンマ線誘発突然変異体を逆遺伝学的に利用出来
る可能性がある。
また非常に大きな欠失が後代に遺伝しないのは
欠失が配偶体形成・生育に必須な遺伝子をも含ん
でいることによると考えられる。したがって動原
体付近等の遺伝子密度が低い領域の巨大蹴る失は
正常に遺伝する可能性が高い。動原体領域では遺
伝的組換えが抑制されているためmap-based cloningが困難であるが，ガンマ線等により引き起こ
される巨大欠失は遺伝子存在候補領域を大きく狭
めることができ，このような領域におけるmapbased cloningに貢献することが期待される。
75
総合討論
座長堤伸浩（東京大学）
堤：昨日，今日とシンポジウムで大変いい勉強を
させていただいたのですが，昨年の１２月にイネの
完全なゲノムの解読が終わりまして，ゲノム情報
という，今まで人間が手にしていなかったよう
な，非常に大きな財産を手に入れたという段階だ
と思います。基礎研究の分野では，すでになくて
はならない研究基盤になっていることは皆さん否
定できないと思うのですが，実際にはバイオロ
ジーの方がそれについていっていません。ゲノム
情報を最も効率的に利用していくにはどのように
すればいいか，いつ何をどれくらいのコストをか
けてやればいいかということを模索している段階
ではないかと思います。そういうことも含めまし
て，シンポジウムでは質問の時間が比較的短かっ
たので，まずそれぞれの先生への個別の質問を受
けまして，あるいは講師の先生方から補足するこ
とがあれば，していただいて，最終的にはゲノム
情報を今後どのように育種に利用する可能性があ
るのかというようなことに議論が発展していけば
と思っております。まず質問等ございませんで
しょうか。自由にどなたにでも結構です。
天野：宮尾先生にお聞きします。レトロトランス
ポゾンをアクティベートして，つまりカルス培養
の後代でチェックしますときに，ファミリー全部
が，例えばロールリーフのミュータント型になっ
てしまっているという経験をしているのですが，
あれはどう考えたらよろしいのでしょうか。仮に
ドミナント・ミューテーションだとしますと，３
対１の１の形で正常なものが残るはずなのです。
ところが，ファミリーが全部変異体という場合な
のですが，いかがでしょうか。昔，大野さんの仕
事のときもその話を伺ったことはあるのですが，
全く見当がつかなかったのです。あるいは，お話
の中でありましたホットスポットの極端な例とし
て，二つのペアになっている染色体の両方に変異
が起こっていたか。だとしますと，分離ではな
く，全構成メンバーが変異体になってしまいます
よね。あるいは，草場先生のお仕事の関係に出て
くるようなRNAiですか，ああいう形で一面にや
られていたのか。いろいろなケースが考えられる
と思うのですが。
宮尾：まさにいろいろなケースが考えられるとは
思います。いちばん最後に言われましたように，
RNAiとか，あのあたりに関しては，まだよく分
かっていない部分が多いとは思うのですが，培養
の過程でRNAiに相当するようなものができてし
まって，その遺伝子が全部抑えられてしまって，
３対１の分離が行かなくなっている場合，可能性
もあると思います。ただ，実験的にはまだ全然そ
ういうところは見られていませんので，そういう
可能性もあるということぐらいしか言えないとは
思います。３対１の分離がうまくいかずに，例え
ば全部黄色になってしまうとか，そういうことは
私も次代解析している中で見掛けたことがありま
す。それは逆にミトコンドリアなどの核外の遺伝
子が，何かのはずみでおかしくなったものが，再
分化してきているのかもしれないと思ったことが
あるのですが，確かにRNAiとか，今までのコー
ディングの遺伝子以外のところで，何か発現がお
かしくなって，そんなことが起こる場合もあり得
るかとは思います。
鵜飼：草場さんにちょっとお伺いしたいのです
が，緩照射の場合に点突然変異が多いと言われた
と思うのですが，それの原理といいますか，なぜ
