Proceedings of 13th International
Genetic Resources
Workshop on Rice Genome and Plant
Genetic Resources
22-23 August 2005
Tsukuba, Japan

National

Instituteof Agrobiological

SciencesC ONTENTS
Pr efac e

1

OkunoK.
Topic 1: Rice Genome: Challenges
Completeand Opportunities

rice genome sequence and its application

to genome biology

Matsumoto T., WuJ. Z.y Katayose Y, Mizuno R, Antonio B., Sasaki
Fujisawa

M. and Namiki N.

Genome comparative
japonicaT., Kanamori R,
3analysisof domesticatedrice

Oryza sativa

indicaandsubspecies

Han B.

8

The Rice Genome Resource Center as the core for cereal genomics
Antonio
Functional
Ashikari
Molecular

B. A, Miyao A, Nagamura Y.and Sasaki
analysisof genes involvingT.

12

culm elongationin rice

M. and Matsuoka M.

17mechanism of broad-spectrum

resistancemediated

by NBS-LRR protein

inricerice
Wang G. L., Qu S. R, Zhou B., Liu G. E, Han B, and Sakai H.

Isolationand functional

analysisof genes controlling

MaJ. E, Yamaji N., Tamai K, MItani N., Konishi
TILLING:

Where functional

Vanavichit

A, Sasoo S., Amorn T., Ruengphayak

Si uptake20

in rice

S. and Yano M.

genomics meets conventional

24

breeding

S., Plabpla

A, Toojinda

T. andTragoonrung S.
Functional

analysis28of the genes controlling

Umemoto T., Aoki K, Horibata

T., Inouchi

eatingand processing

N. and Yano M.

qualityof rice36Topic 2: Global

and Regional

Strategies

Use of Plant Genetic
International

platforms

prospects

and challenges

Sajiseand Sustainable

Resources

for sustained

collaborationin plant

genetic

resources:

EE.

Genomic-based
JiaJ.

for Conservation

crop germplasm research

and Zhang Q.

Genetic

dissectionof resistanceto rice blast

Fukuoka S., Saka K, Kudo S., Shimizu

Genomics and bioinformatics
of plant

genentic

resources

for rice germplasm

enhancementT., Yano M. and Okuno K

approaches

for effective

utilizationand enhancementin RDA-NIAB Genebank

Park Y.J., Rao R. V, KwangJ. G. andKim T. S.

Research and conservation

52of the azuki bean and soybean crop complexes

Vaughan D. A., Kaga A., Tomooka N. and Isemura T.
Ex situ conservationand strategies

of vegetatively

for tropical

propagatedand sub-tropical

58

genetic

resources:

present

statusspecies

Takagi H. and Niino T.
Strategy

for conservation66
and enhanced

use of crop genetic

resources

Gowda C. L L and Upadhyaya H. D.
Compliance,accountability

international

collaborative

Kawase M.

andresearch

ethics71
foron plant genetic

trust-building

partnerships

inresourcesllPreface
Increase in agricultural
production
and productivity
is a recent primary issue to secure
food for growing population worldwide while harmonizing it with sustainability
in agro- andnatural-ecosystems.
However, fruitful outputs of plant breeding have often led to genetic
erosion or loss of crop genetic diversity.
Integrationof plant genomics and biotechnology
with plant breeding is expected to generate innovative enhancement of agricultural
production
and productivity
in sustainable
manner. Bio-resources produced by plant genomic researchand biotechnology
tools give a great impact on breeding science and technology.
On theother hand, geneticallymodified organisms (GMO) have faced to a campaign against GMO
and may pose ethicalproblems and require substantial
debate among policy makers,
researchers and the public at large.
Particularly
in cultivation
and use of GMO, the debate
has become quite polarized and there is therefore an increasing need for high quality researchdata related to these issues.
With this background, the main theme of the 10th International
Congress of SABRAO in
Tsukuba, 2005 is, "How to utilize crop diversity for productivity
and sustainability:
Breeding
science and technology for the new era". The congress comprised plenary sessions, sevensymposium sessions and poster sessions.
Symposium sessions were organized into sevenprimary themes as follows:- Rice Genome: Challenges and Opportunities- Perspectivesof Utilization
and Conservation of Plant Genetic Resources- Germplasm Improvement with DNA Markers in Rice.
- Genetic Improvement ofAbiotic
Stress Tolerance in Crop Plants- Genetic Diversity of Soybean and its Use for High Benefit Production- Sharing the Experience on Transgenic Research and Crop Development in Asia
- Information Sciences for Plant Breeding
National
Instituteof Agrobiological
Sciences(NIAS)
sponsored
the 13th NIAS
International
Workshop on Genetic Resources as parts of symposium sessions during the 10th
International
Congress of SABRAO (The Society
for the Advancement of Breeding
Researches in Asia and Oceania) which was held 22-23 August, 2005 in Tsukuba.
In thisproceedings,
two research highlights
on "Rice Genome: Challenges
and Opportunities"
and"Perspectivesof Utilization
and Conservation of Plant Genetic Resources" are discussed.
I
expect all these research outputs recorded in the proceedings
will contributeto further
advance in breeding science and strategies today and tomorrow.
March 2006

Director

Kazutoshi Okuno
of Genebank, NIAS
Tsukuba, Japan

Topic 1
Rice Genome:
Challenges and Opportunities

COMPLETE RICE GENOME SEQUENCE AND ITS APPLICATIONGENOME BIOLOGY
TakashiMatsumoto,Jianzhong

Wu, Yuichi Katayose, Hiroshi Mizuno, Baltazar
National Instituteof Agrobiological
Sciences
2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602
Japan
mat@nias. afifrc. go.jp
Tel: +81-298-7441
Fax: +8 1-298-7468

Hiroyuki446-1

Kanamori, Masaki Fujisawa,
STAFF InstituteKamiyokoba, Tsukuba, Ibaraki

Nobukazu
305-0854

Antonio,

TakujiTO

SasakiNamikiJapan

Abstract
The International
Rice Genome Sequencing Project (IRGSP), which was officially initiatedin
1998, completed the sequencing ofthejaponica
rice cultivar Nipponbare in December 2004. The
total nucleotide
sequence of ca. 370 Mbp corresponding
to 95% of the entire genome has been
elucidated
and submitted in the public databases. Initial analysis predicted 37,544 protein-coding
genes, about one third of which are clustered in tandem. Similarity
analysis among all the protein
sequences revealed clear segmental duplications
within the genome. The nearly completed
genome sequence also shed light on regions previouslyclassified
as "dark side" due to theabsence of protein coding genes. Analysis of the centromere and telomere regions revealed thepresence of many repetitive
sequences which maybe crucial in chromosome maintenance. Sometranscriptionaly
active genes were also identified
in these heterochromatic
regions. The
high-quality
Nipponbare
genome sequence could be used as a common framework forcomparative analysis of closely related genomes. The end-sequences from BAC clones of an
indica variety, Kasalath have been succesfully
mapped using the Nipponbare
sequence asreference. This could lead to the detailed
sequence comparison between japonica and indica
subspeciesof rice cutivars, both of which are important to modern rice breeding,and could
provide many SNPs and Indels which could become fine genetic markers. Combining the finescale genetic maps, genome sequence and gene annotation could accelerate gene discovery andidentificationof regions responsible
for various biologicalphenomena. This could pave the way
for a genome-based breeding strategy to generate new rice varieties
with highly desirable
agronomic traits. The Nipponbare sequence is therefore a gold standard vital to the understanding
and utilizationof the genomic variation in the genus Oryza and other Graminae species sharing
syntenic relationships
with rice.

Rice is one of the most important crops in the world. It is the staple for 34 countries aroundthe world and it provides 27% of dietary energy supply. Global rice annual production has
steadilyincreasing and reaches to 545 Million tons in 2004. But the predicted
demand for riceproduction at 2025 is 700 Mt. While the late "Green Revolution" from 1966 with IR8 greatly
increased food supply, land and water resources that support our food supply are diminishing.
Breeders from all over the world are trying to produce novel varieties which is characterizedaseven higher yield than modern varieties, resistant to severe diseases which are caused by various
pathogens, global environmental changes cause abiotic stress which might it impossible
for thecrop plants to survive. One of the goals of the current breeding is to produce crops which istolerant to these stresses such as drought,flood,global warning and salinity.Current
dietetics
requiresfor food the balanced
nutrients,
not just calories
in order to live healthy
life.
Supplementationof the nutrients whose lack will cause severe disease is new target for modern
breeding.Thesephenotypicchanges as targets for breeding are governed by the genes and genetic
systems, and genome sequence, which is the blueprintof the whole activities,
will greatly
accelerate
the gene-targetedmolecular breedingby rational design. The International
Rice
Genome Sequencing Project,or IRGSP, completedsequencing
of japonica
rice variety,
Nipponbare
last year (IRGSP, 2005). Establishedin 1998, the IRGSP has set up materials foranalysis,constructed a detailed physical maps by PAC,BAC, and fosmid clones, arid sequenced
the map-based clones by the hierarchal shotgun strategy. In the course, in 2002, a whole genome
sequence with a lOx high-quality
shotgun was revealed, since many rice researchers need therelatively
acuurate rice sequences(IRGSP,
2002).And
after two more years, it could publish thecompleted rice genome sequence which would be invaluable for crop biology and biotechnology.
The IRGSP assigned the twelve chromosomes of rice to each sequencing participants,
and 10countries, devoted to sequence several hundred PAC/BAC clones until it meets Bermuda standard
whose accuracy is 99.99%. 3,453 clones generated 370 million
nucleotides
in total and thesequence has already been open to the public. They are submitted in the public databases such as
DDBJ, EMBL, and GenBank in the appropriate
format. Moreover reserachers can obtain thesequences from the IRGSP website(http://rgp.dna.affrc.go.jp/IRGSP/Build3/build3.html)
by ftp.
These data are called pseudomolecules
as they mimic each chromosome structure except theunsequenced regions.
Adding the sequenced 370Mb length, 36 of the contig gaps, Centromere gaps, rDNA regions,and telomere gaps, the total size is calculated
as 388.8Mb. Sizes of rice genome was calclated as415-430Mb by flow cytometry, 400Mb by the physical map estimation of genomic clones. The
current size is the most precise calculation
concerning japonica rice genome, and it is also
confirmed wehave 95% of them as the sequences.
Next we have predictedgene domains within the sequenced region. First the regions ofvarious repeats such as transposons, retrotransposons,centromere repeats, and other repeats weredetected and removed. The remaining regions were subjected into the gene prediction
by using
FGENESH software trained by monocot dataset. In total, 37,544 genes are predicted.
This
number is almost double the number of genes in Arabidopsis
as 25,000 in the original annotation(AGI, 2000), The gene density or more accurately, gene interval is one gene per 10kb on average,
about twice larger than Arabidopsis.
In fig.l the statistics
on the predictedgenes are briefly
summarized. 61% has some rice transcript
support, 52% has a protein match, 71% has a
Arabidopsis
counterpart , and 62% has at least one motif or domain which is annotated. 17% of
the total predictedgenes have neither transcript
support, nor protein or domain database hit.
Functional assignment of these gene models should be done by experiments. One of the striking
feature of the rice genome is the abundance of the tandemly arrayed genes. About 30 % of ricegenes exists as clusters which consitutes tandem gene arrays. In terms of protein function, manyof these large arrays contain protein kinase genes. Another analysis of protein domain detection
shows that the top 5 domains are all protein kinase or kinase-likedomains showing rice genome
is protein kinase-rich.
Another finding about rice genome evolution is the segmental duplication.
As we have and also Andy Paterson indicatedin 2004 (Paterson et al, 2004), all against all
similarity
searches of the rice predicted
proteins clearly show the conserved giant gene clusters.
Plotting
only the most conserved region pairs indicatedthe the large scale segmental duplicationor polyploidization
which is estimated at about 70MYA.

Rice Predictedfigure 1 Statistics

Genes

37,554on rice predicted

genes

It might be Interesting
to investigate
whether these genes are also diverged In other Grassgenomes, because these duplications
were occurred after divergence of the Poaceae. It might also
be Intriguingthat the duplicatedgenes have same function or have subjected to subfractionation
within the evolution and selection process, because all these genes are predicted
as intact ORFs.
Sequences and gene structures from maize and sorghum, and barley, which will be revealed In thenear future will tell the exact correlation
among these taxons.
Analysis of the complete rice genome by several Investigators,
Inside and outside IRGSP
found that rice genome has some remarkable sequences besides protein coding regions. A large
chroloplastand mitochondria
Insertion were found within nuclear genome. More than 400 cp
Inserts and 1000 mt Inserts were found In all chromosomes, which covers up to 0.4% of ricegenome. 1/3 of the rice genome was occupied by various kinds oftransposable
elements. So far
wedo not exactly know the correlation of transposon and genome function. Moreover, more than
450 non-coding RNAs excluding rDNA? tRNAs are found. These ncRNA are charcterizend
asmicro RNA, small nucleolar RNA, and spliceosomal
RNAs. Moreover many candidates for DNA
markers for breeding,such as SSR and SNPs are found between the two major subspeciesofsativa,non-protein-coding
Oryzajaponica and indica. These
region are current research topics,
because these non-canonical type of gene regulation might Important for the plant systems. Herethe completed genome of Nipponbare could make It possible to assign sequence of other genome
to the japonica sequences accurately.
contigs

Figure 2 shows the sequenced region within rice genome by IRGSP. The green bar Indicatessequence
Although most of the region Is covered by green bars, there are still gaps anddotted regions. Other than the contig gaps shown as the disconnected
bars, sequences from
special part of the genome has not been fully obtained and recognized as "dark side". As theregion which might be transcriptlonallysilent, are centromeres as Indicated by
red triangles,
NORs as dotted green lines,and telomeres at the chromosomal ends. Most of thesesequences were left unseqenced, but since they are Important for the chromosomal maintenanceor chromosomal recognitionand other Important function, we have been continuing sequencingof these regions to complete the rice genome overall sequencing.

Heterochromatin

The

FigureRegions

2 The sequenced

as Dark Sideand gapped region of rice genome.

We have already sequenced and publishedthe overall structure of the 2Mb region ofcentromere and pericentromere
in chromosome 8 in 2004. Furthermore we have been analyzingsome regions within NOR (nucleolar organizing region) where a major rDNA cluster exsits, andtelomere regions to obtain full-scale
image of rice genome.
Since rice is the most studied crop, the sustainable
food supply is the one of the mostimportant objectives
for crop breeding. How rice sequence would accelerate this goal?
There
might be two approaches for that. First functional
analysis of the total rice gene set will lead tounderstand how gene works solely or as part of a network. These analysis will go to theknowledge how to regulate specific gene function without disturbing
cell's other functions. These
technique will enable the targeted gene improvement by human design by either crossing or genemanipulation.
Other approarch to utilize the genome sequence is comparative genomics studies.
There are two information flow in this area. One is to utilize rice sequence and other information
to elucidate
the orthologus gene identificationof Grasses by synteny. The other application
is tolook into Oryza itself. Utilizationof vast Oryza genetic resources worldwide would cause thesecond green revolution to rice breeding to establish
a novel varieties.
The Genus Oryza consists of two cultivars and 21 wild species. But human utilized only two
cultivars, O.sativa and O. glaberrima for agricluture.
For the time being, studies on rice molecularbiology have been concentrated on these two subspecies of O.sativa, japonica and indica. Within
the AA species, in other word, Oryza complex,O. sativa is believed
to be evolved fromO.rufipogon, while O.glaberrima
from O.barthii.
Moreover breeders and researchers of genetic
resources understand there might be vast gene resources to improve modern variety in wild rice.
For example, in 1998, Tanksley's
group found yield QTL in the O.rufipogon species, and in the
recent historyof rice breeding,some novel disease resistancegenes from O.rufipogon,
O.longstaminata,O.officinalis, O.ridely O.minuta, have been introgressed
into rice cultivar byunknownmechanism. Therefore our objectives
for rice comparative genomics will be symbolized
as these two questions. How rice cultivar generated and diverged from the same ancester? Whatis the wild rice? How it is useful to agriculture?
In 2004, we have constructed BAG library of
Kasalath, an indica cultivar, and a Kasalath BAG in silico physicalmaps which covers 70% of
Nipponbare
genome are constructed(Katagiri
et al, 2004). We are in the process of thecomparative work of japonica and indica sequences in the chromosomal wide scale. On the other
hand, the RGP constructed a rice transcript
map for the basis of BAG physical maps in 2002.

There 6,591 ESTs from the unique regions of 3'UTR of rice cDNAs were mapped by PCRscreening(Wu et al., 2002). We applied these Nipponbare EST markers to the wild rice accessions.
By the courtesy ofDr. Vaughaun and Dr. kadowaki at NIAS, DNAs from 45 accession s from 21
species from AA to HHJJ genomes were subjected to PCR amplificationby using the 943 ESTs
throughout the genome as the source of PCR primer. There is a clear difference for amplification
degree for AA species, BB to EE species, and others. This means there is a discrete difference ofsequence similaritiesof3 'UTR among wild rice complexes.

References
AGI Nature408:
796-815(2000)
IRGSP
http://rgp.dna.affrc.go.jp/rgp/Dec
IRGSP
Nature 436: 793-800 (2005)
Katagiri et al. Breed. Sci. 54: 273-279
Paterson et al. PNAS 101:9903-9908
Wuet al. The Plant Cell 14: 525-535

l 8_NEWS.html(2004)(2004)(2002)(2002)

GENOME COMPARATIVE ANALYSIS OF DOMESTICATED RICEORYZASA TIVA INDICA AND JAPONICA SUBSPECIESNational

Bin HanCenter for Gene Research, Shanghai Institutes
for Biological
500 Caobao Road, Shanghai, 200233 China,

Sciences, Chinese
bhan@ncgr.ac.cn

Academy of Sciences,

Abstract
As part of international
efforts to completely
sequence the entire rice genome, the National
Center for Gene Research (NCGR) of the Chinese Academy of Sciences has finished thesequence of rice Oryza sativa japonica chromosome 4 through a map-based strategy. The
finished sequence spans 35.5Mb and represents 98.2% of the chromosome. To characterize theblueprintof intra-specificDNA-sequence variations between Oryza sativa indica and japonica
subspecies,
NCGR has conducted an indica-japonica
chromosome 4 comparison at thenucleotide
sequence level. A 22. 1-Mb of the indica Guangluai 4 (GLA4) chromosome 4 has beensequenced, the largest map-based high-quality
indica sequence, and compared this sequence with
23.2 Mb of the japonica orthologous
chromosome 4. This comparison provided a fine scale
indica-japonica
comparison. A sequence comparison of the collinear
regions from indica andjaponica chromosome 4 has revealed their extensive micro-colinearity
in gene order and content,
but also the intraspecificsequence polymorphisms
in both coding and non-coding regions.
Comparative genome analysis between cultivatedrice subspecies
shows that there is an overall
syntenic relationshipbetween the chromosomes and divergence at the level of single-nucleotide
polymorphisms
and insertions and deletions.
1. Chromosome-wide comparison of indica and japonica subspecies
Rice is the principal
food for over half of the populationof the world. With a compact
genome spanning ca. 430 megabase (Mb) pairs, an extensive genetic map (Harushima et al. 1998),
and establishedsynteny with other cereal crops (Gale & Doves 1998), the cultivatedrice species
Oryza sativa represents a model for cereals and as well as other monocot plants (Shimamoto &Kyozuka 2002). The completionof the genome sequence of rice opens a new and exciting
chapter in our quest to functionallycharacterize
all of the approximately
37,500 annotated genes
in rice (Feng et al. 2002; Sasaki et al. 2002; Rice Chromosome 10 Sequencing Consortium 2003;
International
Rice Genome Sequencing Project, 2005). A systematic approach to characterizingthese genes will allow us to dissect and understand the regulatory
networks and evolutionary
selection
controllingsuch complex traits as yield, grain quality,
biotic and abiotic stresses,
reproductive
barriers,
epigenetics
and flowering
time. The next essential
steps towards
deciphering
the sequenced genome are to develop complete and accurate maps of activelytranscribedregions during rice development,
and to carry out genome comparative analysis oftaxonomicallyclosely related rice subspecies.
These will facilitate
the identificationof all thegenes and proteins encoded in the DNA sequence. Such information will allow further analysis of
their function, regulation,and how they cooperate in complex biologicalprocesses in a systems
manner.
DomesticatedAsian rice Oryza sativa, which consists of two major subspecies
indica andjaponica partially
isolated
in terms of sexual reproduction,
has become the most important rice
crop in the world (Linares et al. 2002; Go ff et al. 2002). Indica-japonica
genome comparison has
been chosen as a model system for understandingof origin, speciation,
domestication
andgenome evolutionof rice. Indica and japonica cultivars
can be classified
based on theiragronomic traits (Oka & Morishima 1997) and the use of molecular markers (Glaszmann 1987;
Cheng et al. 2003). Rapid progresses in both indica and japonica genome sequencing have been
made (IRGSP, 2005; Go ff, et al., 2002; Yu et al. 2002; Yu et al. 2005). The International
Rice
Genome Sequencing Project (IRGSP)
has adopted the clone-by-clone
approach for obtaining afinishedrice genome sequence, because it is modular, allows efficientgap filling,
avoidsproblems arising from distant repetitive
sequences and results in the early completion of larger
contiguous segments of a genome (Sasaki & Ben 2000). As part of international
efforts tocompletely sequence the entire rice genome, the National Center for Gene Research (NCGR) of
the Chinese Academy of Sciences has finished the sequence of rice japonica chromosome 4
through a map-based strategy (Zhao et al. 2002; Feng et al. 2002). This was one of the first tworice chromosomes to have been completely sequenced. The finished sequence spans 35.5Mb andrepresents
98.2% of the chromosome. To characterize
the blueprintof intra-specificDNA-sequence variations
between indica and japonica subspecies,
NCGR has conducted anindica-japonica
chromosome 4 comparison at the nucleotide
sequence level. A 22.1-Mb of theindica Guangluai 4 (GLA4)
chromosome 4 has been sequenced, the largest
map-based
high-quality
indica sequence, and compared this sequence with 23.2 Mb of the japonica
orthologous chromosome 4 (Han et al. unpublisheddata). This comparison provided a fine scale
indica-japonica
comparison. A sequence comparison of the collinear
regions from indica andjaponica chromosome 4 has revealed their extensive micro-colinearity
in gene order and content,
but also the intraspecificsequence polymorphisms
in both coding and non-coding regions (Fenget al. 2002; Han & Xue 2003; Li et al. 2004). Comparative genome analysis between cultivatedrice subspecies
shows that there is an overall syntenic relationshipbetween the chromosomes anddivergence at the level of single-nucleotide
polymorphisms
and insertions and deletions.
The ricegenome has been well mapped both geneticallyand physicallyand has a syntenic relationshipwith other cereals. Identificationof the sequence polymorphismsof the two subspecies
has
produced the most densityof polymorphic
sequence maps of the indica and japonica
chromosome 4. Importantly,
this comparison revealed the dynamic changes that may reflect
major evolutionary
events occurring throughout the domesticationand natural selection of rice.
A complete sequence of a chromosome centromere is necessary for fully understanding
centromere function. The rice chromosome 4 centromere sequence has been completely
determined and its structures
have been analyzed. Complete sequencing of the 124-kb rice
chromosome 4 centromere revealed that it consisted of 1 8 tracts of 379 tandemly arrayed repeats
known as CentO and a total of 19 centromeric retroelements(CRs) but no unique sequences weredetected.
The preferential
insertof the CRs among CentO repeats indicatedthat thecentromere-specificretroelements may contribute to centromere expansion during evolution. The
presence of three intact retrotransposons
in the centromere suggests that they may be responsible
for functional
centromere initiation
through a transcription-mediatedmechanism (Zhang et al.
2004).

2. A rice chromosome 4 tilling array chip: chromosomal level regulation
As a first attempt to decipher the rice genome, computational annotation has been successful,
although improvements are needed (Yuan et al. 2003). Recent efforts to verify experimentallythegene model structure by sequencing CDNA and expressed sequence tags (ESTs) have provided
valuable information toward our understandingof gene structure and genome coding capacity(Wu et al. 2002; Rice Full-Length
CDNA Consortium 2003; Rensink & Buell 2004). An essential
and necessary step in this effort is the determinationof the coding information and expression

patterns of each sequenced chromosome. However, until now only about half of the predicted
genome coding capacity had any CDNA or EST expression
support. Clearly,
experimental
approaches
complementary to computation-based
genome annotation are essential
for anunderstandingof genome structures. Because of the presence of large amounts of unfinishedsequence data, unusual compositional
gradients
in genes, and the large size of the rice genome(Rensink & Buell 2004), there is even greater need for experimental approaches in rice genome
annotation.
A chromosome-scale transcriptional
analysis
will expand our knowledge of possible
chromosomal level transcriptional
regulation.
One prominent feature of eukaryotic chromosomes
is their organization
into heterochromaticand euchromatic regions. Heterochromatin
was first
distinguishedfrom euchromatin cytologicallyas more intensely
staining
nuclear material
throughout the cell cycle in Bryophyta. In fact, heterochromatin
has emerged as a key regulator in
the epigenetic
control of gene expression, chromosome behavior, and evolution. Using tiling pathmicroarray analysis as a tool, it is now possible to perform high-throughput
pro filing of thetranscriptional
activities
along an entire sequenced chromosome to examine potential connections
between transcription
and cytologically-definedchromatin organization.
Xing-Wang Deng's and
Bin Han's laboratories
have developed a tiling path DNA microarray consistingof overlapping
PCR amplified genomic fragments covering over 33 Mb (95.5%) of japonica rice chromosome 4(Figure1). Using this array, they analyzed the transcriptional
activity of chromosome 4 in six
representative
Rice Chromosome 4 Transcriptome
organs or tissues.
Chromosomal-scaletranscription
patterns were analyzed in comparison with cytologicallyobserved chromatinorganizationand the distributionof transposon-related
and various other gene model groups (Jiaoet al. 2005). Six representative
rice organ types were examined using this microarray to catalogue
the transcribedregionsof rice chromosome 4 and to reveal organ- and developmental
stage-specifictranscription
patterns. This analysis provided expression support for 82% of thegene models in the chromosome. Transcriptional
activities
in 1,643 non-annotated regions werealso detected. Comparison to cytologicallydefined chromatin features indicated that in juvenilestage rice euchromatic region is more actively transcribedthan transposon-richheterochromatic
portion of the chromosome. Interestingly,
elevated transcriptionof transposon-relatedgenemodels in certain heterochromatic
regions was observed in mature stage rice organs and insuspension
cultured cells. These results suggest a close correlation
between transcriptional
activity
and chromosome organizationand developmental regulationof transcription
activity at
the chromosomal level (Jiao et al. 2005; Li et al. 2005).
Recent completion of both indica and japonica genome sequence analysis now allowed an
unbiasedinterrogationof transcriptional
activityof the whole genome and experimental
verificationof computational
genome annotation. A comparative tiling analysis of the entire
chromosome 4 of both indica and japonica genomes has also been analyzed. This analysis
detected expression of about 80% of the unsupported gene models without previous experimental
evidence in both subspecies.
This tiling analysis also identified
141 new models for thejaponica
chromosome, representing
a 3.6% increase in the annotated protein-coding
capacity.
This
analysis also revealed that a chromosomal level transcriptional
regulation largely coincides
withthe heterochromatin
and euchromatin domains.
References
Cheng,C, Motohashi,R., Tsuchimoto,S., Fukuta, Y, Ohtsubo, H., & Ohtsubo, E. 2003 Polyphyletic
origin of cultivated
rice: based on the interspersion
pattern of SINEs. Mol Biol.Evol 20, 67-75.
Feng, Q. et al 2002 Sequence and analysis of rice chromosome 4. Nature 420, 316-320.

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M.D. & Devos, K.M. 1998 Comparative
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Glaszmann, J.C. 1987 Isozymes and classificationof Asian rice varieties.
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Goff, S.A. et al. 2002 A draft sequence of the rice genome (Oryza sativa L. ssp.japonica).
Science 296,
92-100.
Han, B. & Xue, Y. 2003 Genome-wide intraspecificDNA-sequence variations
in rice. Current Opinion in
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6, 134-138.
Harushima, Y. et al 1998 A high-density
rice genetic linkage
map with 2275 markers using a single F2
population.
Genetics 148, 479-494.
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793-800.

Jiao,

Y et al 2005 A Tiling Microarray
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Map of the Rice Chromosome 4. Genome Research 12, 817-823.

THE RICE GENOME RESOURCE CENTER
AS THE CORE FOR CEREAL GENOMICS
Baltazar

A. Antonio, Akio Miyao, Yoshiaki Nagamura, Takuji Sasaki
National Instituteof Agrobiological
Sciences
2- 1-2 Kannondai, Tsukuba, Ibaraki 305-8602,
Japan^Corresponding
author: antonio@nias.afrrc.go.jp(Tel: +8 1 29 838 7456)

Abstract
The ultimate goal for rice genomics is to elucidate
the biology of the rice at the cellular,organismal and evolutionary level. With the complete sequence of the rice genome in the publicdomain, researchers can now have access to the sequence of about 40,000 genes that defines arice plant. This must be supplemented with an expanded genetic toolkit that includes biologicalmaterials which could be used for experimental verificationof gene function, viable strategies
toexecute large-scalecharacterizationof the genome and a robust informatics
infrastracturetofacilitate
integrationof all knowledge about the rice plant. Efforts in providing
the researchcommunity with genome resources including
full-length
CDNA clones, mutant lines andbiologicalmaterials for genetic analysis will be describedwith emphasis on how these resources
will shape research in cereal genomics in the future. The next trend in rice genomics will focus in
implementing a whole-systems approach to the identificationof gene function which will require
knowledge of overall gene expression profiles at the cellular, subcellularand organ level under avariety of defined conditions.
Achieving this goal will also require databases and bioinformatics
tools to integrate all information that can be queried from any perspective and provide a platform
for comparative genomics that will lead to better understandingof other cereal genomes.
Introduction
Rice is considereda model cereal crop because of its small genome size and syntenic
relationships
with other major crop species. The completion of the rice genome sequence will
therefore herald a new era for cereal genomics with rice providinga reference sequence forelucidating
the biology of maize, wheat, barley and other cereal genomes (International
Rice
Genome Sequencing Project 2005). However, in order to completely
understand the biology ofthis importantcereal crop and effectively
use it as an experimental
model system forunderstanding
other major cereal crops, the following
challenges
in rice genomics should be
addressed immediately:(1) elucidating
the actual function of about 37,000 genes predictedin thegenome; (2) establishing
the protein profile in rice plant; (3) understanding
the metabolic
pathways and their interactions;(4) clarifying
the cellular mechanics that occur throughout thelife cycle; and (5) deciphering
how rice genome evolves and how it diversifies.
These areas ofresearch basicallycorrespond to the field of functional genomics, which includes understanding
the function from the basic structuralsequence of the DNA, the RNA, proteinsandaccompanying interactions.
Since 1991, the Ministry of Agriculture,
Forestry and Fisheries(MAFF) of Japan has been
continuously
funding comprehensive large-scale
projectsfor rice genome research. Foremost
among these projects
is the Rice Genome Research Program (RGP), which led to theestablishmentof the high-density
genetic map, physical map, transcript
map and eventually thecomplete rice genome sequence. During the same period, other genome related projects havebeen organized to facilitate
efficient utilizationof genome information derived from mapping andsequencing. These include projects on DNAmarker breeding, gene cloning / functional
analysis,proteome, microarray, full-length
CDNA analysis etc., which further generated biologicalmaterials
and genetic resources which could be used for functional
characterizationof thegenome. As part of an effort to ensure that the large investment in these projects will benefit thescientificcommunity at large and encourage more innovative researches in functional and applied
genomics, the Rice Genome Resource Center (RGRC, http://www.rgrc.dna.affrc.gojp/)
wasestablishedin 2003. It also aims to facilitate
efficient management and distributionof genome
resources and to provide access to high-throughput
technologies
in functional
genomics. The
three major genome resources, namely, rice full-length
CDNA, Tosl7 mutant lines and genetic
analysis materials (Table 1) will be describedwith emphasis on their utilizationin functional
characterizationof the genome.
Rice Full-length
CDNA
The full-length
CDNA sequences are important resources to facilitate
the characterizationof gene functions. Such information is important for the confirmation of genes identified
bygenome annotation using various prediction
programs and for identificationof alternative
splicingsites for RNAs. The Rice Full-length
CDNA Project has generated sequence data on
175,642 rice full-length
cDNAs clustered into 28,469 nonredundant clones (The Rice Full-length
CDNAConsortium 2003). The starting materials for library construction
represent various tissues
such as root, shoot and panicle as well as calli subjected
to various treatment and growth
conditions
and therefore represent the complete array of genes expressed in rice. A total of
21,596 clones have been assigned with tentative protein functions through homology searches ofpublicly
availablesequence data. In addition, more than 94% of the clones could be mapped tosubspecies japonica and indica genomic sequences indicatingthat the nucleotide
sequences of thegene-coding regions are very similar in these two subspecies.
All sequence data are available
through the database KOME (Knowledge-based
Oryza Molecular Biological
Encyclopedia,
http://cdnaO1.dna.affrc.go.jp/CDNA/).
The databaseprovidesthe nucleotide
sequence andencoded amino acid sequence information,
results of the homology search with the public
databases,
mapping information,
pattern of alternative
splicing,
protein domain information,transmembrane structure, cellular localizationand gene function by Gene Ontology. Access to
specific information for each full-length
CDNA clone can be made by BLAST search, accession
number of the clone, specific domain name and general key word search.
The availabilityof rice full-length
CDNA clones will greatly enhance the rate of genediscovery, patterns of splicing and the understandingof gene function and protein interactions
inrice. Since the annotation of the sequence simply classifies
various regions of the genome into
genie regions, non-transcribedregion, transposable
elements etc., homology with full-length
CDNAsequence will address whether the predictedgenes are ever expressed or not to facilitate
more informative characterizationof the gene.
Tosl 7 mutant lines
An alternative to the nucleic acid characterization
through full-length
CDNA homology isby direct demonstrationthat the gene has a function. This can be done by disrupting
the genefunction via inserting
either a T-DNA or a transposon sequence. The insertionalmutagenesis
approach is expected to play an important role in determining
the function of the genes predicted
in rice. A transposon tagging strategy, which utilizes
a rice endogenous retrotransposon
Tos17
has been found to be effective in functional
characterizationof rice genes (Hirochika2001).
Mutations due to Tos1 7 transposition
are normally induced under tissue culture conditionsandare inherited in subsequent generations to facilitate
analysis of the mutated gene. So far, a Tosl 7
mutant panel with more than 50,000 insertional
mutant lines carrying about 500,000 insertions
has been generated. These resources would be very useful for forward and reverse genetic
analyses. The Rice Tosll Insertion
Mutant Database (http://tos.nias.affrc.go.ip/)
currently
contains flanking sequences of Tos17 insertion sites from 5,000 lines and associated phenotypes.
By performing a simple BLAST search against these flanking sequences, any gene of interest canbe easily found if the collection
contains a mutation in that gene.
The disruptionof gene function by insertionalmutagenesis is generally applicablefor rice
because it can be easily transformed and regenerated.
The insertion of Tos\7 into the coding
region can induce partial or complete inactivationof the gene that may therefore lead to
identificationof the function of the gene.
Genetic analysis materials
Analysis of agronomicallyuseful genes and complex traits including
QTL have beeneffectively
accomplishedusing a number of resources including
a high-density
linkage map,
appropriate
crosses and subsequent generations of segregating
populations
to allow the mappingof the traits of interest. A strategy that involved developingof specific mapping populations
andfine mapping of many traits proved effective in map-based cloning. The genetic populations
currently available include backcross inbred lines (BILs), chromosome segment substitution
lines(CSSLs)and doubled-haploidlines (DHLs) from japonica/ indica crosses (Table1). Thegenotype of each line was determined using RFLP markers distributed
along the 12 rice
chromosomes. These genetic materials have been successfully
used in QTL mapping of manyagronomic traits including
heading date, seed dormancy and regeneration ability.
Many traits in rice such as flowering time, seed development, disease resistance etc. havebeen extensively
studied in the context of germplasm diversity.
Several QTLs associatedwithsuch traits have been identified(Yano et al. 2003). However there are many key agronomic
characteristics
that remain to be analyzed in rice. In addition
to a wide array of molecularmarkers for rice, the availabilityof genetic populations
with well-characterized
chromosomal
segments will be very useful for fine mapping ofQTLs.
Table 1. Biologicalmaterials

from the Rice Genome Resource Center

R eso u rce s
R ic e fu ll‑len g th CD N A
T o s 1 7 in sertio n m u tant lin es
N ip p o n b are/K asalath B IL s
N ip p o n b are/K asalath C S S L s
K o sh ih ik ari/K asalath B IL s
K o sh ih ik ari/K asalath
S asan ish ik i/H ab atak i B IL s
S asan ish ik i/H ab atak i C S S L s
A k ih ik ari/K o sh ih ik ari D H L s

Sto ck s
DN A

see d sN o o f clo n e s/lin e s
3 2 ,0 0 0 clo n es
5,0 0 0 lin es
9 8 lin es
54
1 82
3 9 lin e s
85
3 9 lin e s
2 12High-throughput technologies for functional genomics
Muchof the researches in the post-genome sequencing era will depend on high-throughput

technologies.
The Rice Genome Resource Center is providing
access to microarray technologythat will allow a much larger number of investigators
to apply global expression pro filing into
their specificresearch programs on rice. A rice oligonucleotide
microarray with 22,000
non-redundant
probeswas constructedin collaboration
with
Agilent
Technologies(http://www.agilent.co.jp).
A 2-day protocol for microarray analysis was also developed to allow
researchers
to explore expression
pro filing. After RNA extraction,
sample labelingandhybridization
can be performed on the first day. Subsequently,
washing and data spot analysis canbe performed on the second day. It is a highly reliable protocol eliminating
most of the limiting
factors that affect microarray expression analysis such as sensitivityof the quantity of RNA andbackground intensity. Imaging analysis using MFP-3D atomic force microscopy was performed
to determine the quality of each DNA spot in this microarray. At 90 |um 5 90 |um scale each DNA
spot in this array is nearly circular in shape. Magnificationof a 3|um 5 3|um section as well as
lpm 5 l|um section showed a clear boundary between the glass surface and the DNA spot. Thissuggests that hybridizationof target genes can be performed with high reproducibility
andenhanced sensitivity.
This microarray chip is also available
commerciallythrough Agilent
Technologies.
A platform for rice functional
genomics initiative
The effort of the Rice Genome Resource Center in providing access to genome resources isa major step towards developing
a genetic platform for functional
genomics. The international
community is responding
as well as reflected in the formation of the International
Rice
Functional
Genomics Consortium, which was patterned after the International
Rice Genome
sequencing Project (Hirochika
et al. 2004). The collective
goals of the consortium include sharingof genomic materials,
integrationof databases,
bilateralor multilateral
partnerships,
implementing
initiatives
for the cooperative
elucidationof gene function and accelerating
deliveryof research results to benefit rice production(http://www.iris.irri.org:8080/IRFGC/).
Specifically,
the consortium has set a timeline for various functional
genomics programs asfollows: 1) making genome-wide oligo chips publicly
available
by 2005; 2) tagging 90% of ricegenes using a variety of mutant collections
by 2005; 3) establishinga global internet network ofrice functional
genomics databases by the end of 2005; 4) developinga high-throughput
verification
system by 2007; and 5) characterizing
the function of 50% of rice genes by 2010.
With the rapid accumulation of genomics resources and the growing interest of researchers in rice,these goals could be easily achieved. The current progress on how the rice scientificcommunity
is moving towards these goals can be reflected from the resources available from various groups
worldwide (Table 2).