そうなるかということをちょっとご意見を伺いた
いのですが。
草場：大きい欠失が起きる原因というのは，恐ら
く一つの染色体上に二本鎖切断が複数できて，そ
の修復時にある断片を欠失した形で結合が起きて
しまうというようなことで，大きい欠失が起きる
のだろうと想像しているのですが，そうだとしま
すと，染色体上に二本鎖切断の数が少なければ，
大きい欠失が起きる可能性は減ってくるだろうと
考えています。緩照射の場合ですと，同じ線量を
76
当てたとしても，長い時間をかけて同じ線量を当
てることになりますから，その間に修復がどんど
ん起きているわけです。そうすると，ある時点だ
けを取ってみれば，二本鎖切断が起きている頻度
は，急照射に比べて低いということになりますの
で，そういう巨大欠失は起きにくいのではないか
と考えました。
鵜飼：実験データはありますか。まず，その二本
鎖切断が少ないということと，もう一つは，それ
によって，結果的に点突然変異というか，小さい
欠失が多いという，それはどこまで実験的な裏づ
けがあるのでしょうか。
草場：まず，二本鎖切断が少ないかどうかという
ことについては，全くデータは出しておりませ
ん。緩照射で点突然変異が多いのではないかとい
うのは，まだ始めたばかりで，たった３例しか同
定しておりません。たった３例ですが，３例とも
点突然変異だったという状況です。
堤：急照射の場合には大きい欠失の方が９割位を
占めているというお話でした。
草場：後代に遺伝しないものを含めれば，巨大欠
失が非常に多いです。
堤：ただ，急照射の場合も巨大欠失に比べて小さ
な欠失はもっと多くなるような気がするのです
が，いかがでしょうか。
草場：実際をいうと，観察した現象としては，明
らかに巨大欠失の方が完全に多いのですが，
ちょっと複雑な話になりますが，実際に巨大欠失
の方が点突然変異より多いかどうかというのは少
し議論があるところで，巨大欠失というのはひと
つのイベントでたくさんの遺伝子をいっぺんに欠
失させているということがありまして，逆にいえ
ば，検出されやすいということがあります。少し
バイアスがかかっていると考えた方がいいと思う
のですが，現象的には巨大欠失が多いと言ってい
いと思います。
久保山：逆遺伝学的に遺伝子をつぶす場合，放射
線を使ったときに得られる次世代に伝わる突然変
異というのは，点突然変異が多く，しかも，１
ベースとか，２ベースとか，そういう短いデリー
ションが多いというお話でした。逆遺伝学的に利
用する場合，よくケミカル・ミュータジェネシス
（chemical mutagenesis）だと，塩基置換が多いとい
うことだと思うのですが，どちらが検出し易いか
とか，どちらが結局少ない数で多くの突然変異を
得られるかということをお聞きしたいです。
草場：私自身ケミカル・ミュータジェン（chemical
mutagen）でやったことがないので，感触的には
ちょっと分からないのですが，普通の処理法でし
たら放射線とEMSなどのケミカルで，それほど極
端な差はないと思います。九大の佐藤先生などが
やっているような，受精卵にケミカルを働かせる
ような系はかなり変異率が高いと聞いています。
久保山：デリーションのような点突然変異と塩基
置換とは，どちらが検出しやすいとお考えですか。
草場：それは基本的には変わらないのではないか
と思っています。
奥本：何か草場さんばかりになってきてしまって
申し訳ないのですが，修復エラーによるミュータ
ントであるとすると，修復系が壊れた突然変異な
どを使うと，当然出てくる欠失や大きさについて
も変わってくると思います。その場合，いろいろ
なミュータントを使ってやってみるというご計画
はおありですか。
草場：私自身はそういう計画を持っていないので
すが，多分，田中先生にちょっとコメントをお願
いした方がいいのではないかという気がします。
田中：私たちも始めたばかりでやはり修復系が変
わると突然変異の分布も変わるだろうし，感受性
も変わるということでやり始めました。植物の修
復系自体がまだはっきり分かっていないというこ
とと，草場先生のご研究からやはりNonhomologous End-Joiningが多いとなると，ヒト細胞では
Ku80をはじめとした系が知られているのですが，
どうも植物はそれだけではなさそうだということ
で，まずはKu80マイナスになると感受性が高く
なって，Nonhomologous End-Joiningを取りにく
い。取りにくいということは突然変異が起こりに
くいというような傾向にはあるかと思うのです
が，それ以上まだやっておりません。
堤：今回モデル植物，あるいはモデル作物以外で
も，野菜とカンキツのゲノム情報を利用したとい
うお話があったのですが，その辺に関するご質問
なり，コメントなり，ございませんでしょうか。
近藤：大村さんにお伺いします。今回講演を聞き
まして，非常に進んでいるので敬意を表したいと
77
思いますし，私どもの林木も非常にゲノムは大き
いですし，同じ木本ということで，うらやましい
限りです。BACライブラリーまで作られまして，
今後どのような方向にゲノム研究を持っていかれ
ようとしているのでしょうか。
大村：カンキツのゲノム研究をどうするかとい
う，ちょっと難しい問題で今残念ながら私はカン
キツのゲノムの全責任を持っている人間ではない
のでその辺を素直には答えにくいのですが，当面
の問題としてBACライブラリーをどのように使う
かということになります。これは，一つは昨日申
し上げましたように，育種，当面のアウトプット
の最も近いところで考えているマーカーの実用化
に向けて，BACの情報を使いこなしていくという
ことがあります。たまたまESTをベースにして
マーカーを作っていますのでそれに当たっている
BACを拾い上げて，その周辺の塩基配列情報から
新たにマーカーができないかという探し方をする
方向があります。当然のことながら，選抜形質遺
伝子座により近づいたマーカーを作るという意味
では，その辺の領域のコンティグを作るというこ
とがどうしても重要な仕事になります。マーカー
を作るにしても，あるいはカンキツ独特の有用な
遺伝子を単離するにしても，BACコンティグを
作っていろいろな仕事に使えるようにするという
ことを一つの方向として考えています。
別な面になりますが，昨日いちばん最初に
ちょっと申し上げたのですが，主力のウンシュウ
ミカンでいろいろな枝変わりが品種として成立し
て，そのうちのどれを作るのかということがあり
ます。そういうときにその違いが何なのかを知り
たいということがあります。それはゲノム構造か
ら知るという方法もあるかもしれません。宮川早
生という系統から出た盛田温州というウンシュウ
ミカンがあるのですが，それは果面につやのある
品種です。それは単に果皮の光沢における変異だ
けではなくて，果皮が非常にしっかりしていて，
温度が上がっても浮き皮になりにくいという特性
を持っているので，そういったものを親に使うと
きに，原品種である宮川早生を使ったときと，ど
のように違ってくるかという予測をしたいという
ことがあります。そういう場合にその二つの品種
での遺伝子発現上の違いをマイクロアレイなどを
用いてあらかじめ押さえておくことを近い将来の
取り組むべき課題と考えています。
堤：福岡先生，野菜の方では何かいかがですか。
福岡浩之：今後の展開としては，一つには，やは
りイネのアナロジーもありますが，主要なQTL解
析などがある程度詳細に行えるようなゲノム情報
を，主要な野菜では確立したいということがあり
ます。いろいろな野菜で，例えば白菜とかメロン
といったもので，最近DNAの解析がローコストに
ハイスループットでできるようになってきたとい
うことは，やはりモデル植物でゲノムプロジェク
トを先行してやったことの一つの波及効果だと思
うのです。つまり，イネとか，あるいはArabidopsis
でやった頃ほど今では大変な作業ではなくなっ
た。イネのレベルまで行くのは大変ですが，ある
程度のレベルまで行くためのコストや負担が少な
くなってきたということがありますから，幾つか