Table 2. Rice genome resources available
R e s o u rc e s
C o m p lete g e n o m e s e q u e n c e
D r a ft g e n o m e se q u e n c e
D r a ft g e n o m e se q u e n c e
G en eM u ta n t lin e s
M u ta n t
M u ta n t l i n e s
G e n etic p o p u la tio n sworldwideD e sc r ip tio n
J ap o n ic a v a r.N ip p o n b a reIn d ic a v a rie ty
J a p o n ic a v a r.N ip p o n b a reF u ll‑le n g th C D N A
7 b 5 1 7 in s e rtio n lin e s
T ‑D N A 1
T ‑D N A 1
B IL , C S S L , D H L

A c cess
IR G S P
B e ij in g G e n o m ic s In st.
S y n g e n ta
RG RC
RG RC
P o h a n g U n iv . S c i. T e c h .
IR R IRGR C

G ene array
G ene array
G ene array

22,000 full‑length CD N A
60,000 E ST / genom ic sequence
20,230 TIG R gene m odels

RGR C
B eijing G enom ics InstU niv.C alifornia D avis

The Rice GenomeResource Center will continue to take part in this initiativeby providing
access to more biologicalmaterials
as they become available
and developingmore
high-throughput technologies to assist rice researchers.
Acknowledgement
The Rice Full-length CDNAProject was led by Dr. Shoshi Kikuchi, the Tosll mutant lines
were developed by the research group of Dr. Hirohiko Hirochika and the genetic analysis
materials by the research group of Dr. Masahiro Yano. We also acknowledge the RGRC staff
including Ms. Jianyu Song, Ms. Kazue Ito, Ms. Reiko Kise, Ms. Ritsuko Motoyama and Ms.
TomokoOosaki.
References
The Rice Full-length
CDNA Consortium, 2003. Collection,
mapping, and annotation of over
28,000 CDNAclones fromjaponica rice. Science 301 :376-37
Hirochika, H., 2001. Contribution of the Tosll retrotransposon to rice functional genomics. Curr.
Opin. Plant Biol. 4:118-122.
Hirochika, H. et al., 2004. Rice mutant resources for gene discovery. Plant Mol. Biol.
54:325-334.
International
Rice Genome Sequencing Project, 2005. The map-based sequence of the ricegenome.Nature 436:793-800.
The Rice Full-length
CDNA Consortium, 2003. Collection,
mapping, and annotation of over
28,000 CDNAclones from japonica rice. Science 301:376-379.
Yano,M. et al., 2003.Marker-assisted
dissection and pyramiding of complex traits in rice. Rice
Science: Innovations and Impact for Livelihood (edited by Mew,T.W.et al.) p257-263. IRRI.

FUNCTIONAL ANALYSIS OF GENES INVOLVING
CULM ELONGATION IN RICE
Motoyuki Ashikari
and Makoto Matsuoka
Bioscience& Biotechnology
Center, Nagoya University, Furouchou, Chikusa, Nagoya, Aichi, 464-860
Corresponding
author: makoto@agr.nagova-u.ac.ip,
Tel: 8 1-52-789-5225,Fax:
8 1-52-789-5226,

1 , Japan.

In the 1960s, the rapid accelerationof the world population
growth rate and dramatic
decrease in cultivated-lands
raised fears that food production would not meet the growing demand,
leading to a global food crisis. To address the problem, in 1966, the International
Rice ResearchInstitute(IRRI) bred a semi-dwarf and high yielding variety IR8, well known as "miracle rice", by
the crossing between a Taiwanese native semi-dwarf variety Dee-geo-woo-gen, which carries thesemi dwarf1 (sdl) gene, and an Indonesian good taste variety, Peta. The yield improvement ofIR8
was mainly due to the reduction of plant height caused by the sdl allele. In general, nitrogen
fertilization
is essential
to increase grain production, but it also induces culm elongation, resulting
in an overall increase in the height of crop plants. Such tall crop plants are easily damaged by windand rain and consequently
yield losses occur. The IR8 semi-dwarf rice variety dramaticallyresolved this problem, mainly because it responded to fertilizer
inputs to produce an increased
yield without culm elongation. Widespread adoption of IR8 led to major increases in rice grain
production and a famine was averted. It was the so-called rice "green revolution". Like IR8, thehigh yielding
varieties Taichung Native 1 in Taiwan and Tongil in Korea, which also contained thesdl allele from Dee-geo-woo-gen, contributed
to food security in these countries. Similarly,aJapanese native semi-dwarf variety Jikkoku,
and y-ray induced variety Reimei in Japan, and they-ray induced variety Calrose 76 in the United States of America, also carried different sdl alleles
and were widely used in the rice breeding programs in these countries. The fact that such different
sdl alleles have been used as cross parents and contributed to numerous rice breeding programs forboth indica andjaponica
species demonstrates that the sdl locus is the most suitable for controllingthe height of the rice plant.
There are various reasons for the dwarf phenotype
in plants, but one of the mostimportant factors for determining
plant height are gibberellins(GAs) known as one of the plant
hormones. GAs are a large family oftetracyclic
diterpenoids
and are associated with a number ofplant growth and developmental
processes such as seed germination,stem elongation,floweringand fruit development. To date, we have studied several rice GA-related mutants and isolated thegenes involved in GA biosynthesis
and signal transductionin the rice plant. Through these studies
on rice GA-related mutants, we recognized that the plant morphology of the sdl mutants is similar
to that of GA-deficient mutants with weak allele. To investigate
the possibility
that the SD1 gene isrelated to GA, we examined the GA response in the sdl mutant. As a first step, we compared thesecond leaf sheath elongation in the sdl and wild type plants in response to various amounts of GA3
treatments. The sdl seedlings responded better than the wild type and the sheath length of sdl wasrecovered to that of the wild type at 10"6 M ofGA3. This result suggests that sdl is GA-deficientmutant.
To identify which enzyme the SD1 gene encodes, we directly examined the intermediate
GA levels in the sdl mutant. The amounts of GA2o, GAi, GAg and GA29 in the sdl mutants werelower than that of the wild type plants, whereas the amounts ofGA44 and GA19 in the mutants wereequal level those of wild type. According to GAi is mainly used as active GA in rice, these resultssuggest that the activityof GA 20-oxidase(GA20ox),which catalyzes
the 3 steps from
GA53-GA44-GA19to GA2o, is weaker in the mutants than in the wild type, and therefore the SD1gene may encode GA20ox.
So far, one gene encoding a GA20ox (GA20ox-l)
has been isolated from the rice plant,
but it does not correspond to the SD1 gene since GA20ox-l was mapped on chromosome 3 of therice genome whereas sdl was mapped on the long arm of chromosome 1. With this in mind, we
suspected that SD1 may encode another GA20ox since the Arabidopsis
genome carries four GA
20-oxidase genes and the products function in a redundant manner. To isolate other G^20ox-related
genes in rice, we designed degenerated
primers based on the conserved domain between the riceand Arabidopsis
GA20ox genes. Two amplified DNA fragments were obtained from the ricegenome (japonica
cultivar, Taichung 65); one corresponds to the previously
identified
GA20ox-l
and the other is a novel GA20ox gene (GA20ox-2). The GA20ox-2 was located on the long arm ofchromosome 1, tightly linked to the sdl locus. We isolated the full length of the GA20ox-2 geneand the CDNA clones. The deduced amino acid sequence of GA20ox-2 showed 47.8% or 49.5%
identity
to that of GA20ox-l or Arabidopsi GA5, respectively.
We cloned and sequenced theGA20ox-2 genes from four sdl mutants and found that one sdl allele contained 383 bp deletion(Dee-geo-woo-gen),and other three sdl alleles had single nucleotide
exchanges to induce aminoacid exchange respectively(Jikkoku, Reimei and Calrose 76). Various mutations in the gene ofsdl
mutants strongly indicate that SD1 encodes GA20ox-2. The introductionof the GA20ox-2 genefrom the wild type plant rescued the dwarf phenotype of sdl, confirming that SD1 encodes
GA20ox-2. To demonstrate that GA20ox-2 encodes an active GA20ox, we subcloned the coding
region in an expression vector and expressed its product in E. coli. The recombinant protein
catalyzed the conversion of GA53 to GA20. These results show that GA20ox-2 encodes an active
GA20ox.
Recently, the wheat green revolution gene, Rht, was identified
and revealed that it
encodes a transcriptional
factor that works as a negative regulator of GA signaling. As wheat has ahexaploid
genome, recessive alleles
such as sdl in rice are not available
for producingasemi-dwarf strain of wheat. Actually the Rht gene was a gain-of-function
allele caused by an
N-terminal truncated product which is specific region related to the perceptionof GA signaling.
Even though the genetic and biochemicalfunction of the rice SD1 and wheat RHT proteins arecompletely different, that is, recessive and dominant, loss-of-fimction
and gain-of-function
events,enzyme and transcriptional
factor, respectively,
the products of both genes are related to GA. The
fact that both green revolution genes are related to GA may not be a coincidence,but may be anatural consequence. Consequently, manipulationof GA biosynthesisor perception may be a goodtarget for regulationof crop height.
The rate of world population growth has now once again exceeded the rate of growth infood-grain production,and prompt measures and action for a second green revolution have beencalled for to avoid widespreadfood scarcity in the future. As our present study revealed thatmodulating active GA levels during the vegetative
stage can produce a suitable plant architecture
for high crop yield, the genetic manipulationof GA-biosynthesis
using a molecular biologicalapproach may provide us with an opportunity to address food security concerns as was the case forthe previous green revolution.
References
Ashikari M, Sasaki A, Ueguchi-Tanaka M, Itoh H, Nishimura A, Datta S. K, Ishiyama K, Saito T,
Kobayashi M, Khush G S, Kitano H, Matsuoka M. 2002 Loss-of-functionof a ricegibberellinbiosynthetic
gene, GA20 oxidase (GA20ox-2), led to the rice green revolution.

Breeding Science.52:
143-150.
Khush G S. 1999 Green revolution:
preparing for the 21st century. Genome 42: 646-655.
Peng J, Richards D. E, HartleyN.
M, Murphy G P, Devos K. M, Flintham J. E, Beales J, Fish L. J,
Worland A. J, Pelica F, Sudhakar
D, Christou P, Snape J. W, M. Gale D, HarberdN. P.
1999 'Green revolution'
genes encode mutant gibberellin
response modulators.
Nature.
400: 256-261.
Sasaki A. Ashikari M, Ueguchi-Tanaka M, Itoh H, Nishimura A, Datta S. K, Ishiyama K, Saito T,
Kobayashi
M, Khush G S, Kitano
H, Matsuoka
M. 2002 A mutant ofgibberellin-synthesis
gene in rice. Nature. 416: 701-702.

MOLECULAR MECHANISM OF BROAD-SPECTRUM RESISTANCE
MEDIATED BY NBS-LRR PROTEIN IN RICE
Guo-Liang Wang1'4'*, Shanghong Qu1, Bo Zhou1, Guifu Liu1, Bin Han2, Hajime Sakai3
1. Department of Plant Pathology,
Ohio State University,
Columbus, OH 432 10 USA
2. National Center for Gene Research, Chinese Academy of Sciences, Shanghai 200233,
China
3. DuPont Crop Genetics, Delaware Technology Park 100/200,
DE, USA
4. Rice Genomics Laboratory, Hunan Agricultural
University,
Changsha, Hunan, 4 10 128 China* Corresponding
author: Guo-Liang Wang, Department of Plant Pathology,
The Ohio State University,
20 1 Kottman
Hall, 2021 Coffey Rd, Columbus OH 43210 USA Tel (614) 292 9280 or 1375, Fax (614) 292 4455, Email:wang.620@osu.edu

Abstract
To understand the molecular basis underlying
the broad-spectrum
resistanceto ricepathogens, we cloned and characterized
the rice blast resistancegenes Pi9 and Pi2. Both of theresistancegenes confer high level of resistanceto diverse isolatesof Magnaporthe grisea
collected
from different rice growing areas. Sequence analysis indicatedthat the two resistancegenes are allelic to each other and encode highly homologous NBS-LRR proteins. Each locus
contains multiple
family members spanning about a 100 kb region. Comparative sequence
analysis of five haplotypes
revealed that the LRR region is under diversification
selection.
Theorthologous
region of the Pi9 and Pi2 genes in three wild species is being sequenced which will
provide important information
about the origin and evolution of the complex resistance genecluster in the genus Oryza.
Introduction
Broad-spectrum disease resistanceis defined as a type of resistancethat is effective
tomany strains of the same pathogen or to strains of two or more unrelated pathogens. It not only
provides a useful resource for breeding of durable resistance in crop plants but also an excellent
material for the understandingof the molecular basis of resistance specificity
in plants. To date,
several broad-spectrum resistancegenes have been cloned in plants. Xa21 is the resistancegenecloned with a high level of resistance to many Xanthomonas oryzae pv. oryzae strains (Wang etal. 1996).
The recessive mutations {mid) of the barley Mlo locus mediate a broad-spectrum
resistance to all known isolates of powdery mildew fungus (Blumeria graminis f sp. hordei)(Buschges
et al. 1997). m/o-based disease resistanceinvolves a spontaneous-lesion
phenotypeand cell-wall depositionin epidermal tissues preceding pathogen attack. The Arabidopsis RPW8
gene (Xiao et al. 2001) belongs to a new family of broad-spectrum resistance gene and confers
resistanceto different
isolates of the same powdery mildew pathogens as well as to different
powdery mildew fungi. The protein it encodes is a small membrane protein with a putative
coiled-coildomain that has limited homology to the N-terminus of an NBS-LRR gene (Xiao et al.
2001). RPW8 is not involved in the gene-for-gene interaction
and may interact with different A VR
genes from different pathogens. Recently, the same resistance gene that was denoted KB by Songet al. (2003)
and RPI (Rpi-blbl)
by Van der Vossen et al. (2003) was cloned from an NBS-LRR
gene cluster. RB/RPI confers a broad-spectrum resistance to all known races of the late blightp athogen Phytophthora infestans.
Rice blast, caused by the fungal pathogen Magnaporthe grisea is a major disease in riceproduction
worldwide. To elucidate
the molecular basis of broad-spectrum
resistanceto thepathogen, we cloned the blast resistancegenes Pi9 and Pi2 genes using a map-based cloning
strategy. Both Pi9 and Pi2 confer high level of resistance to diverse isolates collectedfrom many
countries and have been widely used in rice breeding programs. Detailed characterizationof theresistance cluster provides new insight into the molecular basis of broad-spectrum resistance toplant pathogens.
Results and Discussion
An NBS-LRR gene cluster was identified
by sequence analysis of a 76-kb genomic region
from the Pi9 locus:
A BAC (bacterial
artificial
chromosome) was constructed using the DNA
isolatedfrom the Pi9 donor line 75-1-127.
Using the P/P-linked
marker pB8 as the hybridization
probe, three BAC clones spanning the locus was identified that were used to build a BAC contig(LIU et ah 2002). To obtain sequence information at the Pi9 locus, we fully sequenced two of the
BAC clones, 75-1-127BAC12
and 75-1-127BAC3.
The gene prediction
program GENSCAN was
first used to identify the putative coding sequence (CDS) in the 76-kb region, and then the
BLAST program was used for homology searches to confirm the gene prediction
results. Seven
putativegenes were identified
from the 76-kb sequence. The first gene in the 76-kb region(between 10489-12966bp), located at the SP6 end in 75-1-127BAC12,
is a homolog of themaize gene that encodes a putative nitrate-induced
NOI protein. The other six genes, denoted
Nbsl-Pi9 to Nbs6-Pi9, were considered to be Pi9 candidate
genes because they all have highhomology to the NBS-LRR disease resistancegenes cloned from various plant species (BENT,
1996; DANGL and JONES, 2001; MARTIN etah 2003).
Rice transformation
with individual
candidategenes determinedthat Nbs2-Pi9 is Pi9. From
the Pi9 deletion mutant analysis, we narrowed down the Pi9 gene to either Nbs2-Pi9 or Nbs3-Pi9.
To pinpoint the Pi9 gene in the genomic region, we made two genomic constructs of the Nbs2Pi9 and Nbs3-Pi9 genes. The pNBS3 construct contained a 14.6-kb (18395-33070bp) genomic
fragment comprising the Nbs3-Pi9 gene. Ti plants of 19 independently
transformed lines weresusceptible
to PO-6-6, and all of the T2 plants from nine transgenic lines were highly susceptible
to PO-6-6. For rice transformationof Nbs2-Pi9, we developed the pNBS2 construct, whichcontaining a 13.5-kb Sail genomic fragment (32363^45848
bp). This 13.5-kb fragment contained
1362 bp of the Nbs2-Pi9 5'-UTR region (32962-33724
bp), the whole Nbs2-Pi9 coding sequence(33,725^2,313bp), and 1804 bp of the 3'-UTR sequence (42314-44117
bp). Of the 19 Ti
transgenic
plants we inoculatedwith rice blast isolate PO6-6, 13 were resistantand six weresusceptible.
We subsequently
evaluated T2 plants from three resistant Ti lines (#10, #12, and#77) and one susceptible
Ti line (#8) for resistanceto rice blast isolate PO6-6 and observed thesegregationof resistant and susceptible
plants in all three resistant lines.
Co-segregationof the transgene and blast resistancewas confirmed in transgenic
line #12
using Southern blot analyses. Genomic DNA was extracted from ten T2 plants of the #12 line,
digested with Ec6Sl9 and probed with a 928-bp fragment from Nbs2-Pi9 (40350-41278
bp). The
928-bp fragment was found to be located within the second exon of the putative Nbs2-Pi9-coding
sequence and corresponds to the sequence encoding the last 151 amino acids of the NBS domain
and the first 158 amino acids of the LRR domain. All seven resistant T2plants (#12-13,#12-14,#12-18, #12-20, #12-21, #12-29, and #12-30) contained the expected ll-kb Nbs2-Pi9 band, while
the three susceptible
T2 plants (#12-5, #12-17, and #12-23)
did not, suggestingthat the resistancein the transgenic lines is truly due to the Pi9 transgene.