の園芸作物ではもうすでにそういった動きが出て
います。そういうことが世界各地で起こり，だん
だん情報が集まってきますと，マップベースク
ローニングぐらいのことがいろいろな作物ででき
るようになっていくだろうと思います。それは一
つの研究所で全部やることはできないわけですか
らそれの一端を私たちも担えればと考えています。
堤：平井先生にお願いしたいのですが，Arabidopsisを使った非常にエレガントな仕事だと思うの
ですが，先生の内容だと，プロテオームはスキッ
プしてもいいということでしょうか。
平井：おっしゃるとおりでプロテオームのところ
が抜け落ちているのですが，昨日ちょっと申しま
したとおり，メタボロミクスのところが技術的に
まだ発展途上ですので正直そこでいっぱいいっぱ
いということはあります。やはり応用科学という
ことで，このあと，いわゆるシステムバイオロ
ジーというような展開の仕方を一つ考えるわけで
すが，そこではやはりプロテオミクスは避けて通
れない問題になってきていると最近改めて思って
おります。いずれ考えていきたいと思っています。
堤：昨日のお話では硫黄化合物と色素という，限
られたというか，硫黄がついているからという特
殊なものがあったのですが，メタボローム解析と
いうのが現段階でどれくらい一般化できるものな
のかということを知りたいのですが。
78
平井：ファンクショナル・ゲノミクスということ
で考えますと，昨日お話しした色素やグルコシノ
レートといったものは二次代謝産物であり，二次
代謝産物というのは生存に必須ではないですか
ら，恐らく転写レベルで生合成系全体をレギュ
レーションして，最終産物をわっと上げるような
レギュレーションがかかっているのだと私は思っ
ているのです。そういう系ですと，多分トランス
クリプトーム，転写物のプロファイルが最終メタ
ボライトのプロファイルを規定しているだろうと
私は思っておりまして，そういうところでは非常
に対応がつけやすくて，解析がしやすいと思って
います。
一次代謝の方をこれから理解していきたいと思
うわけですが，そこは転写レベルのみならず，も
ちろん酵素活性のレベルや翻訳レベルなど，いろ
いろなレギュレーションが文字どおり複雑にか
かっていて，変化に対して一方向に変わるという
よりは，新しい平衡状態に向けて，わーっと動い
ているという感じだと思うのです。ですから，
言ってしまえば，一次代謝の理解はまだちょっと
難しいように思いますので普遍的な一次代謝の理
解という部分では解釈が難しいような気がしてい
ます。分析技術としては，一次代謝の方はイオン
性の物質などが多くて，これは技術的な話です
が，キャピラリー電気泳動マスみたいなものが多
分有効で，微生物の方では慶応大の先端生命研で
先駆的な研究がされています。
堤：モデル植物に関する何かご質問はございませ
んか。
奥本：平井先生に質問ですが，昨日，何個体か
取ってこないとやはり安定した結果は出てこない
という，個体のレベルの差が結構あるのだという
話をされていたと思います。しかし，純系の植物
を使って，かなり制御した環境であれば，見た目
にはどの個体も同じような反応をしているのに，
例えば５個体，あるいは３０個体ぐらい集めてこな
いと結果が安定しないというのは，何か抽出とか
物理的な問題なのか，もう少し高次の制御があ
り，植物の方が何かしているからそうなっている
のか，個体レベルでやると抽出効率がばらつくの
でということなのか，どちらの方なのでしょう。
平井：なるべく均質にということは，分析する側
で非常に注意を払っていますので，多分抽出のば
らつきよりは，やはり個体が持っている個体差だ
と思っているのですが，それがどうして生じるの
かというのはまだ分かっていないと思います。
奥本：そうすると，メタボロミクスとか，いろい
ろ言っているけれども，それを超えた何かもう少
しよく分からないところがまだあるということで
すか。
平井：そうですね。どう申しましょうか。メタボ
ロミクスでよくやりますのは，昨日も申しました
ように，検出されたピークに化合物としての同定
が難しいということがあって，現時点ではプロ
ファイル，フィンガープリントみたいな利用をす
ることもまだ多いのです。そういったときに，全
体的な代謝のプロファイルの傾向を把握するよう
な解析，主成分分析などの手法があるのですが，
そうしますと，例えば１遺伝子の変異のものと，
それに対する親株，野生型のものとをそれぞれ何
個体かずつ分析したときに，それぞれスキャッ
タープロットのようなものを書かせますと，ばら
つくのですが，野生型はこの辺にばらついてい
て，変異型はちょっと違うところにばらついてい
る。場合によっては多少重なりもあるのですが，
全体として分かれてくるということがありまし
て，そういう解析の仕方をするのが現在主流かと
思います。
福岡浩之：ひき続いて平井先生にお願いしたいの
ですが，昨日のご講演の最初の方で，実験の考え
方としまして，スクリーニングという考え方と
ちょっと違うということがあって，あの辺が
ちょっと私はうまく理解できなかったものですか
ら，単に反復を取らなくてもいいというようなイ
メージの話なのかと思ってしまいましたが，その
後やはり３０個体ぐらいはやらないと傾向が見えな
いということでした。このことについてもう少し
ご説明いただければと思うのですが。
平井：すみません。多分ちょっと言葉が足りな
かったのですが，スクリーニングしないようにと
いうのは，私の個人的な考えと思っていただいた
らいいかと思います。質量分析の方で３０個体と申
しましたのは，分析する側のといいますか，実は
私自身はあまりメタボローム分析ということはし
ないのです。私個人は，アベーラブル（available）
79
な技術を利用して解釈する方が仕事と思っており
まして，実際に分析している側では，やはり反復
を取りたいということです。あくまで私個人の考
えなのですが，例えばトランスクリプトーム解析
ということがまずアベーラブルになってそちらか
ら始めたわけですが，やはりあれは金額的なネッ
クがありまして，十分な反復を取りたくても取れ
ないのです。そういうときに限られた予算の中で
反復を増やすよりは，たった１回だけれども，実
験区をたくさん増やすことで全体的な傾向を見た
いということがありました。それで，昨日のよう
な言い方をさせていただいたのです。もちろんト
ランスクリプトミクスの方もメタボロミクスの方
も，技術的に１００％確実，あるいは９９．
９％確実な方
法を確立する必要があると思うのですが，一方
で，私個人の立場としてはまだ不完全なところが
あっても，現状アベーラブルなもので何か新しい
ことが分かっていけないかという，全く個人的な
立場で昨日はお話しさせていただきましたので，
そのようにご理解いただけたらと思います。
草場：同じく平井先生にお願いしたいのですが，
先ほど堤先生もおっしゃいましたが，プロテオー
ムとメタボロームの融合というのは多分重要だと
思いまして，多分たんぱく質の活性が変わること
による制御もかなりあると思うのです。そのとき
に，世界的に見てですが，例えばリン酸化たんぱ
くの消長とメタボローム解析の融合とか，そうい
うことはすでに試みられているのでしょうか。
平井：プロテオームと結びつけてという話は，ま
だ世界的にも多分出てきていないと思います。
奥本：川口先生にお聞きします。マメ科の作物は
二次代謝物が非常に多様になっているというお話
と，それから進化の過程でそういうことから根粒
菌の共生を進化的に獲得してきたのではないかと
いうお話をされていたのですが，根粒菌との共生
を達成したから多様な二次代謝物を作れるように
なったとお考えでしょうか。それとマメ科の中に
は共生しないものもあったと思うのですが，こう
いうものもやはり多様な二次代謝物があると考え
られるのでしょうか。
川口：マメ科植物は非常に二次代謝産物が豊富で
すが，根粒菌との共生がその多様性を生み出す原
動力になったかということに関しては分かりませ
ん。ただ，共生の初期認識のところでフラボノイ