Pi9 encodes a putative NBS-LRR protein containing
1,032 amino acids: To isolate the CDNA
fragment ofPi9, we prepared total RNA using leaf tissue from 75-1-127 collected 24 h following
blast inoculation.
The Pi9 CDNA was 4009 bp, including
3099 bp of Pi9 coding sequence and910 bp of 3'-UTR. Protein translationof the CDNA sequence revealed that the Pi9 gene encodes
a predicted1032-amino acid polypeptide
with a molecular weight of 117.05 kDa and an
isoelectric
point (pi) of 7.55. The deduced Pi9 protein belongs to the NBS-LRR class of R
protein.
The three sequences in the NBS domain, GMGGLGKT (positions
193-200),
KRYFVILDDLW (positions
277-287),
and GSRIVITTRNVDL (positions
307-3 19), correspondto kinase la (P-loop),kinase 2, and kinase 3a, respectively.
At the C-terminal region is the LRR
domain, which is composed of 17 imperfect LRR repeats.
Pi2 is allelic to Pi9 and encodes an NBS-LRR protein: Using the markers tightly linked to Pi9,a high-resolution
map at the Pi2 locus was established.
A BAC/TAC contig that covered the Pi2
region was constructed and subsequently
sequenced completely.
Sequence analysis showed that
there is a gene family in the Pi2 region that comprises nine NBS-LRR resistance gene-likesequences (named Nbsl-Pi2 to Nbs9-Pi2). The Pi2 candidate gene was delimited into both NbslPi2 and Nbs2-Pi2 based on results from the high-resolution
mapping in the Pi2 region. Molecularanalysis of the Pi2 susceptible
mutants further narrowed the candidates
to Nbs2-Pi2, which was
finally confirmed by gene complementation
test. Pi2 encodes a novel nT-NBS-LRR protein andhas a unique gene structure in respect to the intron position. Pi2 was constitutively
expressed at
low levels in both resistantand susceptible
rice after blast infection.
Physical
mapping andsequence analysis revealed that Pi2 and Pi9 are allelicalthough they were introgressedfromdifferent donor lines. Cloning and characterizationof the Pi2 gene provide us additional
material
to elucidate
the molecular mechanisms of host-pathogen
interaction,
evolution, race-specificity
and signaling pathway of broad-spectrum resistance to rice blast.
Reference
Bent, A. F., 1996 Plant disease resistancegenes: function meets structure. Plant Cell 8: 17571771.
Buschges, R., K. Hollricher,
R. Panstruga,
G. Simons, M. Wolter et al, 1997 The barley Mlo
gene: a novel control element of plant pathogen resistance.
Cell 88: 695-705.
Dangl, J. L., and J. D. G. Jones, 2001 Plant pathogens
and integrated
defense responses
toinfection.
Nature 411: 826-833.
Liu, G., G. Lu, L. Zeng and G. L. Wang, 2002 Two broad-spectrum
blast resistancegenes, Pi9(t)and Pi2(t),are physicallylinked on rice chromosome 6. Mol. Genet. Genomics 267: 472-480.
Martin, G. B., A. J. Bogdanove and G. Sessa, 2003 Understanding
the functionsof plant disease
resistance
proteins.
Annu. Rev. Plant Biol. 54: 23-61.
Song, J., J. M. Bradeen, S. K. Naess, J. A. Raasch, S. M. Wielgus, et al, 2003 Gene RB cloned
from Solarium bulbocastanum
confers broad spectrum resistanceto potato late blight.
Proc.
Natl. Acad. Sci. USA 100: 9128-9133.
Van Der Vossen, E., A. Sikkema, B. L. Hekkert, J. Gros, P. Stevens etal, 2003 An ancient R
gene from the wild potato
species
Solanum bulbocastanum
confers broad-spectrum
resistanceto Phytophthora
infestans in cultivatedpotato and tomato. Plant J. 36: 867-882.

Wang GL, Song W, Ruan D, Sideris S, Ronald PC. 1996 The cloned gene, Xa21, confers
resistanceto multiple Xanthomonas oryzae pv. oryzae isolates in transgenic
plants.
Molecular Plant-Microbe
Interactions
9:850-855.
Xiao, S., S. Ellwood, O. Calis, E. Patrick, T. LI et al9 2001 Broad-spectrum
mildew resistanceArabidopsis
thaliana mediated by RPW8. Science 291: 1 18-120.

inISOLATION

AND FUNCTIONAL ANALYSIS OF GENES CONTROLLINGSI UPTAKE IN RICE

Jian Feng Ma1*, Naoki Yamaji1, Kazunori Tamai1, Namiki Mitani1, Saeko Konishi2, Masahiro Yano3
1 Research Institutefor Bioresources,
Okayama University,
Chuo 2-20- 1 , Kurashiki
7 10-0046,
Japan
2 Instituteof Society for Techno-innovationof Agriculture,
Forestry and Fisheries,
Kamiyokaba, Tsukuba, Ibaraki
305-0854,
Japan
3 Department of Molecular
Genetics, National
Instituteof Agrobiological
Sciences,
Tsukuba, Ibaraki
305-8602,
Japan*Corresponding
author: maj@rib.okayama-u.ac.jp(Tel: +8 1 86 434 1209)

Beneficial
effect of silicon on plant growth
Silicon (Si) is the second most abundant element in soil.
In soil solution, Si occurs mainly as
monosilicic
acid (H4SiC>4) at concentrations
ranging from 0. 1 to 0.6 mMand is taken up by plants
in this form (Epstein 1994; Ma and Takahashi 2002).
After the uptake, Si accumulates on theepidermisof various tissues mainly as a polymer of hydrated amorphous silica.
All terrestrial
plants contain Si in their tissues although the content of Si varies considerablywith the species,
ranging from 0.1% to 10% Si on a dry weight basis (Ma and Takahashi 2002).
Silicon has notbeen recognized as an essential element for plant growth from viewpoint of physiology,
however,a lot of beneficial
effects of Si on plant growth have been observed in a wide of plant species.
The beneficial
effects of Si reported include increased photosynthetic
activity, increased insect
and disease resistance,
reduced mineral toxicity,
improvement of nutrient imbalance,
andenhanced drought and frost tolerance (for reviews, see Ma and Tahakashi, 2002, Savant et al.,1997).
Overall, the beneficial
effects of Si show two characteristics.
One is that the beneficial
effects vary with the plant species.
Beneficial
effects are usually obvious in plants that
accumulate high levels of Si in their shoots (Ma et al. 2001).
One typical example is rice, whichaccumulates Si up to 10% Si on a dry weight basis in the shoot.
High accumulation of Si in rice
has been demonstrated to be necessary for healthy growth and high and stable production.
Forthis reason, Si has been recognized as an "agronomicallyessential element" in Japan and silicate
fertilizers
have been applied to paddy soils.
The other characteristic
is that the beneficial
effectsof Si are usually expressed more clearly when plants are subjected to various abiotic and biotic
stresses.
Silicon is probably the only element which is able to enhance the resistance to multiple
stresses.
Silicon uptake mechanism in different
plant species
The accumulation of silicon (Si) in the shoots varies considerably
among plant species, andthis variation has been attributed to the different capacity of the roots to take up Si. The uptake
system of Si was recently investigatedin terms of radial transport from external solution to root
cortical cells and release of Si from cortical cells to xylem in rice, cucumber and tomato, which
differ greatly in the shoot Si concentration(Mitani and Ma, 2005). The concentrationsof Si in the
root-cell
symplast in all species were higher than that in the external solution, although theconcentration in rice was 3- and 5-fold higher than that in cucumber and tomato, respectivelyAkinetic study showed that the radial transport of Si was mediated by a transporter with a Kmvalueof 0. 15 mM in all species, but with different Vmaxvalues in the order of rice> cucumber >tomato.
In the presence of the metabolic
inhibitor
2,4-dinitrophenol
and at low temperature, the Siconcentrationin the root-cell
symplast decreased to the similar level of apoplasmic
solution.

These results suggest that both transporter-mediatedtransport and passive diffusion of Si are
Involved In the radialtransportof SI and that the transporter-mediatedtransport Is an
energy-dependent
process (Fig. 1).
The SI concentrationof xylem sap In rice was 20- and 100-fold higher than that In cucumber
and tomato, respectively.
In contrast to rice, the SI concentration
In the xylem sap was lower
than that In the external solution in cucumber and tomato (Mitani and Ma? 2005).
A kineticstudy showed that xylem loading of SI was also mediated by a kind of transporter In rice (Tamal
and Ma, 2003), but by passive diffusion
In cucumber and tomato (Fig. 1). These results Indicatethat a higher density of transporter for radial transport and the presence of a transporter for xylem
loading are responsible
for high SI accumulation in rice.

Fig. 1 Schematic representationof Si uptake system In different plant species. Radial transport of
SI includes transporter-mediatedtransport and passive diffusion in rice? cucumber and tomato.
Xylem loading of Si Is mediated by a kind of transporterin rice, but by diffusion in cucumber andtomato.
SIT1, SI transporterfrom external solution to corticalcells; SIT2, Si transporterforxylem loading. From Mitani and Ma (2005).
Isolationand characterizationof a gene controlling
Si uptake in rice
To Identify the genes encoding SI transporterIn rice9 a rice mutant (Isil) defective
in SI uptake
has been Isolated, by using Ge tolerance as an index (Ma et al, 2002).
This mutant had a plant
type similar to the wild type except that the leaf blade of Isil remained droopy when Si was(Lsil)supplied.
The Si concentrationof the tops was much lower in the mutant than in the wild type,while that of the roots was similar.
A short-term uptake experiment showed that the Si uptake
by the mutant was significantly
lower than that by the wild type (Fig. 2), while there was nodifference in the uptake of other nutrients such as P and K. Further, Si uptake by the wild-type
rice was inhibitedby metabolic inhibitors
including
NaCN and 2, 4-dinitrophenol
and by low
temperature, whereas Si uptake by Isil was not inhibitedby these agents.
The Si concentrationin the xylem sap of the wild-type
rice was also much higher than that of Isil. These results
suggest that an active transport system for Si uptake is disrupted
in the mutant (Ma et al., 2002).
Analysis ofF2 populations
between Isil and WT showed that roots with high Si uptake and roots
with low Si uptake segregatedat a 3:1 ratio, suggestingthat Isil is a recessive mutant of Si
uptake.

Fig. 2 Uptake of Si by WT rice (cv Oochikara)
and a mutant (lsil)
Twenty-day-old seedlings
were placed in a nutrient solution containing
Si as silicic acid.
Error bars represent±SD (n=3).

defectivein Si uptake.
0.15 (A) and 1.5 mM(B)Fig. 3 Linkage
xylem loading

markers and a gene for Si

relationshipbetween EST-based PCR and microsatellite
on chromosome 2 in rice.

To map the responsible
gene, we performed a bulked segregant
analysisby using both
microsatellite
and EST-based PCR markers in a mapping population
derivedfrom a crossbetween lsil and Kasalath,.
As a result, the gene was mapped to chromosome 2, flanked by
microsatellite
marker RM5303 and EST-based PCR marker E60168(Ma et al., 2004) (Fig. 3).
Fine mapping resulted in the isolationof this gene.
The CDNA was 1409 bp long and the
deduced protein
consistedof 298 residues,
and the program PSORT predicts
that the Lsil
encodes a membrane protein.
Real-time PCR analysis showed that this gene was not induced by
Si and constitutively
expressed mainly in the roots.
To investigate
cellular localizationof Lsil,
Lsil with its promoter was fused with GFP and introduced
into rice.
Microscopic
observation
showed that LSI1 was localized
in endodermis
and exodermis, where Casparian strips exist.
Furthermore, staining with anti-Lsil
polyclonal
antibody revealed the localizationof Lsil in theplasma membrane of the endodermis and exodermis.
References
Epstein E 1994: The anomaly of silicon in plant biology. Proc. Natl Acad. Sci., 91, ll-17
Ma JF, Miyake Y, and Takahashi, E 2001a:
Siliconas a beneficial
element for crop plants. In
Siliconin Agriculture.
Ed. LE Datnoff, GH Snyder, and GH Korndorfer, p. 17-39, Elsevier
Science, Amsterdam
MaJF and Takahashi 2002: Soil, Fertilizer,
and Plant Silicon Research in Japan. Elsevier Science,
Amsterdam.
Ma JF, Tamai K, Ichii M, and Wu K 2002: A rice mutant defectivein active Si uptake.
Plant
Physiol,
130, 2111-2117
Ma JF, Mitani M, Nagao S, Konishi S, Tamai K, Iwashita T and Yano M 2004 1Characterizationof Si uptake system and molecular mapping of Si transporter
gene in rice.
Plant Physiol 136,
3284-3289
Mitani M, Ma J F 2005: Uptake system of siliconin different
plant species. J. Exp. Bot 56,
1255-1261
Savant NK, Snyder GH, and Datnoff LE 1997: Silicon
management and sustainable
rice
production.
Advan. Agron., 58, 151-199
Tamai K and Ma JF 2003: Characterizationof silicon uptake by rice roots.
New Phytol, 158:
431-436
Corresponding
author: Jian Feng MaResearch Institute for Bioresources, OkayamaUniversity, Chuo 2-20-1,
Kurashiki 7 10-0046, Japan. mai@jib.okavama-u.ac.jp; Tel/Fax 086-434-1209

TILLING:

WHERE FUNCTIONAL GENOMICSCONVENTIONAL BREEDING

MEETS

Vanavichit
A1 *., S. Sasoo-7 ,T. Amorn - , S. Ruengphayak-7 , A. Plabpla-7, T. Toojinda-7,
S. Tragoonrung-7
i *Agronomy Department, Faculty of Agriculture,
Kasetsart Univeristy,
Kampangsaen, Nakhon Pathom, 73 140,
Thailand,apichart@dna.kps.ku.ac.th,Tel: 66-34-355192-7Rice Gene Discovery, National Center for Genetic Engineeringand Biotechnology
at Kasetsart University,
ThailandIntroduction
In a new challenging
world, rice has now been the focal point as a medium for alleviating
poverty and a hidden hunger.
Taking one example, a new paradigm for rice breeding
is toimprove overall grain nutritional
status that have been overlooked since the green revolution.
Inthis situation,
genetic diverisityfor grain quality may not be available to drive a successful rice
breeding
program.
Mutation breeding
has been successfully
used to generate new genetic
variations
from any genetic background. However, the limitationof any mutation breeding is thelack of efficient and high throughput
screening.
TILLING, Targeted Induced Local Lesion in
Genome, a new reverse genetics strategy to discover allelicseries of induced mutations in genes
of interest was first developed in Arabidopsis
http://tilling.flicrc.org:9366.
The original TILLING
was induced by ethyl methane sulfonate (EMS) followed by PCR-based screening of coding
regions of interest (Colbert
et al., 2001; McCallum et al., 2000a,b).
A target 1 kb region of acoding fragments are amplified
using 5'-end-labeled
primers on DNA extracted from EMSmutagenized M2plant. A mismatch occurs at the site of the point mutation, which can be detectedusing mismatch-specificendonucleases
such as CELI from celery (Apium graveolens; Yang et al.,2000)
and detectionof cleaved
fluorescentproducts
on denaturing
polyacrylamidegelelectrophoresis(Kulinski
et al., 2000; Oleykowski
et al., 1998). Genetic mutation is a powerfultool that establishes
a direct link between the biochemicalfunction of a gene product and its
phenotypes. This TILLING project allows the identificationof allelic variation in a target gene ina high-throughput
manner. Any useful mutants can readily be used to add new traits to a target
cultivar.
Materials
and Methods
Plant materials
JHN is a non-photosensitive/purple-seedrice cultivar developed for nutrition purposes. Its
grain contains up to 1.5 mg/100 g of Fe, 2.5 mg/100 g ofZn and 12.5% of protein, and highly
enriched in antioxidants,
vitamins B and E. Several mutations observed naturally when grown inlarge production,reflecting its mutable nature of this rice. JHN matures approximately
105 daysand grown to about 65 cm to the panicle top.
Radiation mutagenesis
JHN was irradiatedwith fast neutron at 33 Gy. The total of 400,000 seeds was allocatedinto 26 vacuum aluminum vessels containing approximately
300 g or 15,000 seeds. Each vessel
was then soaked into a radioactive
pond for 30 min and then air-driedfor 1 day before
germination.
The output seeds were irradiatedup to 0.1 Krad as measured by a Giger counter.
The mutagenized populationconsisted of approximately
125,000 Mi and M2.
Chemical mutagenesis

JHN was also treated with 1.0 mM Sodium aside (NaN3) for 8 hr in the dark. This
treatment was selected from pre-testingchemical mutagen that had four treatment: 0.5 mMNaN3
for 2 hr., 0.5 mMNaN3 for 8 hr., 1 mM NaN3 for 2 hr., and 1 mMNaN3 for 8 hr. The
mutagenized population consisted of 8,000 Mi (Maintenance of the M2-3populations).
For each Mi plant, all panicles
were harvested, dried and refrigerated
for long-term
storage. In the M2 generation, a single panicle from each Mi was seeded and thinned out leaving
eight plants for observation.
Genomic DNAextraction
All rice plants were grown as a 96-well-plate
format in the experimental
field. Leaves
from two-week-old plants were randomly collected,
following
the ID and 3D protocols, for theisolationof genomic DNA. The original CTAB extraction procedure (Rogers and Bendich, 1994)
was modified slightly
DNAp ooling strategies
The 3D pool: Plants were grown in a 96-well Falcon plate style. Each plot (plate)consistsof96 hills arranged in 8 rows of 12 Mi plants (or 12 columns of 8 Mi plants). Each sample unit
consisted of 10 plots (plates).
Leaves were excised from two-week old Mi plants were pooled as
three dimentions in 8 rows (120 plants each) and 12 columns (80 plants each), and 10 plates (960plants each). To equalize the contributionof each genotype in the pool, cares were taken to excise
equal amount of leaf tissue from each plant. The Genomic DNA was isolated from each pooledleaf and stored at -20°C freezers as references. In total, there were 4,050 pools representing
the135,000 Mi lines.
The ID pool: For the M2 generation,
leaves were collectedfrom 24 Ml-derived
M2
sublines to increase the probabilityof detecting
mutation in the M2. This ratio was determinedfrom a sensitivity
test using different
mixtures of known mutation in JHN wild type. Fivemixtures of the wild type to amutant were 95:1, 47:1, 23:1, ll:1 and 1:1. Based on the outcomeof this sensitivity
test, the 23:1 ratio was used for developing
genomic pools for the M2
generation.
In total 208 genomic pools were isolated from 40,000 M2 sublines.
Mutation detection
SSCP analysis:
PCR products denatured at 95°C were separated on 8% polyacrylamide
gels.
Allelic strands were dissociatedunder specific temperature profiles conditions
according to theirconformation characteristics.
Gels were stained with silver staining
accordingto the protocol
recommended by the manufacturer.
DHPLC analysis:
Immediately
prior to DHPLC analysis, PCR products were denatured byheating at 96°C for 5 min and then graduallycooling to 25°C over a period of 30 min. The
samples
were kept at 4°C, 3 ul was applied
to a preheatedreversed-phasecolumn(Eclipse,dsDNA
preparative
column; 4.6x75mm; Hewlett Packard). DNA was eluted with thefollowing
gradient consistingof buffer A (0. 1 mol/L triethyammonium
acetate) and buffer B (0. 1
mol/L triethyammonium
acetate containing 250 mL/L acetonitrile): 60% A-40% B for 30s; 50%
A-50%B for 5.5 min; 25%A-75%B
for 10 s; 5%A-95%B
for 1 min; and 60%A-40%B
for1.33 min. The mobile phase was heated to the respective
denaturing temperatures. The gradientstart and end points were adjusted
accordingto the size of the PCR amplicon. Melting
temperatures were analyzed by DHPLC Melt Program that multiple temperatures were tested for
optimal resolution.
Sequencing

Overlapping
PCR strategy was applied to sequence the whole gene. The PCR products
were sequenced from both directions
using ABB77 XL with Big-dye terminator chemistry (ABI).
The fragments were assembled by program on web (http://rice.kps.ku.ac.th/hconnect.html).
The
multiplesequence alignment and clustalW programs were used comparing the assembled
sequences.
Amplified Fragment Length Polymorphism (AFLP)The AFLP proceduce was practiced by following the protocol of VOS et al.(1995)
withsome minor modifications.
The best combinationsof AFLP primers for mutation detection in the
FA population were CT-CAG (72%), CT-CTG (2%) and CT-GCC (2%).