ドという物質が重要なのですが，種特異的に認識
されるフラボンがあるのでホストとシンビオント
の共進化プロセスで多様化が生じた可能性はあり
ます。それ以外にも，例えばマメゾウムシとの相
互作用や，それ以外の昆虫との相互作用など，い
ろいろとありますので，そのような中でさまざま
な二次代謝産物が進化していったということが考
えられます。
もう一つの質問の方に関してですが，マメ科植
物はすべて根粒菌と共生して窒素固定できるわけ
ではなくて，ジャケツイバラやネムノキの仲間で
は共生しないものもかなりあります。そのような
共生しない植物の二次代謝産物にどのようなもの
があるかということに関しては，まだあまり知見
が得られていないと思います。我々になじみが深
いのは特にソラマメの仲間で，大豆やインゲン，
小豆，落花生など，身近なマメでは多くの解析例
はあるのですが，ジャケツイバラやネムノキの仲
間においてはあまり情報がありません。ですので
今後共生を行わないマメにおける二次代謝産物の
プロファイルをいろいろ調べていくことによっ
て，初めて比較ができると思います。
堤：オルガネラのゲノムなどを見てもマメ科だけ
ちょっと特殊であり，ゲノム上に残っている遺伝
子を比較すると，マメ科だけかなり違っていたり
するのです。昨日もたしかCLAVATAでしたか，
同じような遺伝子が違う機能を持っているという
ことで何かマメ科は変なのですか。
川口：マメの植物の遺伝子機能を解析してきて本
当に予想外だったのですが，メリステムというの
は植物の地上部を作る組織なので非常に重要でそ
れを制御する遺伝子はすべての植物において共通
に維持されているだろうと思っていたのです。例
えばイネでFon1というメリステムを制御する遺
伝子が平野博之先生らによって明らかにされて，
それはCLAVATA1と非常に相同性の高いものでし
た。ところが，マメにおいてCLAVATA1と最も相
同性の高い遺伝子の機能が明らかにされると，そ
れは根粒形成に関わっており，メリステムとは全
く異なる機能を持っていたわけです。
話しは変わりますがシロイヌナズナでは
WUSCHELという転写因子がメリステムでの細胞
80
分裂を促進する重要な因子で，それはイネでも同
様に働いているかと思っていました。それについ
て，昨夜，長戸先生に伺ったところ，今のところ
イネの分子遺伝学的な解析からは，それを示唆す
るデータは得られていないということを教えてい
ただき，興味深いと思いました。
マメ科だけが変わっているかどうかというのは
まだよく分からなくて，ひょっとすると，基本的に
重要な遺伝子であっても，ファミリーが変わると
機能が異なっているのかもしれません。それに関
しては，マメでは一つ一つ遺伝子機能が分かりつ
つある状態で，今後検証していきたいと思います。
中川：そこに関係するのですが，当初Medicago truncatula（タルウマゴヤシ）でゲノム解析が行わ
れて，最近またLotus（Lotus japonicus：ミヤコグサ）
でやろうという話ですよね。Lotusの利点は昨日
たくさん伺ったのですが，Medicago truncatulaでは
できないことがLotusではできるのかということ
と，今の問題に関連して，MedicagoとLotusとの共
通点から，イネ科とか他のArabidopsisが見えてく
るのかどうかという，その２点を伺いたいと思い
ます。
川口：MedicagoでできることでLotusでできないこ
とはほとんどなくて，逆に，Lotusでできることで
Medicagoでできないこともほとんどありません。
基本的にマメの場合，モデルは一つでよかったと
は思うのですが，実際はミヤコグサとMedicagoの
両方でゲノム解析が進展しています。２つのマメ
科のゲノム情報が最近急速に出てきて，来年の１２
月ごろには両者のゲノムのドラフト配列が得られ
るだろうといわれています。特にジーンリッチ・
リージョン（gene-rich region）に関してです。そ
こを比較してみると面白いのは，例えばMedicago
に，２コピーある遺伝子がLotusでは一つである
とか，逆にMedicagoで一つの遺伝子がLotusで三
つタンデムに並んでいるとか，そのようなゲノム
の構造です。
例えばケミカル・ミュータジェン（chemical mutagen）処理で突然変異を導入した場合，ゲノムに
遺伝子が一つしかない場合は表現型に出やすく
て，同じような機能を持っている遺伝子がタンデ
ムに複数存在している場合は出にくいのです。し
たがって同じ変異処理をしてもMedicagoとLotus
で違うものが出てきます。それは染色体上の遺伝
子構成の違いによっています。ですので，恐らく
一つのモデルだけではなくて，二つのモデルを使
用することによって，よりマメの遺伝子機能の解
明が進展することが期待されます。
もう一つ，今回のシンポジウムに絡めて私が特
に期待を持っていることを述べたいと思います。
昔，ショウジョウバエやゼブラフィッシュを使っ
てNşslein-Volhardtらが試みたように，ケミカル・
ミュータジェンによるスクリーニングを大規模に
やれば，最終的にミューテーションを飽和させる
ことができるのだと思っていました。しかし，実
はそうではなくて，植物ゲノムを見てみると遺伝
子がタンデムに並んでいる場合が多いのでそれら
をごっそり削らないと，表現型が出てこないとい
うことが予想できます。
先ほどゲノム領域に，二つ，三つタレデムに並
んでいるという例を挙げましたが，実際ミヤゴグ
サのゲノムを見てみると１
０個ぐらい並んでいるよ
うなところがある。その場合EMS等のミュータ
ジェンによるフォワード・ジェネティクスは機能
しないので，
100 kbとか200 kbぐらいの大規模なゲ
ノム欠失を伴うミューテーションが有効だと考え
られます。そのような意味で，今後ガンマ線やイ
オンビームが変異原として優れているのではない
かと思っています。ゲノムを効率よく削る核種や
照射量の検討。これが，将来的な遺伝子機能解析
に重要かと思いました。
堤：ガンマフィールドの存在意義がまた高まった
ようです。
田中：今の川口先生とも関係あるかと思うのです
が，私は宮尾先生のTos17の系を用いたものが，
フォワードでもリバースでも，どちらでも非常に
いいのではないかと思います。イオンビームを
やっていますが，内在性のトランスポゾンは非常
にいいなと思っている次第です。なぜかという
と，佐々木先生の要旨にも書かれてありますよう
に，T-DNAや外来性のトランスポゾンは遺伝子組
み換えになる。レトロトランスポゾンは内在性な
のでまず遺伝子組み換え体にならない。外来性の
トランスポゾンは，標的遺伝子にインサートして
も，また抜けてしまって，標識にならないことが
結構あったり，近傍にしか飛ばなくて，ゲノム全
81
体に飛ぶようなことがなかったりすることが多い
と思うのです。でも，宮尾先生のご発表を聞きま
すと，Tos17はほとんど遺伝子に漏れなく入る。
逆に放射線のほうは，非常にさまざまな，今言わ
れたような幾つかのタンデムの遺伝子を飛ばすこ
とはできるのですが，最大のネックはやはり標
識，タグがないということで，ミュータントは取
れるのですが，それに作用する遺伝子をつかむの
に非常に時間がかかります。
ということで，質問したいのは，イネではTos17
が成功しているのですが，ほかの作物で同じよう
に内在性のトランスポゾンを動かせば，よりフォ
ワードもリバース・ジェネティクスもこれから使
えるのではないかと思うのですが，その可能性に
ついてお伺いしたい。
宮尾：私はイネに特化していますのでほかの作物
について内在性のトランスポゾンで，うまくいっ
ているという話はあまり聞いてはいません。探し
ているという話や，相談を受けたりすることはあ
りまして，こうすれば見付かるのではないかとい
うようなことをアドバイスなど差し上げたりする
ことはあるのですが，今のところはレトロトラン
スポゾンでというと，そんなにはないかもしれな
いです。ただ，トウモロコシなどはAc/Dsの系と