Ph en otyping
Phenotypic data was collected on specific M2 generation while mutation is detected in Mi.
M2 seeds of each Mi plants stored systematicallyand supplied
upon request by collaborators.
Phenotypic
data collectedfrom collaborators
was recorded in a relational
database to facilitate
publicaccess. Characterizationof phenotype in M2 generation, were collectedon database,correlatedwith candidate
gene approach that is the important quality traits. We recorded acharacteristic
phenotypeas 7 major group: awn, rice grain, culm angle (tiller),panicle, glume(hull),
stem, and leaf. In addition,
we can be inspecteda characteristic
phenotypes
by indirect
methods. For example, measurement of phosphorous
content was inspectedby HIP method.
Other phenotypes can be measured with lipid, protein, and antioxidant
content.
HIP (High Inorganic Phosphorous) testing
Example extraction, to measure an each seed grain. Next step, aliquot HCL solution 0.4 M per
10-fold of each seed grain weight content. Seed grain was incubated in HCL solution at 25 °C for
5 hr and grinned seed rice, and then incubates seed was ground at 4 °C for 8 hr. Extraction
solution was taken 20 ul. Put 80 ul ofdH2O in the sample. Added 100 ul of colorimetricreagent(Component:
3 M H2SO4: 2.5 % (w/v) ammonium molybdate :10 % (v/v) ascorbic
acid(incubation
at 4 °C) and dH2O ratio as 1 : 1 : 1 : 2 respectively)
into sample solution. Incubatedmixer solution at 25 °C for 1 hr. Then, compare color of mixer solution with standard solution.
Standard solution is a component of 1 mM K2HPO4 : level 0 = 0.0 ug/ml Pi, level 1 = 0.15 ug/ml
Pi, level 2 = 0.46 ug/ml Pi, level 3 = 0.93 ug/ml Pi and level 4 = 1.39 ug/ml Pi (freshly prepared).
Results

and Discussion

Mutant library construction
To construct a mutant library , JHN wild type was treated with Fast neutron radiation at
33 Gy and NaN3 mutagen at 1 mM. Mi seeds were planted. These pooled seeds (materials
andmethods) were planted on a 96-well-plate
format, and whole leaf rice was collected
between 20 30 days according to 3D method. Genomic DNA was isolated from the rice tissue. M2 seeds werecollected
from individual
Mi. In M2 plant were pooled as ID. Because the limited sensitivityofdetection
method ID adapted pooling was in M2. All mutant lines were represented
by DNA
samples into pools of complexity DNA samples were inspected by PCR-base method, SSCP and
DHPLC strategy. The mutant libraries
consist of 136,000 Ml and corresponding
M2 lines.
Mutation

Screening
Several pooling strategies
screening of the targeted
codingof genomic DNA were implemented
sequence. Three screening methods

to facilitate
for mutation

the rapid
detection

including
DHPLC, SSCP, PAGE, and AGE, were compared. The DHPLC is the most sensitive
mutation detection.
This method of mutation detection
was 2.76 folds more efficientwhengenomic DNA is pooled as 24-fold (1-D) than 96-fold (3-D).
Estimationof Mutation Rate
A mutation rate was estimated by using sequencing in M2 as 1.54 x 10~7 per nucleotide.
Inaddition,frequency of mutation was estimated as 8.04 x 10" 4 per plant basis while frequency ofmutation analyzed by AFLP technique was 17%. When compared frequency of mutation in Fastneutron radiation (FA) populations,
AFLP technique has proven to be an efficient technique for
estimating
frequency of mutation in the rice TILLING project.
The Thai TILLING project is the largest TILLING project so far establishedworld-wide(Table 1). In the first year, at least six target mutant genes were identified
in the Thai TILLING
project (Table 2). This is considered
a good start because all mutants are viable and will play
important roles in understanding
nutritional
blocks in rice breeding. From the breeding point ofview, these productive mutants are invaluablegenetic materials for gene pyramiding
aiming for
improving nutritive values. For examples, crossing between original JHN and low phytic acid, Fe
chelate mutants are underway to improve Fe density and bioavailability
in rice.
TILLING is useful for isolating
QTLs. We demonstrated herein the case of high inorganic
phosphate (HIP) mutants that AFLP tagging in M3 can be used to isolate the responsible
genes.
Two successful
cases for the isolationof mutants were reported for myo-inositol
phosphate synthase (IPS) and ketoacyl-ACP-synthase(KAS III). For the IPS gene, two aminoacids were changed that affect the gene functioning.
For KasIII on the other hand, large numberof single nucleotidechanges had no effects on functioningof Kas III. Therefore,Table 1 Rice mutant resources developedby the Internationaland National

Rice Research Community.

Table 2 Gene specificprimer were designed

for mutation screening

Table 2 Gene specificprimer were designed

for mutation

screening(continued)

Table 3 Types of mutation detectedin the Thai TILLING

project

FUNCTIONALANALYSIS OF THE GENES CONTROLLING
AND PROCESSING QUALITY OF RICEEATING

Takayuki Umemofo1*,Noriaki Aoki1, Tetsuya Horibata2, Naoyoshi Inouchi2, Masahiro Yano3
1 National
Instituteof Crop Science, Tsukuba, Ibaraki 305-85 18, Japan
2 Dept. of Applied Biological
Science, Fukuyama University,
Fukuyama, Hiroshima 729-0292,
Japan
3 National Instituteof Agrobiological
Sciences, Tsukuba, Ibaraki 305-8602,
Japan^Corresponding
author: ume@affrc.go.jp(Tel: +8 1 29 838 8952)

Introduction
In the rice breeding
program, eating quality is the one of the most Important targets.
However, the sensory evaluation of cooked rice quality is time consuming and need a large
amount of rice samples. Therefore, DNA marker-aided breeding Is favorable strategy to select theeating quality.
To perform marker-aided breeding,we have to know the genetic factors that
control eating quality of rice. Since the major component of rice Is starch, variations
In starch
composition,amylose to amylopectin
ratio, or molecular structure of amylose and amylopectin
could affect cooked rice quality (Juliano,
1998). Thus, the genetic factors that control starchvariations
also could control eating quality of rice. In this study, we examined effects of naturalvariations in the three starch synthase genes on the starch properties
and cooked rice quality.
Amylose and amylopectin
are both glucose polymers. Amylose Is basicallya linearmolecule with tiny amount of branched chains. On the other hand, amylopectin
is a highly
branched molecule. Three sorts of enzymes; starch synthase, branching enzyme, and debranching
enzyme are indispensable
to form starch (Fig. 1). Each enzyme has several Isoforms (Hirose andTerao, 2004). Among them, this study deals with granule-bound starch synthase I (GBSSI,
Wx
protein)working in amylose synthesis,
Starch synthase I (SSI) and starch synthase Ha (SSIIa,
Alk) those elongate short chains of amylopectin(Nakamura, 2002). In the later half of this study
will focus on the natural variations In SSIIa of rice to Identify the responsible
SNPs Important for
the enzyme's starch-binding
function and starch properties.

Figure1.

Starch biosynthesis

pathway in rice endosperm

Effectsof starch synthase variations
We used near isogenic lines

on starch properties
and cooked rice quality
for each starch synthase locus (Fig. 2). The threestarchsynthase genes are all on the short arm of chromosome 6. These NILs have chromosomal
segments from an indica cultivar
Kasalathwith a japonica
cultivar
Nipponbare
genetic
background. ^lL(Wxa) produces several times more Wx protein compared to Nipponbare with
Wxhalkie. NIL^/)
has starch synthase I with slightly
stronger activity and higher mobility on
the native-gel containing glycogen. ~NlL(AIk) has tolerance against alkaline solution of potassiumhydroxide. Recently, starch synthase Ha gene was proven to be the alk gene that controls alkali
disintegration
property of rice grain by map-based cloning (Gao et al 2003).

Figure 2. Near isogenic lines for three starch synthase loci
The NILs have indica Kasalathchromosomal segments
Nipponbare genetic background.

(closedrectangle)

withjaponica

Amylose content was measured by gel permeation
chromatography
after starch samples
were debranchedwith isoamylase.
The amylose content of Nipponbare
was 19.9%, while
Kasalath was 31.5%. The ~NIh(Wxa) inherited
the Wx allele from Kasalath and the amylose
content was similar to that of Kasalath (20.1%).
Thus, the natural variation
in *SS7 between
Nipponbare and Kasalath did not largely affect amylose content. The WL{Alk) had slightly
lower
amylose content (17.4%) than Nipponbare.
Using the NILs9 we confirmed that the Wxaallele had
the largest effect on amylose content among the natural variationsin the three starch synthasegenes.
Chain-length
distributionof amylopectin
was analysed by an HPAEC-PAD after theamylopectinbranches
were releasedby isoamylase.
The chain-length
distributionsofamylopectinof Kasalath and NILs were compared that ofNipponbare(data not shown). Kasalath
has less short chain of degree of polymerisation(dp) around 8 and more middle chains dp around
17 compared to Nipponbare.
Very similar
profileswas observed between WL(Alk)andNipponbare.
This means that the differencesin chain distributionof amyiopectin
betweenKasalath and Nipponbare are almost explained by the alk locus or the chromosomal region close
to the alk. The chain distribution
of NIL(FFxa)
was also differedfrom that of Nipponbare.
However, this difference is probably
duo to the differences in temperature during grain filling.
The relative amount of short chains dp around 6 to 13 was increased while that of dp around 20
to 27 was decreased when the temperature during grain-fillingdecreased (Umemoto et al. 1999).
Actually, flowering period of this NIL was about one week later than Nipponbare,and matured
under cooler temperature compared to Nipponbare.
Howdo starch syntiase variations affect cooked rice quality?
Cooked rice ofNILs was evaluated in the stickiness,
softness, and overall preference with
Nipponbare
as the standard just after cooking (Fig. 3). WL(Wxa) with high amylose was lesssticky and had hard texture and overall preference for Japanese testers were very low. These weresimilar to the evaluationof Kasalath. The natural variation
in aSS7 between Nipponbare
andKasalath did not largely affect eating quality judging
from the evaluation of cooked NIL^/).
Interestingly,
Alk variation clearly affected chain-length
distributionof amyiopectin
and alkali
disintegrationof rice grain, however, eating quality of WL(Alk)
did not differ from those of
Nipponbare.
Wealso conducted the evaluation after the cooked rice has been cooled at 5°C over night(Fig. 3). Compared to Nipponbare
after cooling, Kasalath, NIL(ffxa)
and WL(SSf)showedsimilar textures and preference to the results just after cooking. However, the results ofWL(Alk)greatly differed from those of just after cooking. Once cooled NIL(^/i)
was less sticky and had
harder texture, and overall preference was clearly decreased.

Figure 3. Sensory evaluation of NILs
Cooked NILs were evaluated with Nipponbare

as the standard.

Functional
analysis of starch synthase lla (SSIIa, Alk)
It was reported that SSIIa is the gene responsible
for the varietal differencesin chain-length
distributionof amylopectin
and alkali disintegrationof rice grain (Umemoto et al, 2002; Gao etal., 2003). SSIIa in the alkali tolerant cultivars has the function bind to starch granule in vivo, but
SSIIa in alkali susceptible
cultivars
lacked this function even though SSIIa was produced in theendosperm (Umemoto et al., 2004).
Which specific variation in SSIIa gene is critical one for the enzyme and starch properties?
We compared SSIIa sequences available
at that time and found four single nucleotide
polymorphisms
predicting
amino acid substitution(Umemoto and Aoki? 2005). These SNPs were
genotyped with 60 rice cultivars world wide, and five haplotypes
were found. We conducted an
association
study between the SSIIa haplotypes
and the starch binding of SSIIa protein, shortchain ratio of amylopectin,
alkali spreading score, and pasting temperature of rice starch (Fig. 4).
It was very clear that haplotypes
1, 2, 3a were functional haplotypes
producing SSIIa protein thatcan bind to starch granule, had fewer short chains of amylopectin,
more alkali tolerant, and had
higher pasting temperature compared to nonfunctional
haplotypes,
haplotype3b and 4. Then,
which specific SNP is really important? Compared to haplotype
3a, haplotype
3b only differ at
SNP4. Thus, SNP4 is criticalone for the enzyme function and starch properties.
Compared to
haplotype3a, hapotype 4 only differ SNP3. This shows that SNP3 is also a critical
SNP. As aresult, we identified
two responsible
SNPs for the enzyme function and starch properties. These
SNPs in rice SSIIa are probably responsible
for the eating and processing qualities
of rice.

Figure 4. Association
ratio of amylopectin,of the SSIIa haplotypes
with the starch-binding
alkali spreading score, and pasting temperatureof SSIIa protein,of rice starch.

short chain

Conclusion
Using NILs, we have confirmed the effects of natural variationsin the three starchsynthases on starch properties
and cooked rice quality The variation in Wx (GBSSI) gene (Wxaand Wxh) affected amylose content and cooked rice quality both just after cooking and after
cooling.
Effectsof SSI variation
between Nipponbare
and Kasalath on starch and eating
properties
were not clear in this study Currently, we are conducting precise evaluation of the SSI
variation on rice quality using Koshihikari/Kasalath
recombinant lines. Variation in Alk affected

39-the chain-length
distributionof amylopectin
and the eating quality of rice, but only after thecooked rice became cool. Okamoto et al. (2002) revealed that this variation in SSIIa also affected
the hardening speed of rice cakes, one of the important characters in waxy rice processing.
Finally, we have proposed two functional
SNPs for SSIIa and starch properties,
which in turn
alter the eating and processing qualities
of rice.
References
Gao Z, Zeng D, Cui X, Zhou Y, Yan M, Huang D, Li J, Qian Q (2003) Map-based cloning of the
ALK gene, which controls the gelatinization
temperature of rice. Science in China (Series C)
46, 661-668.

Hirose T, Terao T (2004) A comprehensive
expression
analysis of the starch synthase gene family
in rice (Oryza sativa L.). Planta 220, 9-16.
Juliano BO (1998) Varietal impact on rice quality. Cereal Foods World s, 207-222.
Nakamura Y (2002) Towards a Better Understandingof the Metabolic
System for Amylopectin
Biosynthesis
in Plants: Rice Endosperm as a Model Tissue. Plant and Cell Physiology
43,718-725.
Okamoto K, Kobayashi K, Hirasawa H, Umemoto T (2002) Structuraldifferencesin amylopectin
affect waxy rice processing.
Plant Production Science 5, 45-50.
Umemoto T, Nakamura Y, Satoh H, Terashima K (1999) Differences in amylopectin
structure
between two rice varieties
in relation to the effects of temperature during grain filling. Starch
51,58-62.

Umemoto T, Yano M, Satoh H, Shomura A, Nakamura Y (2002) Mapping of a gene responsible
for the differencein amylopectin
structure
between japonica-type
and indica-type
rice
varieties.
Theoreticaland Applied Genetics 104, 1 -8.
Umemoto T, Aoki N, Lin HX, Nakamura Y, Inouchi N, Sato YI, Yano M, Hirabayashi
H,
Maruyama S (2004) Natural variationin rice starch synthase Ha affects enzyme and starchproperties.
Functional Plant Biology 31, 671-684.
Umemoto T, Aoki N (2005) Single-nucleotide
polymorphisms
in rice starch synthase Ila thatalter starch gelatinisationand starch associationof the enzyme. Functional Plant Biology 32,
763-768.

Topic 2
Global and Regional Strategies
for Conservation and Sustainable
Use of Plant Genetic ResourcesINTERNATIONAL PLATFORMS FOR SUSTAINED COLLABORATIONIN PLANT GENETIC RESOURCES: PROSPECTS AND CHALLENGES
Percy E. Sajise
Regional Director
International
Plant Genetic Resources InstituteAsia, Pacific and Oceania (APO)
P O Box 236, UPM Post Office43400 Serdang, Selangor, Malaysia
Tel : 603-8942-3891
Fax : 603-8948-7655
p. sajise@cgiar.
org

Brief Background
Plant genetic resources before the 1980's was considered
as a common good andheritageof humankind and were exchanged freely. The International
Undertaking
on plant
genetic resources (IU) of the Food and Agriculture Organization(FAO) which served as basis forthis paradigm was adopted in 1983 (Hawtin 1999). During this same period, the applicationofIntellectual
Property Protection(IPP) on plant genetic resources was also starting. For example,
in 1961, seven countries in Europe signed an agreement to protect new plant varieties.
This groupbecame recognized as the Union for the Protectionof New Varieties of Plants (UPOV). The
UPOV agreement was revised in 1978 to allow each member state to legislate
its own Plant
Variety Protection
Act (PVPA) that provides protectionof new plant varietiesthat meet therequirements of novelty, distinctiveness,
uniformity and stability.This
was again revised in 1991
and received independent
status from UPOV-1978 because many developing
countries resistedchanges made to the original agreement, demanding a continuation of UPOV-1978. In addition,
there is the Treaty on Trade-Related
Aspects of Intellectual
Property Rights (TRIPS)
under thepurview of the General Agreement on Tariffs and Trade (GATT) which is the most comprensive,
multilateraltreaty which deals with types of IPR with the minor exceptions of breeders rights andutility models or petty patents. In the context of PGRFA, the most important clause of TRIPS is
article 27.3(b)under which "any WTO member may exclude plants and animals other than
micro-organisms and essential biologicalprocesses for the production of plants or animals otherthan non-biologicaland microbial
processes"
from patentability
under TRIPS. However,"members shall provide for the protectionof plant varieties either by patents or by an effective
sui generis system or by any combination thereof "(Bragdon and Downes, 1998).
These conflicting
IPR regimes on PGRFA reached a climax in 1993 when theConvention on Biological
Diversity(CBD) came into force and Agenda 21 was put into place.
Both the CBD and Agenda 21 stress the importance of developing
and strengthening
the capacityof countries to benefit fully from the biologicalresources available to them within their borders.
They also promote access to these resources to others on mutually agreed terms. In return for
gaining access to genetic resources, parties are required to provide access to new technologies,
aswell as training,
information and financial resources, to enable developing
countries to better
conserve and use their biodiversity
and strengthen their capacity to reduce hunger and poverty(Hawtin et al 2000). In the face of these conflicting
international
protocols and platforms onaccess and benefit appropriation
on PGRFA which slowed down the needed exchanges of PGRmaterials, information and technology, FAO in 1994, initiatedan intergovernmental
negotiatingprocess to revise the IU of 1983 and to make it a legallyprovisions of the CBD.

bindingagreement,

harmonized with the

The new and CBD-harmonized version of the IU took shape in the form of theInternational
Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA) which was
ratified in June 2004. The Treaty involves a multilateralsystem to facilitate
access to key genetic
resources with minimal procedural and administrative
costs. The multilateralsystem is intended
to be efficient, effective and transparent. It aims to ease access not only to plant genetic resources
for food and agriculture
but also to information
about those resources, and to share fairly andequitably
any benefits that may arise from the use of these resources (Frison 2005).
Those
benefits go beyond the merely financial
to include information exchange and access to andtransfer of technology, as well as facilitatedaccess to the genetic resources themselves. Under the
benefit sharing mechanism, the "owners" of a commercialized
product that incorporates
materials
from the multilateralsystem will pay a royalty into a speciallydesignatedfund. The payment is
mandatory if the products are not available for further research and breeding, as a result of theapplicationof measures to protect intellectual
property. It is voluntary when the product can be
freely used for breeding and research. While details of the level of royalties, the organization
andgovernance of the fund, and disbursementsfrom it have yet to be agreed, the Treaty clearlyenvisages that benefits will flow primarily to farmers in developing
countries who conserve anduse crop diversity (Frison 2005). Another funding support for the Treaty will be coming from the
Global Crop Diversity Trust (GCDT) which is an independent
global body set up to generate thefunds in perpetuity
to provide support to secure the most important ex situ collectionsof Annex 1
crops of the Treaty. The Establishment
Agreement for the Trust was signed in early 2005 and it is
now in the process of funding the development of crop and regional conservation strategies
aswell as selectively
providingfunds to some important ex situ collections
which require urgent
support in some parts of the world.
Another landmark platform on PGRFA is the Global Plan of Action (GPA) for theconservationand sustainable
use of PGRFA which was adopted during the International
Technical Conference on Plant Genetic Resources in Leipzig,
Germany in June 1996. The
objectivesof the GPA are to ensure the conservationof PGRFA for food security, promotesustainable
use of PGRFA and to promote a fair and equitable sharing of the benefits arising fromthe use of PGRFA. It comprises 20 priority
activities
and is based on the principlethat only
through the use of PGRFA can the social and economic benefits of the conservation of PGR be
realized(Cooper
et al 1998).
Currently,
a monitoringand evaluation
system on theimplementationof the GPA in some selected countries is going on through the development of an
information sharing system on PGRFA. This will be implemented on a global basis in the coming
months.
In summary, the international
platforms for PGRFA has been oscillatingfrom theseresources being considered
as a common heritage in the IU to a mixed platform of mixed
property rights, i.e., protected to common. These changes in the property rights regimes for
PGRFA has been primarily due to both scientificadvancements in the field of molecular biology
especiallyon how traits are transferredto the science of "biologicalengineering"as well as agreater recognitionof the importance that biodiversity
materials
can provide in the face ofdwindlingnatural resources. It has also been a result of declining
public investments in thisbiologicalarea of research and the increasing assumption of investments by the private sector.