か，大昔からありますし，そのあたりはもっと
大々的にされてもいいのではないかとは思ってい
ますが。野菜や果物に関してはいかがでしょうか。
福岡浩之：私の方は，むしろ今の質問を，もしど
なたも質問なさらなければ，したかったなと思っ
ている次第でして，これから勉強します。
堤：最後に佐々木先生に今後ゲノム情報をどのよ
うに利用するのか，可能性というか，展望という
か，そういうことが何かありましたら，ぜひお聞
かせいただきたいのですが。
佐々木：何も偉そうなことを言える立場ではない
ですが，今回のこちらの組織委員会の先生方で，
「ポストゲノム」というタイトル，これについて私
はいつも不満に思っていて，実は今日ここに並ん
でおられる先生方のお話を伺っても，まだゲノム
研究まっただ中なのです。別に「ポスト」（次の）
ではなくて，入り口かもしれない。放射線育種に
関しても，草場先生のお話で，いろいろ原因は推
察されていますが，まだ証拠はなかなかない部分
が多いわけです。そういうところは今後ますま
す，特にイネやシロイヌナズナであれば，手掛か
りになる情報が多いわけですから，そういうもの
を使えばもっとサイエンティフィックに語れるの
ではないかという気はしています。もちろん，そ
れを支えるためには，山崎先生が作られているよ
うなデータベースが非常に役に立つわけで，そう
いう資源を私たちは大いに使って，
「ポスト」では
なくて最前線のゲノム研究をますますやっていき
たいと思っています。
もう一つ，私たちが心掛けなければならないの
はこのように研究が進んでいきますと，もちろん
育種の方々は応用利用を考えておられたので社会
との接点は多かったわけですが，ゲノム研究自体
が社会との接点が非常に増えてきて，いろいろな
意味で責任が生じている点を意識しなくてはなり
ません。ゲノムと聞いただけで，少し顔を背ける
かたがたも結構おられて，そういうところをいか
に説明するかが重要になってきます。説明して分
かっていただけない方もおられますが，いずれに
しても説明責任が生じているわけで，そういうこ
とを行うには，このシンポジウムはちょっと内容
が難しいですが，一般の方にゲノム研究を理解し
てもらう話し方を常日頃心掛けなければいけない
と思っています。ゲノム研究に対する理解を広め
るためには，ここに集まっていただけたような広
い専門範囲の方々の協力のもとで，また知恵を出
し合っていかなくてはいけないのではないかと，
最近は強く感じています。例えば米国等では，
ごく最近４人の優れた科学者の業績をたたえる記
念切手が出たのですが，そのうちの１枚はバーバ
ラ・マクリントックの業績が取り上げられていま
す。その切手には中にバーバラ・マクリントック
の肖像と，彼女が発見したトランスポゾンを説明
する，私はちょっと意味が分からなくて米国の友
人から説明を受けたのですが，有名な図がプリン
トされているのです。日本でももう少し，このよ
うな植物研究の成果を社会的に宣伝していかない
と，それでなくても研究者のコミュニティが小さ
いわけですから，ぜひ皆さんで知恵を出し合って
社会にもっと広げていくように，
「ポスト」ではな
くて，最前線のゲノム研究の意義を広めていくよ
うな広報活動を是非やっていきたいと思っており
82
ます。
堤：ありがとうございました。そろそろ時間にな
りましたので，総合討論をこれで終了させていた
だきます。どうもご協力ありがとうございました。
平成１９年２月９日印刷
平成１９年２月１５日発行
独立行政法人農業生物資源研究所
放
射
線
育
種
場
〒３１９−２２９３
茨城県常陸大宮市私書箱３号
TEL ０２９５−５２−１１３８
FAX ０２９５−５３−１０７５
印刷所いばらき印刷株式会社
那珂郡東海村村松字平原３１１５−３
TEL ０２９−２８２−０３７０

No.44 (2005年/平成17年)

Transcription

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