The Need for Sustained
PGRFA Collaboration
The continuous evolution of living organisms and ecosystems, manifestingin one form
as the existing biodiversity,
is a basic phenomenon that provides the requirementsof human
existence for today and in the future. Inherent in this process is the exchange of plant genetic
materials through natural or human-mediated processes, which allow not only the use of thegenetic diversity
but also its enhancement which in turn becomes the source of variability
forcrop improvement, as a natural defense mechanisms against genetic vulnerability
and even
providinga buffer for environmental
risks (Council
1972; Brown 1983; Prescott-Allen
and
Prescott-Allen 1990). For example, in 1993, taro blight destroyed 95% of the taro crop in Samoa
where it is a staple crop. The only reason for the recovery of the taro crop in Samoa is theavailabilityof leaf blight resistanttaro varieties
from the Philippines
and Palau which provided
the genetic resources which enabled Samoa to recover the productionof their staple crops. New
threats are constantly
emerging; among the newest is Asian soybean rust, which arrived in the
United States in November 2004. Projected
losses is around US 2B and it is already costingBrazil and China millions of dollars in fungicides
and lost harvests (Prison 2005). Changes in
global climate will require new materials for more adaptable species to biotic and abiotic stresses.
Historical
records are replete with similar examples that befall important crops illustrating
this
important rationale and emphasizing that PGR is a common heritage of humankind (Lebot 1992).
The global interdependenceof countries
in terms of crops for their food economy
was reported
by Palacios(1998).
For
example the following
countries
havebeen dependent
on outside sources ofgenetic
materials
for their food crops;
China 46% to 55%, Colombia,
84% to94%? Republicof Congo, 82% to 94%,
Cuba, 87% to 97%, Germanay, 83% to98%, India, 35% ti 47%,
Iraq, 83% to89%, Nicaragua,
62% to 75%, Nigeria,
46% to 61%, PapuaNew Guinea, 76% to100%, Philippines,
28% to 38%, Rwanda,
80% to 94% and the United States, 84%
to 99%. The same pattern can be seen in
the improvementof individual
crop
species.
For example, with modern rice
varieties
releasedin 15 countries,
thelandraces used in breeding to produce thevarietyoverwhelmingly
comes fromother countries (Prison 2005).

In summary, the agricultureof today has been made possible
through substantial
exchanges of PGRFA in the past until now. It is also a fact that no single country is self-sufficientfor its present and future needs of plant genetic resources in general. Additionally,
centres ofdiversity of plants and animals do not recognize country boundaries and important food crops andanimals are already widely distributed.
It is, therefore,
important that the exchanges of genetic
resources should continue in a more rational and acceptable way, through various international,
national and local platforms to ensure food security while at the same time recognizing
thecreativity
and innovativenessof stakeholders
and the fair and equitable
sharing of benefits forthese stakeholders
and users including the farmers and local communities who are custodians ofthese resources and knowledge system.
Challenges

and Prospectsof Current

International

Platforms

The Convention on Biological
Diversity
and Agenda 21
Article 3 of CBD recognizes the sovereign rights of nations over their biologicalresources.
While the CBD recognizes these rights, it also places certain responsibilities
and obligations
oneach party. The onus for conserving biodiversity
is on the countries themselves. It recommends
use of biologicalresources sustainably
and the equitable
sharing of benefits. Prior informed
consent is a requirement in obtaining access to these materials and the knowledge and technology
associatedwith them. Exchanges of PGRFA can take place under this platform but will beinefficientand less transparent compared with the provisions
in the ITPGRFA. It can take placemost practicallyon bilateralbasis instead of a multilateralsystem of arrangements. Both the CBD
and Agenda 21 stress the importance of developing
and strengthening
the capacity of countries tobenefit fully from the biologicalresources available to them within their borders. CBD offers
perpetual
protection
to the country's biologicalresources not covered by other international
treaties which exempts some species otherwise.
Trade-Related
Property Protection
Platforms
UPOV 1978 and 1991 as a platform offers definite periods of protection to products offormal breeders and has both full exemption (1978 UPOV) and limited exemption (1991 UPOV)
to farmers right. It give strong protection and IPR's over improved PGRFA to formal breeders.
TRIPS, on the other hand, both under the Utility Patent Act and sui generis system,
provide strong IPR protection
to formal breeders and their products of improved PGRFA. It haseither conflicting
or opposite provisions
as well as options for maneuvering with regards toinformal farmer breeders as well as recognizing
community and individuals
ownership over local
PGRFA.

Overall, these platforms provides protection and incentives to formal breeders and others
who produce novel and commerciable plant varieties
but have been criticized
for monopoly
control, lack of recognitionof materials produced by farmers and communities over generations
and its effect in hindering
exchanges of PGRFA for agriculturaldevelopment in general andgreater marginalizationof low input farmers in particular.
The International
Treaty on Plant Genetic Resources for Food and Agriculture(ITPGRFA)
and the Global Plan of Action (GPA)
The ITPGRFA aims to both simplify the procedures for access to and exchange ofgenetic resources, and to ensure that benefits that may accrue from the use of such resources is
equitably
shared through an original multilateralmechanism. The objectivesof the Treaty
parallels
those of the CBD. However, while CBD deals with biodiversity
in general, the Treaty

Idealapplies only to "plant genetic resources for food and agriculture" which is specified in Annex 1 of
the Treaty. The Treaty, through the Material Transfer Agreement (MTA), governs access andbinds recipients
includingthe in trust collectionof almost 600,000
accessions of the Future
Harvest Centres. The ITPGRFA recognizes the most important food and forage crops as acommonheritage which should be exchanged among countries through an agreed arrangement,
the MTA, and just like the CBD, its period of protection
is perpetual.
It offers exemptions toFarmer's and Breeder's varieties and specifies benefit sharing and assurance for access to newtechnology.
Challenges

and Prospects
Today, there exist several international
platforms of cooperation which provides a rangeof property regimes to PGRFA from being acommonheritage to a strictly property protected
regimes (Figure 1). What is needed is to be able
to balance the need for public goods, especiallyfor the most important and basic food and humanwelfare necessity
crops and plant
genetic
resources and the protectionof private rights.
The combinationsof these platforms must be
Figure 1. Current international
platforms in terms of species and spectrum ofable to balance the rights and recognition
toproperty rights regimes
rightsof various stakeholders
especiallytheguardians
and stewardsof these
genetic
resources, i.e., plant breeders,farmer-breeders,traditional
knowledge holders and others. At thispoint in time, these platforms of cooperation has
not even defined what constitutedistinctively
farmer's rights. There are even fewer countries
which have attempted to define and implement
these farmer's rights. The biggest challenge of all
for the global society is to swing the pendulum infavor of granting more easier access on a fair andequitablemanner to more crops and plant genetic
Figure t.
international
platforms in terms of species and spectrum of property
rights regimes
resource needed for basic human needs for foodand healthsecurity
as well as ensuring afunctional ecologicalsystems to provide the needed commonservices to ensure human welfare
such as clean air, water and landscapes/seascapes(Figure 2).

This would mean expanding the list of species
covered by the ITPGRFA while
cautiously"opening up" the provisionsof the CBD on what species can be delegated to theTreaty. This will also mean that the parties to the Treaty must quickly resolve the arrangements
for the MTA and the mechanisms for benefit sharing which are at the heart of the Treaty. The
parties to the Treaty must also resolve the scope of article 12.3d "in the form received from themultilateralsystem". As of now if it is interpreted
to exclude the genes and materials of the genes
in the materials received. In this context, the door to an open access to the most important crops
listed in Annex 1 of the Treaty can be closed as this can be interpreted
that genes and materials

derived from the modification
Treaty received in genebanks.

can be patented

even if it is with crops belonging

to Annex 1 of theConclusion
Biodiversity,
particularly
plant diversity
for food and agriculture,
still offer one of thebest ways of meeting present and future needs of humankind for basic food and nutritional
security as well as ecosystem services. Diversity within and among plant species remains perhaps
as our best insurance that we will be able to continue to meet these basic needs in the future. As
we are continously losing these valuable plant diversity,
there is an urgent need to conserve thembut conserving them alone is nothing without our ability to use it. A food secure future will benecessary for the attainment of global peace and security.
A food secure future for all demands not only that the world's plant heritage is accessible
to all but that all have an equal abiity to use and benefit from it. Global platforms by which thesebasic principles
will be allowed to be nurtured and applied is needed and will probably
be acombination of what international
platforms already exist but should evolve more in terms ofspecific group of crop species and a set of more effective, efficient and transparent multi-lateralsystems of access and benefit sharing.
References
Brown, W.L.1983. Genetic

diversityand genetic

vulnerability-

An appraisal.

Economic Botany

37,4-12.

Council, National Research. 1972. Genetic vulnerabilityof major crops. National Academy of
Sciences. Washington D.C. 1996. Report on the State of the World's Plant Genetic ResourcesInternational
Technical
Conference on Plant Genetic Resources, Leipzig,
Germany, 17-23
June 1996. FAO, Rome, pp 56.
Cooper, H.D., C. Spillane,
I. Kermali and N. Murthi Anishetty.
1998. Harnessing
plant genetic
resources for sustainableagriculture,
PGR Newsletter 1 14: 1-8.
Frison, E. 2005. Agricultural
biodiversity
and sustainabilityof agriculture.
Paper presented
during the International
Symposium on "Human Centered Sustainable
Development"tocommemorate 80th birth anniversary of MS Swaminathan, Chennai, India, 7 August 2005.
Hawtin, G. 1999. Management of plant genetic resources in the CGIAR: problems, prospects andquest for equity. Paper presented
to the World Trade forum, 27-28 August 1999, Berne,
Switzerland.
Hawtin, G, RE. Sajise and V. Ramanatha Rao. 2000. The conservation
and use of plant genetic
resources for a food secure future. Introductory
address to the International
Symposium on
Oil Palm Genetic Resources and Utilization,
8 June 2000, Hilton, KL, Malaysia.
Lebot, V. 1992. Genetic vulnerabilityof Oceania's traditional
crops. Experimental
Agriculture
28:309-323.

Palacio,
XF. 1998. Contribution
to the estimationof countries'
interdependence
in the area ofplant genetic resources. Rep. 7, Rev. l, UN Food and Agriculture
Organizaing
Committee on
Resources, Food and Agriculture.
Rome, Italy.
Prescoot-Allen,
R. and C. Prescott-Allen.
1990. How many plants feed the world. Conservation
Biology 4:365-373.

GENOMIC-BASEDCROP GERMPLASM RESEARCH

Jizeng Jia1 and Qifa Zhang2
1. Key Lab of Crop Germplasm & Biothechnology,
MOA, China; InstituteBeijing,
100081,China
2. National Key Lab of Crop Genetic Improvement; Huazhong Agriculturalof Crop Sciences,
University,

CAAS,

Wuhan, China

Abstract
Plant genomics is one of the fields
in which new developments
emerge in endlessly.
Genomics-based germplasm research, known as"Genoplasmics",
is a new intersecting
discipline
resultingfrom the hybridization
between plant genomics and germplasm research. Genoplasmics
refers to germplasm research at the global genomic level, which include the five topics:(1)
genoplasmics
platform; (2) studies on genetic diversity
and core collections;(3) gene discovery in
germplasm resources; (4) gene cloning;(5) germplasm enhancement. The rise and development ofgenoplasmics
will promote germplasm research to a new stage through determining
structures andfunctions of genes carried in germplasm and making"germplasm banks" worthy of the name of "genebanks'Mhese endeavors will greatly accelerate the establishmentof foundations for agriculture
andother related industries.
A technology strategy of genomics-based
germplasm research has been development, in which,the groundwork is the core collection,
the approach is to develop genomic stocks, and the key target isgene discuvery. This strategy
combined crop genomics, germplasm and crop breeding
together.
Following this research route, we have obtained primary achievements:
1) Establishedcore collection
and mini-core collectionsof rice, wheat and soybean, and investigatedgenetic diversityof germplasm resources for above three crops;2) Establisheda new strategy for genomic stock development, germplasm enhancement and breeding.
By using the strategy and the core collection,
hundreds of recombination(RIL), thousands of nearisogeniclines (NIL),
introgression
lines (IL) were generated. These materials
have become
important germplasm resources in the new era. Some of them have been used as breeding parents,
even as new varieties to be released. The results proved that this strategy is a efficient strategy to
combine genomics, germplasm research and plant breeding.
3) Based on the above mapping population,
hundreds of novel genes/QTLs have been discovered byusing genetic mapping approach.
4) More than forty agronomic important genes have been cloned by using map-based cloning andcomparative genomics. Detection of natural allele variation is on the way.
The above results have proved that this strategy is a high efficient to germplasm research. We
anticipate
that there will be great achievement in the future.

Corresponding
Crop Sciences,author: Jizeng
CAAS, Beijing,

Jia, Key Laboratory of Crop Germplasm & Biotechnology,
100081,Chine, jzjia@mail.caas.net.cn

InstituteofGENETIC DISSECTION
OF RESISTANCE TO RICE BLASTFOR RICE GERMPLASM ENHANCEMENT
Shuichi Fukuoka1, Norikuni
Saka2, Satoru Kudo2, Takehiko Shimizu3, Masahiro Yano, 1 Kazutoshi
Okuno1
1 National
Instituteof Agrobiological
Science (NIAS) Kannondai 2- 1-2, Tsukuba, Ibaraki 305-8602
Japan
2 Aichi Agricultural
Research Center (AARC), Mountainous Region Agricultural
Research Institute,
Inabu, Toyota,
Aichi 441-2513,
Japan
3. Instituteof Society
for Techno-Innovationof Agriculture,
Forestryand Fisheries(STAFF),
Ippaizuka,
Kamiyokoba ,Tsukuba, Ibaraki 305-0854, Japan^Corresponding
author: fukusan@affi-c.go.jp (Tel: +8 1 -29-838-7458)

Introduction
Rice has a great diversitythat is recognized
as morphologicaland physiologicaltraits.
Beneficial
traits found in germplasms have been introduced into desirable
genetic backgroundthrough cross breeding.
Since most agronomic traits are controlled
by multipleloci, genetic
improvement is time consuming especiallywhen introducing the traits from exotic germplasms.
High-density
genetic linkage map and physical map are powerful tools for targeting genes foragronomic traits. DNA marker-based analysis makes us possible to determine genotype at eachlocus that was difficult
in conventional
method. Recently,
complete genome sequence
information ofNipponbare,gene annotation database and CDNAlibraries have been developed as
the tools that will accelerate the genetic studies for practical breeding as well as establishingitsconnection with basic science. These rice genome resources have broadened the use of the rice
genepool.
Rice blast is a destructive
disease of rice worldwide and improvement of resistance toblast is an important breeding objective.
Two types of resistance to blast, complete resistance andfield (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 forcomplete blast resistancehave been identified
in rice (McCouch et al. 1994). In spite of theirsignificant
effect against rice blast, complete resistance genes have been overcome by compatible
races of the pathogen several years after the resistant varieties
with this type of resistancewere
released. Field resistanceis usually incomplete and characterized
by a susceptible
infection type,although it does limit the proportionof diseased leaf area. Field resistanceis considered
to bedurable, but the genetic basis and mechanism is not fully understood. Present study was initiatedto understand genetic control of field resistanceof the germplasm containing high level of field
resistance for enhancement.
Detectionof chromosomal regions conferring field resistanceto rice blast
Japanese upland rice is in the primary gene pool of Asian cultivated rice, being used as asource of the genes for field resistanceto rice blast. Previous studies have suggested that field
resistance in Japanese upland rice is under complex genetic control (Higashi and Saito 1985), andconventional
genetic analysis
did not allow researchers
to identify the genes and theirchromosomal locations for field resistance.
Major part of the resistance in Japanese upland rice
was not used in irrigatedrice varieties,
possibly
due to the close linkage between the gene forfield resistanceand certain undesirable
characteristics.
Quantitative
trait loci (QTL) analysis

using DNA markers allows for improved understandingof the genetics of the traits controlledbymultiple
genes (Tanksley1993).
To determine
the chromosomal regions involved
in field
resistanceto blast in Japanese upland rice, QTL analysis was carried out using 146 F4 lines of across between Japanese
irrigated
rice cultivar
Nipponbare
and the uplandcultivar
Owarihatamochi(Fukuoka and Okuno 200 1). Nipponbare
and Owarihatamochi
have no gene forcomplete resistanceto rice blast, based on the screening using Japanese differential
blast race.
Nipponbare
has a lower level of field resistancethan does Owarihatamochi.
We assessed field
resistancein F4 lines based on the diseased leaf area (DLA). of 40- to 50-day-old plant, scoredusing a rating from 0 (highly resistant)
to 10 (highly susceptible).
The scores for Owarihatamochi
and Nipponbare were 2.8 and 8.0, respectively
The frequency distributionof the field resistancescore in the F4 lines was continuous and ranged from 1 to 10. We used 111 restriction
fragment
length polymorphism(RFLP) and 5 single sequence repeat (SSR) marker loci to detect QTLs byusing software MAPMAKER/QTL ver. 1.0 in a logarithmof the odds (LOD) thresholdof 2.0.
The analysis revealed four QTLs located on three chromosomes (Figure 1). The resistant allele on
chromosome 9 is derived from Nipponbare, while the resistantalleles on chromosomes 4 and 12
come from Owarihatamochi.
The two QTLs on chromosome 4, close to RFLP marker loci G271
(pi2J) and G1 77 (qBR4-2),
explained 45.7% and 29.4% of the phenotypicvariation, respectively,
while the QTLs on chromosome 9 (qBR9-l) and 12 (qBR12-l)
explained
7.9% and 13.7% of thetotal phenotypicvariation, respectively.
All together, the four QTLs explained 66.3% of the total
phenotypicvariation.

Figure 1. Location of the QTLs for field resistance to rice blast in F4 lines
irrigated rice Nipponbare and upland rice Owarihatamochi.

of the cross between

Development of backcrossed
Hues to confirm the effect of putative QTLs
One strategy for analyzing QTL as single Mendelian factors and for characterizing
each QTL isthe development of backcrossed
progeny lines for each QTL by marker-assisted
selection (Yanoand Sasaki, 1997). To confirm the effect of putative QTLs, backcrossed
progeny lines weredeveloped from three F3 plants identified
as having resistant alkies from upland rice based on the
result of QTL analysis. Aichiasahi,
a highly susceptible
cultivar, was used as the recurrent parent.
The same set of DNA markers in QTL analysis was used to select relevant chromosomal regions.
During backcrossand 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 contain just one out of three blast resistancealleles from OwarihatamochL The field
resistance was assessed in 98? 44 and 46 BC2F3 lines forpi21,
qBR4-2 and qBR12-l, respectively.
Wecompared the levels of field resistanceamong the lines with Qwarihatamochi-homozygous
alleles and Aichiasahi-homozygous
alleles. Genotypes at each QTL were estimated based on the
genotype of DNA markers around QILs.Ihe average DLA of the lines with the Owarihatamochi
allele was significantly
higher than those with the Aichiasahi
alleles at all three QTLs (Figure 2).
The difference between two genotypes was largest at the QTL on chromosome 4 and smallest at
the QTL on chromosome 12, in good accordance with the result ofQTL analysis.

Figure 2. Proportionof the decrease of the diseased
compared with those with susceptible
alleles at the three
the average DLA of the lines with the Owarihatamochi
indicated.

leaf area in the plants with resistantalleleQTL for blast field resistance.
The ratio fromallele to those with the Aichiasahi
alleles is

Map-based cloning of field resistancegene,|ii2i
Map-based cloning of target genes is one of the strategies
for identifying
gene. As apreliminary
survey, 82 BC1F3 lines in which pill was segregatedwere subjected
to genetic
linkage analysis to determine the map location of pill. The genotypes at the pill were readily
determined based on the difference in diseased leaf area and its segregation
among plants within
a line. A resistancegene for this QTL, was mapped as a single recessive loci between RFLP
marker loci G271 and G317 at a distance of 5.0 cM and 8.5 cM, respectively(Fukuoka andOkuno 2001). Further analysis using expressed sequence tag (EST) clones developed by the RGP(Rice Genome Research Program, Japan) identified
RFLP marker locus RA3591 between RFLP
marker locus G271 andpi2l,
at a distance of 0.6 cM topill.
To develop DNA markers aroundthe pill locus, PI artificial
chromosome (PAC) clones were screened from a Nipponbare PAC
library
constructedby the RGP using the sequence-tagged-site(STS) for RA3591 and C975,
which is proximal to G31 7. A polymorphism
survey of twelve PAC-end fragments or subclones
by RFLP and SSCP (single strand conformation polymorphism)
analysis identified
eleven DNA

markers around the /?z27 locus. We developed a mapping population consistingof 1014 lines forfine genetic mapping of pi21 and selected
182 recombinants
between RFLP marker locus
RA3591 and G317 for a field resistancetest to determine genotypes at HhQpi21 locus. Linkage
analysis revealed that pi21 is located between the two RFLP marker loci 14T1 and 13S1. The
analysis narrowed downed the pi21 region as 29kb where six genes are predictedin theannotation database. Further genetic analysis using the mapping populationconsisting2703
plants successfully
identified
one predictedgene as candidate gene for pi21. The sequence ofpi21 gene does not have homology with genes of known function but had partial homology withthe genes in sugarcane and sorghum. These observations
imply that field resistance gene/?/27
is anovel type of resistance gene. To characterize field resistance,
histologicalstudy and expression
analysis were carried out by using near-isogenic
line for pi21. The response of the plant cellagainst fungus invasion was different from that in hypersensitive
reaction. The expression ofpi21
gene is constitutive
and the levels of expression
did not change after fungus inoculationin
RT-PCR analysis.
These results provide useful information
for the mechanism of the field
resistance.
Conclusion
Genetic analysis using DNA markers efficiently
identified
the chromosomal regions
associatedwith beneficial
trait under complicatedgenetic control. The genetic analysis using
backcrossed lines are useful to limit the regions for QTLs and identify DNA markers for theirselection.
Characterizationand evaluationof these lines allow us to collectinformationtoeliminate
undesirable
characteristics
as well as for understanding
the mechanism of trait
expression. Map-based cloning of the gene for beneficial
traits has a great impact on not only
understanding
the gene function but also allowing us to screen allelic variation,,enhancing theuse of the wide range of germplasm. Selecting
the lines with beneficial
traits under complicatedgenetic control is difficult
by conventional rice breeding. After selecting the lines with each QTL
in desirable
genetic background,combining them in one plant is the strategy to enhance rice
germplasm. Rice genome resources will accelerate
to discover novel and beneficial
genes andcontribute to efficient use of rice gene pool.
Reference
EzukaA (1972) Field resistance of rice varieties to rice blast disease. Rev Plant Prot Res 5: 1-21
Fukuoka S and K Okuno (2001) QTL analysis and mapping ofpi21,
a recessive gene for field
resistance to rice blast in Japanese upland rice. TheorAppl Genet. 103: 185-190
Higashi T, Saito S. 1985. Linkage group of field resistancegenes upland rice variety Sensho toleaf blast caused by Pyricularia
oryzae CAV.Japan J Breed 35 : 438-448
McCouch SR, Nelson RJ, Tohme J, Zeigler RS (1994) Mapping of blast resistance gene in rice.
In Rice Blast Disease (edt by Zeigler RS, Leong SA, Teng PS) pp167-186,
IRRI, Manila
Tanksley SD 1993 Mapping polygenes. Annual Rev Genet 27 =205-233
Yano M and Sasaki T. 1997. Genetic and molecular dissectionof quantitative
traits in rice. Plant
Mol Biol 35: 145-153

GENOMICS AND BIOINFORMATICS
APPROACHES FOR EFFCTIVEUTILIZATIONAND ENHANCEMENT OF PLANT GENETIC RESOURCES
RDA-NIAB GENEBANK

IN

Yong-Jin Park1'2*, V. Ramanatha Rao2, Jae-Gyun Kwag1, Tae-San Kim1
National
Instituteof Agricultural
Biotechnology,
Suwon 44 1 -707, R. of Korea
2 IPGRI-APO, PO Box 236, UPM Post Office, Serdang 43400, Malaysia*Corresponding

author:y.park@cgiar.org

(Tel:

+603

8942

3891)

Abstract
With the efforts of International
Rice Genome Sequencing
Project (IRGSP),
completesequence
information of whole genome of Oryza sativa L cv. Nippon bare is publicallyavailable
now, which can be
exploited
for allele mining of diverse germplasm to identify useful variants for many important traits such as
resistance to biotic stresses, tolerance of abiotic stresses, enhanced yield and improved quality. Identificationofallelic
series of variants of such genes and their association
with phenotypicor biochemical
characters will
provide a potential tool for crop improvement. The database of DNA stocks and profiles covers 15 crops and19,000 accessions such as 7,500 rice, 5,000 soybean, 600 barley, 500 wheat, 700 mungbean, 600 apple, 500
Vigna species, 500 sesame, 500 perilla, 500 Italian millet, 500 buckwheat, 500 pear, 400 maize, 400 red pepper
and 300 Job's tears accessions. The DNA stock along with tissue bank provides the base for developing
their
DNAprofiles and molecular characterizationin the RDA gene bank. For DNA pro filing of under-utilized
crops(UUCs) such as sesame, perilla,
Job's tears, buckwheat, amaranth, finger millet, mungbean and Chinese
cabbage, around 30-50 marker sets of simple sequence repeats (SSR) have been developed for each crop by the
RDA gene bank. This marker development work is going to be extended to other UUCs including fruit species
such as zinger, several millets, Zoysia, Allium, persimmon, Korean plum, Citrus etc. A web-based software has
also been developed in order to provide DNA profile information
with passport and characterization
data forgermplasm. It includes
several functions
such as facility
to calculate
genetic distances
using standard
algorithms,
generating matrix tables of target accessions and clustering
analysis using the DNA profile data.
This DNA profile database has potential
uses in identificationof duplicate
accessions and for management oflarge germplasm collections.
Using genomic and bioinformatics
tools, the RDAGB attempted a case study on
gene mining of useful traits from the rice accessions of the RDA gene bank. We are targeting
the genes
involved to grain quality of rice for association
analysis between phenotypic and allele variation, more focusedon Korea originated landrace and weedy collection.
For the association
analysis between alleles and amylose
contents in 394 accessions of Korean landrace using the combination of alleles of microsatellite
markers within
the genie regions of GBSS (Granule-Bound
Starch Synthase),
SBE (Starch Branching Enzyme) and SSS(Soluble
Starch Synthase)
was sought with the other physiochemical
properties
includingamylopectincomposition.
In addition, the association
analysis by highly saturated DNA pro filing with microsatelliteand
SNP markers selected and designed from the 82 genes associatedto embryo & endosperm development,
screened from the various database of rice genome including
TIGR and NCBI, is undergoing for increasing
germplasm uses.
Keywords
Gene bank, high-throughput
genotyping,
SSR markers, core collection, allele mining

I. Introduction
Plant genetic resources of agriculture include landraces, wild species, bred lines & varieties and their wild
relatives. The release of new varieties and changing to monoculture of cropping system led drastic replacementof landraces. Rapid industrializationand expansion of civil areas also destroyed habitats of wild species andlandraces of many crops. Therefore, there is an urgent need to collect and conserve landraces and wild species
together with other important artificial
lines and varieties
for future uses in plant breeding
includingbioindustries.
The RDA (Rural Development Administration)
gene bank has 151,287
seed accessions of 1,939
species or sub-species.
About 30,000 accessions are landrace of diverse crops collectedfrom local farms or
farmers' houses before 1986. As for the vegetatively
propagating
crops, the gene bank has 22,625 accessions of
996 species on the fields of 1 5 different research stations or institutes.
With the availabilityof genomic information and new tools of biotechnology,
the development of DNA
stocks of seed & clone germplasm is needed for their characterization
using DNA markers and gene-based usesof germnlasm (Ortiz 1998: Uvoh et al. 2003). The informationof each accession at DNA level may becompounded with the phenotype
data. Recently, the RDA gene bank has started to develop the DNA banksystem and took molecular characterization
for diversity
assessment, development of core collection,
allelemining of useful charactersand gene discovery within germplasm collections.
These biotechnologicalapproaches will provide a better management and uses of germplasm.
II. A new approachfor developing
core sets with maximized
genetic
diversityand minimized
redundancy
using a heuristic
algorithm
in rice (Oryza sativa L.)
Since the development of a concept for core collection by Frankel (1984),
many strategies for sampling anddeveloping
method of core set using cluster analysis have been studied. But skew population
and unequal
diversity
among accessions makes the difficultyin constructing
diverse and representative
core collection.
Tofacilitate
the management and to analyze the genetic resources in RDA gene bank, the method for maximizing
genetic diversity and minimizing genetic redundancy had been studied. As a result, an advanced M strategy, thatuses admissible
heuristic algorithm, was designed. In brief, after designing the classified
matrix according to thedescriptionsof the characters, each accession was stacked into matrix by means of evaluation of A* algorithm.
Admissible
heuristicalgorithm
evaluates the path at each node by the sum of the node number and thecorresponding
value to be filled with the expected accession.
168 accessions were selected from 10,368 characterizedaccessions using 1 1 qualitativeand 27 quantitative
characters by this algorithm.
This core set showed 22% higher genetic diversity value (the results of ShannonWeaver Diversity Index value) than that of the initial collection
due to the diverse constitutes
with minimum
accessions. Also, the heuristiccore set showed the highest diversity
in comparison with core sets developed byusing clustering
analysis and different sampling strategies.
There was significant
difference in coincidence
rate(CR %) between the entire collection
and core sets developed by the method of clustering analysis. But, heuristiccore set showed 100% CR value, which represented a full retention of the entire collection's
characters. But,
even distributionof characters at heuristic core set resulted in the difference from the entire collectionby t-test
and x2-test.
These results showed that Advanced M strategy designed by heuristic
algorithm
provides the efficientsolution for reducing the redundancy and effective representing
character of the collection
with small number ofcore set. Especially,
since enormous SSR DNA profile data in various crops was accumulated in RDA database,
Advanced M strategy will be useful to develop the core set. Although it remains to study the further adaptationof various data like genotyping
data, the advanced M strategy of designing admissible
heuristic method might bean efficient way to retain the genetic diversity
and reducing the redundancy of the entire collection.
And ifcombined with cluster analysis, it maybe offers a good explanation of characteristics
of resources.

Fig 1. Diagrammatic

illustration

for developmentof heuristic

program to select the entriesof core collection.

Fig 2. Status of accession composition of rice core collection
developed heuristic
program by the RDA genebank.
III.

of rice developed

using SSR profile

data and newly

High-throughput
DNA pro filing of Gene bank accession and developmentof SSR markers onneglected/under-utilized
crop germplasm
The database of DNA stocks and profiles
covers 19,000 accessionsof 15 crops. The crops species, which
are currently being subjectedto the DNA pro filing are, rice, soybean, barley, wheat, maize, cowpea, sesame,
perilla,sorghum, mungbean, buckwheat, Italian millet, pear, apple, persimmon, red pepper, Brassica spp. andAllium spp. For providing
DNA profile informationon the web-site, the RDA gene bank developed a new webbased program to analyze genetic similarity
and clusteringof accessions, which are based on DNA profile data.
The digital data of DNA profiles can be visualized
by their fragment size like gel image with different
colors byprimer sets.
Realizing
the importance of many under-utilized
crops for their use as significant
complements to themajor cereals, we have initiateda program to evaluate levels of genetic diversity
within germplasm collectionsof many UUCs by developing
new microsatellite
markers.
In this regard we have developed around 30-50
microsatellite
marker sets for eight different
UUCs namely, Amaranthus, Brassica, fingermillet,
Job's tears,
mungbean, Perilla,jadish
and sesame.
Screeningof microsatellites
from huge sequence data is very laborious
and time consuming. Several
microsatellites
finding
programs were already developed,
but most of them has no function for screening
interrupted
repeat units or inefficientand inconvenient
to use. To overcomethese problems, a new program'ARGGS ' was designed for detecting
microsatellites
includinginterrupted
or split SSRs in cloned sequences andcan also design primers from the flanking
sequences of repeat units for supporting SSR marker development
program.

Fig 3. The current status of high-throughput
DNA profilingwork carried out at RDA-NIAB gene bank. So
far, total 19,000 accessionsof main as well as under-utilized
crops were analyzed with DNA markers(SSRs & AFLPs).

IV. Allele

mining and gene discoveryof rice collection
The association
analysisof rice germplasm using microsatellite
markers associatedwith starchsynthesizing
genes of GBSS, SBE and SSS was successfully
applied for characterizationof grain quality in rice
(Bao et al. 2002).
This analysis
was conducted with 394 Korean landraces.
The original
type of allele
combinationin 3 genes is deduced as (CT)i8
in GBSS, sbe c in SBE and sss c in SSS. The various
recombinations
on these genes had been studied regarding
changes in amylose contents and ADV (Akali
Digestibility
Values). The association
analysis on these genes is undergoingwith the 1,955 rice accessions
including
Korean weedy collection
and selected world collection.
The association
analysis using microsatellite
markers will be extended with 100 SSR makers within 2005 and with expected 500 SSR markers in 2006. The
selection
based on grain quality and disease resistances
for blast and bacterial blight is sought for the Korean
originatedaccessions through core collection
strategy.

Fig 4* The example of association

analysisof the alleles

with amylose/ADV(Akali

Digestibility

Value)

in thenon-waxy accessions of Korean landraces.
The above photos show long-size
grains from 4 different
accessions of Indica type Korean landraces, which are rarely found in Korea with very high amylose contents.
Allele mining is exploitationof DNA sequence of one genotype to isolate useful alleles from related
genotypes. With the efforts of International
Rice Genome Sequencing Project (IRGSP),
complete sequence
informationof whole genome of Oryza sativa L cv. Nippon bare is publicallyavailable
now, which can be
exploited
for allele mining of many important traits including
starch quality. Identificationof allelic series ofvariants of starch biosynthesis
genes and their association
with quality of starch will provide a potential tool forcrop improvement. Single nucleotide
polymorphisms(SNPs) are the most commontype of sequence differences
between alleles. These polymorphisms
could be used as simple genetic markers, which may be identified
withinor in the vicinityof virtually
every gene. There is a great potential
for the use of SNPs in the detection of
associations
between allelic forms of a gene and phenotypes. In this regard, several SNPs have been identified
within different genes responsible
for grain quality in rice including
a granule-bound starch synthase I (GBSS- 1)
or waxy gene, which plays a critical role in the synthesis of amylase in all the cereal crops including rice. Basedon these SNPs, we have developed CAPS/ dCAPS markers and demonstrated their successful utilization
in DNApro filing of different rice accessions.

Fig 5. An example of developmentof SNP- based dCAPS markers and their use in genotyping

Endosperm and embryo mutants with different
genetic backgrounds
were selected after the chemicalorradiation
mutagenesis
using MNU (M-Methyl-N-Nitroso
Urea) and y-ray. So far, mutants composed of 5,800
lines of 'Sindongjin',
837 lines of cIlpum', 293 lines of 'AranghyangchaF,
1,066 lines of 'Vandana' and 314
lines of 'Hyangnambyeo'.

Fig.6. An example for detecting
sequence variations
embryo developmentfrom mutants of rice.

withinthe genes,

responsible

for endosperm

andFor analyzing associationof phenotype to genetic variation in mutants, 82 candidate genes have been
identified
after searching the TIGR database by the criterion of genes responsible
for embryo and endosperm
development including
starch. These genes code for the enzymes related with carbohydrate
synthesis,
starchsynthesis (amylose and amylopectin),enriched proteins during embryo development, and dehydrins related todehydrationof seeds in Rice. Each primer set was designed from the 5' flanking region including
exons andintrons of the selected genes based on rice BAG sequences and verified the location of primer sequences by ePCR in Rice genome sequences.
Wehave to determine the sequence variationsin these genes' and to identify the SNPs or indels within thesequences among mutations. After the designingof markers such as dCAPS and STS, each F2 population will bescreened the relation of phenotypes with genes.

V. Conclusion
A new trial has been conducted to develop an integrated
system for utilizing
informationof theconserved germplasms.
This integrated
system includes
passport
information,
characterizationdata,
GIS information,
web-based core-set development,
DNA profile data and their compounding
data. Thissystem is more focusing on the utilizationof PGRs. The RDAGB is going to provide a processeddataof each crop as a case study for increasing
utilizationof stored germplasm. Mutant accessions
as a new
type of germplasm such as T-DNA or As/ Ds insertional
mutants need managing system for flanking
sequences as passport
data in gene banks. The temporary system for mutant germplasm has alreadybeen developed
for managing 1 0,000 accessionsof mutants.
The development of software program to provide better information on characterization
including DNA
profile data for germplasm users has been attempted through developing core collection and web-based genetic
analysis system. Using genomics and bioinformatics
tools, the RDAGB has initiated program for gene mining of
useful traits from the gene bank accessions. Also, the association
analysis by highly saturated DNA pro filing isplaned during next 5 years. To develop DNA profile databases for under-utilized
crops (UUCs) such as sesame,
Perilla, Job's tears, buckwheat, amaranth, finger millet, mungbean, Chinese cabbage, around 30-50 marker setsof simple sequence repeats for each crops have been developed by the RDAGB now.This marker development
work is going to be extended for other UUCs and fruits such as zinger, several millets, Zoysia, Allium,
persimmon, Korean plum, Citrus etc. Some case study through molecular characterizationin rice collection
provided a new type of database.
The need for capacity building in the RDAGB is to secure properly educated researcheres and to provide
proper training to the staffs on molecular technology and bioinformatics.
The development of a mutant genebank needs hands on experience at functional study. The bioinformatics
may help in providing network systemof gene bank materials for the purpose of biotechnologicalresearch. The RDAGB is preparing for developmentof functional data of the preserved accessions such as DNA micro array analysis including
protein pro filing ofspecific tissues or those expressed during specific conditions such as cold and salt in future.
References
Bao S, Corke H, Sun M 2002. Microsatellites
in starch-synthesizing
genes in relation to starch physiochemical
properties in waxy rice (Oryza sativa L.). TAG 105: 898-905.
Dixit A et al. 2005. Development of polymorphic microsatellite
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Kim YC, Park YJ et al.2004. Screening of Bacterial Leaf Blight Resistance Genes (xa5, xal3, Xa21) using Sequence Tagged Site (STS) Marker in Korean Varieties and Landraces. Korean J. of Plant Resources 7(3): 187194.

Kwon SJ et al. 2005. Isolationand characterizationof microsatellite
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EA, Nkang AE, Eneobong EE 2003. Biotechnology,
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RESEARCH AND CONSERVATION OF THE AZUKI BEAN AND
SOYBEAN CROP COMPLEXES
D. A. Vaughan, A. Kaga, N. Tomooka, Y. Kuroda, T. Isemura
National
InstituteofAgrobiological
Sciences (NIAS)
Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602,
Japan
Corresponding
author: Duncan Vaughan. Kannondai 2- 1-2, Tsukuba 305-8602,
Ibaraki, Japan. Tel:
Fax:8 1-298-38-7408,
Email: duncan@afrrc.go.jp

8 1-298-38-7474,

Abstract
Crop complexes occur where wild, cultivatedand intermediate
forms are sympatric andrepresent the primary gene pool of crops. Crop complexes are the principal
genetic diversity forcrop evolution and breeding. Each crop has a unique crop complex. The conservation of cropcomplexes poses particular
problems as the wild and weedy components are found in ruderal
habitats
subject to frequent, usually unpredictable,
disturbances.
Over the past decade we havebeen studying the azuki bean crop complex and more recently the soybean crop complex. In thispaper three questions are addressed in relation to these two crop complexes, (a) What are thecomponents of the crop complex and where are they found? (b) What genetic variation exists inthe components of the crop complex and what are their genetic relationships?
(c) What are theimplicationsof crop complex research for conservation of the crop complex?
The azuki bean crop complex is distributed
across much of East Asia but wild and weedy
components seem to be most commontoday in Japan. A distinct center of diversity for this cropcomplex exists in the Himalayan region and adjacent areas. While the azuki bean complex
consists of three recognizable
components wild, weedy and cultivated forms many intermediatetypes exist suggesting
continuous variation. The inter and intra-population
diversity of this cropcomplex in Japan are greatest in central parts of Honshu. In this region the various components ofthe crop complex show a high level of genetic diversity and complex patterns of pollen and seed
flow. Currently the components of this crop complex in Japan are well represented
in ex situ
genebank collections.
However, pollen flow during regeneration means that isolation is necessary
to maintain accession genetic integrity.
Overall in natural environments in Japan this cropcomplex is not threatened but populations
in some areas are isolated and thus in danger of local
extinction.
The soybean crop complex is confined to East Asia and Far Eastern Russia Despite the global
importance of soybean little is known about the components of this crop complex particularly
weedy forms. In China weedy forms are reported but no good morphologicalcharacters can beused to accurately distinguishbetween wild and weedy types. In Japan a weedy ecotype ofsoybean has not been recognized although recently hybrid individuals
or small populations
havebeen found. It appears that gene flow among components of the soybean crop complex does occur
but that in natural habitats hybrids quickly revert to wild type habit. Wild soybeans in Japan are
more commonthan wild azuki and they are only locally threatenedsuch as where there aredevelopment projects.
In China, however, the threat to wild/weedy
populations
is high. The
components of the soybean crop complex are well represented in ex situ genebank collections.
Aswith the azuki crop complex regeneration of this crop complex requires attention to the potential
for pollen flow among different accessions.
Introduction
Crop complexes constituteall or part of the primary gene pool of a crop (Harlan and
DeWet, 1971). Components of the primary gene pool can generally form fertile hybrids withgood chromosome pairingand normal gene segregation.
The crop complex is thereforeessentiallyequivalent
to a biological
species.
For most crop complexes there are threemorphologicallyrecognisable
components - the cultigen, the close wild relativesof the cultigen
and weedy forms. Where two or more of these components grow in the same region they exist as
a complex. The importance of crop complexes lies in the fact that most crop evolution occurs
within them and plant breeders can readily use the genes from crop complexes.
In this paper we focus on two important East Asian legumes and their crop complexes theazuki bean crop complex and soybean crop complex. Both azuki bean and soybean belong to thePhaseoleae that includes many hot weather legumes of importance to agriculture.
While they are
phylogeneticallyclosely related they have different chromosome number (azuki bean 2n=2x=22
and soybean 2n=2x=40). Both azuki bean and soybean were domesticatedin East Asia where
they are important for human nutrition.
The azuki bean crop complex
a. What are the components of the crop complex and where are they found?
The azuki bean crop complex consists of the cultigen Vigna angularis var. angularis and wild
form V angularis var. nipponensis and intermediateor hybrid forms between these two varieties.
In addition,a recently described species from the Himalayan region, V nepalensis, is considereda member of the azuki bean complex (Tomooka et al, 2005). There are several other species in
the azuki bean primary gene pool these species do not grow in the same region as V angularis
hence are not part of the azuki bean complex (Vaughan et al., 2005) (Fig. la). The main
production areas of azuki bean are China, Japan, the Korean peninsula, Nepal and Bhutan. In
Japan azuki beans are grown mainly in Hokkaido where the wild form does not occur. In the restof Japan azuki bean is grown primarily on a small scale for home consumption. In Japan a weedy
form has been described(Yamaguchi, 1992). This weedy type seems to form a stable ecotype insome parts of Japan. However, detailed observationsof weedy type plants reveal that there iscontinuous variationfrom the wild type var. nipponensis to the cultigen var. angularis (theauthors, personal observations).
b. What genetic variation
exists in the components of the crop complex and what are their
genetic relationships?
Several studies have been conducted using different
molecular techniques to determine
the genetic relationships
among components of the azuki bean crop complex. Analysis of theazuki bean complex accessions from across its entire range has been performed using AFLP
(Zong et al, 2003) and SSR analyses (Xu et al, in preparation).
These analyses indicatedthatgeographic
origin, primarily,and status wild, weedy or cultivated,secondarily,
are the major
basis for genetic variation. These studies reveal a high level of genetic diversity
in germplasm of
the Himalayan region.
It is not known where azuki bean was first domesticatedor whether it was domesticatedmore than once. The process of domestication
can be deduced from the way in which wild Asian
Vigna are gathered today. Pod picking is rather easy for Asian Vigna since the pods are relatively
large and clustered (Tomooka et al, 2003). In Japan the intermediate(or weedy) type has been
harvested in the recent past (Egawa et al, 1990).
The Vigna angularis complex in Japan
Vigna angularis in Japan forms a true crop complex with three types of natural population
readily recognisable- wild, weedy and mixed or "complex" populations
that are composed ofwild, weedy and intermediatetype plants. Complex populations
tend to cover a larger area thanwild or weedy populations
and sometimes evidence of hybridisation
among members of the
population
can be observed based on variation in plant morphology.
Analyses of germplasm accessions do not provide information on the diversity
withinpopulations
at a single site. Morphologicaland molecular (SSR) studies of many individuals
andpopulations
in Tottori prefecture have enabled the site-to-site
variation in the azuki bean complex
to be dissectedin this prefecture(Miranda-Jonson
et al, in preparation;
Wang el al.? 2004).
Population size, spread, gene cluster (patch) size in populations
and introgression
from cultivatedto wild or complex populations
are influenced by the time of population
establishment
and local
ecology of sites. Thus a wild azuki populationthat established
on a steep landslip
establishedapatch size similar to populations
establishedon non-sloping land for a longer time. These studies
suggest that seed flow is more important than pollen flow in explaining
the genetic diversity
inthis crop complex center of diversity.
c. What are the implicationsof this crop complex research for conservationof the cropcomplex?
Ex situ conservation of the azuki bean complex was reviewed by Tomooka et al (2002).
The wild and weedy relativesof azuki are poorly conserved for most areas except Japan andcollectionsof cultivatedazuki from the Himalayan region are deficient.
Genetic analysis of the azuki bean complex population
types suggests that particularattention
should be placed on in situ conservationof populations
that consist of mixed
populations(complex populations)of wild and weedy types in proximity to the cultigen. These
complex populations
have a higher level of genetic variation than populationsof wild or weedy
azuki bean. To understand the changes occurring in populations
scientificmonitoring both atdifferent times during the year and from year to year is necessary. Monitoring of the immediate
environment of the populationand its general vicinity are important components to observe.
The soybean crop complex
a. What are the components of the soybean crop complex and where are they found?
The taxonomy of the soybean complex has a rather confused history. Two species are widely
recognized the cultigen {Glycine max Merr.) and its presumed wild progenitor
G soja Sieb. &Zucc. (formerly
called G ussuriensis Regal & Maack). In addition,
there is a type of wild
soybean in northern Taiwan, China that has been called G formosana Hosokawa. G formosana
has been show by RAPD analysis to be more diverged from G max than G soja (Thseng et al,2000) and to have a heat soluble protein profile different from Glycine max (Hsieh et al, 2001).
In addition,
to these species an intermediatetype has been named G gracilis that is presumed toresult from hybridization
between G soja and G max(Hymowitz and Newell, 1977). G gracilis
falls within the range of variation of G max based on RAPD polymorphism (Xu and Gai, 2003).
While these four species names have commonly been used for members of the soybean complex
they are one biological
species. Soybean workers have not reached a consensus on the taxonomic
rank or status of these taxa (Fig. lb).
The distributionof wild soybean is restricted
to East Asia and adjacent parts of Russia along
the Amur River. In China G soja is reported from as far west as Tibet (Dong et al, 2001; Lu,2004). Thus the soybean crop complex is only found in this region.
Glycine soja is widelydistributed
across Japan from southern Hokkaido in the north to Kyushu in the south (Shimamoto
et al, 1998). In Japan wild soybeans occur in either naturally or human disturbed habitats. The
natural habitat appears to be river sides. These are disturbed sites as a result offlooding.
Chinese and Korean populationsof intermediate
or weedy soybean occur but it is notclear how commonthey are (Oka, 1983; Lu, 2005). In Japan a weedy type of soybean has notbeen describedbut individual
plants that are presumed hybrids between cultivatedsoybean andwild soybean have been reported (Kuroda et al, 2005a).
The habitatof presumed wild
soybean/soybean
hybrids were with wild soybean populations
that were adjacent to cultivatedsoybean fields.

b. What genetic variation exists in the components of the crop complex and what are theirrelationships?
The present day diversity of and similarity
between wild and landrace soybean germplasm
based on cytoplasmic genome diversity
suggests the Yangtze River valley of southern China iswhere soybean most likely was domesticated(Shimamoto,2001).
Vavilov suggested that thegreatest diversity
for a species can correspond with its center of domestication.
Based on analysisof 15 morphologicaland biochemical
traits in 22,695 accession Zhou et al (1999) proposed a
diffuse area from southwest to northeast China as the likely region of soybean domestication.
It is clear that in commonwith other crops soybean underwent a genetic bottleneck
duringdomestication
as several studies show much higher diversity
in wild soybean than cultivatedsoybean (Xu etal, 2002; Maughan etal, 1995, 1996, Powell etal, 1996).
Compared to other crops of Chinese origin, such as rice, soybean appears to be a relatively
recent domesticate with first dates related to soybean appearing in 1 100 BC literature (Hymowitz
and Newell, 1980).
It is not known how soybean was domesticatedfrom wild soybean,
particularly
how wild soybeans were initiallyharvested. The pods of wild soybeans are small anddifficult to pick suggesting
plants may have been cut or uprooted, piled up and dropped seeds
gathered after the pods shattered. Alternatively
wild soybeans may have been cultivatedas forage
and then dropped seeds around farmhouses gathered. There are many forage types of cultivatedsoybean all having small seeds.
There are no crossing barriers between soybean and wild soybean, G soja. However,recognizableintermediatetypes are rare in Japan. In China they are many reports of intermediatetypes (Oka, 1983; Lu, 2005) and germplasm from China in the USDA soybean collection
arerecognized as intermediatetypes. In experimental
crosses it is very difficult
to obtain progenythat are similar to G max in F2 populations
derived from G maxand G soja crosses (Carter et al,
2004a).

Molecular techniques enable cryptic gene flow from cultivated soybean to wild soybean to be
estimated. Results of SSR analysis of 77 wild populations(616 individuals
deliberately
selectedfor small seed size) and cultivatedsoybean (53 cultivars)
showed that a small percentage ofsamples (about 6%) appeared to have introgressed
genes from cultivatedsoybean. However,variation from population to population
was high. In most populations
outcrossing
could not be
detected due to small population
size, however, 23 populations
had variable outcrossing
rates
some in excess of 10%. These accessions with presumed introgression
from soybean were foundin all regions of Japan where wild soybeans occur. Thus gene flow between cultivatedand wild
soybeans in the field in Japan appears to be occurring. The results of this study suggest
intermediatetypes quickly revert to wild type habit even though retainingsome cultivatedsoybean genome fragments (Kuroda et al, 2005b).
c. What are the implicationsof this crop complex research for conservationof the cropcomplex?
The current status of ex situ conservation of the soybean crop complex has recently beenreviewed (Carter et al, 2004b). This reveals that there are about 156,000 accessions of soybean
and 10,000 accessions of wild soybean in the world's genebank collections.
The maintenance ofthe genetic integrity of wild soybean populations
ex situ is difficult as the plants can grow over alarge area and thus require much land and wide spacing and cross pollination
can occur where
their natural pollinators
are found.
There has been a proposal for in situ conservation of wild soybeans in China (Lui Liang
personal communication).
In Japan wild populations
are very commoneven in heavily built up
areas such as wasteland between newly built roads. In Japan this species is not threatened and is
only likely to suffer from local extinct on small populations.

Conclusions
Azuki bean and soybean crop complexes overlap to a large extent in their distributionin
Asia. Below is a summary of the main similarities
and differences
between these two cropcomplexes.
1.

2.

3.

4.
5.
6.

The azuki bean complex in Japan consists of the cultigen,wild progenitor
and a readily
recognisableweedy type and mixed populationsof wild, weedy and intermediatetypes.
The soybean complex consists of the cultigen and wild populations
but intermediatetypes
are apparently not common.
Azuki bean has complex primary, secondary and tertiary gene pools that seem to reflect arather recent and continuing evolution of the Asian Vigna. Soybean has a simple primarygene pool consistingof the soybean complex only. There is no secondary gene pool andthe tertiary gene pool consists of subgenus Glycine.
Wild azuki bean seems to be less aggressive
than wild soybean since it is rarely found in
highly disturbed
built up areas. Wild soybeans are commonall across Honshu, Japan, but
wild azuki bean are less common than wild soybeans north of the Kansai region(Os aka/Kyoto).
The haploid chromosome number ofazuki bean is ll and that of soybean is 20.
Reported outcrossing
rates among components of both crop complexes while usually very
low includes
a high degree of variation.
Some high levels of outcrossing
have beenreported among members of both crop complexes (>10%).
The process of domestication
for azuki bean can be inferred from present day harvestingof wild Vigna species. The process of domesticationof soybean is unknown.

Acknowledgement
This research was supported
Japanese Ministry of the Environment.

in partby Global

Environment

Research

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Fig.

la. Gene pool system for azuki bean (Vigna angularis)(modifiedfrom Tomooka et at,2002);
lb. Gene pool system for soybean (Glycine max) (based on Hymowitz, 2004)

EXSITU CONSERVATION OF VEGETATIVELY PROPAGATEDGENETIC RESOURCES: PRESENT STATUS AND STRATEGIESFORTROPICAL AND SUB-TROPICAL SPECIES
Hiroko Takagi **and Takao Niino 2
1 Okinawa Subtropical
Station, Japan International
Research Center for Agricultural
2 Genebank, National Instituteof Agrobiological
Sciences, Tsukuba, Japan^Corresponding
author: takagiw@affrc.go.jp(Tel: +8 1 980 88 6301)

Sciences,

Ishigaki,Japan

Introduction
Ex situ conservation means the conservation of components of biologicaldiversity
outsidetheir natural habitats(Engelmann & Engel 2001) and is generally
used for safeguardingcrop
species and their relatives that are at risk of destruction,
replacement,
or genetic deterioration.
Ex situ conservation activities
at each genebank consist of collecting,handling and managementof germplasm, conservation,
regeneration,
characterization/evaluation,
documentation,anddissemination
to users, including
qualantine
procedure. All of these activities,
as routine
operation, are under the responsibilityof curators of genebanks, or breeders holding germplasm
collections.
To implement sustainable
ex situ conservation,
all the activities
need to be welloperatedand managed. For the case of clonallypreserved
species,conservation
techniques/systems
of germplasm itself still remains as a basic problem to be ameliorated.
This paper presents a brief overview of present status and strategies
of conservation
techniques of ex situ collectionsof the species for which conservation in seed form is problematic,
i.e. vegetatively
propagated and non-orthodox seed species.
Present status of ex situ conservationof plant genetic resources
In a broad sense, ex situ conservation of germplasm is a practice that humans used since thebeginningof agriculture.
International
united efforts were initiatedin early 1960s. In 1963,
FAO set internationalguidelines
for the collection,conservation and exchange of germplasm topromote ex situ conservation of crop genetic resources (FAO 1997). Moreover, major advances inplant breedingbrought about the 'Green Revolution'and continuous destructionof naturalenvironments by developments
made deteriorationof plant genetic diversity
as a common
concern worldwide. Great emphasis was placed on global germplasm collecting
during the 1970s
and1980s.
As a result, it is estimatedthat existing
global ex situ collections
contain approximately
6
million accessions at over 1,300 genebanks and germplasm collections.
Of all the accessions in
ex situ collections,some 600,000 are maintained within the Consultative
Group on International
Agricultural
Research (CGIAR) system, and the remaining 5.5 million accessions
are kept innational or regional genebanks (FAO 1997).
Ex situ collectionsof clonally preserved species
Collecting
and conservation activities
focused largely on cereals and some legumes andmost conservation efforts were concentrated on ex situ conservation particulary
seed genebanks.
Approximately
90% of accessions in the ex situ collections
are stored in form of orthodox seeds
under dry condition combined with low temperatures. It is estimated that, worldwide, only 9% i.e.
527,000
accessions
are stored in field genebanks, while fewer than 38,000 accessions
areconserved in vitro (FAO, 1997).

The species which conservationin seed form is problematic
are mainly vegetatively
propagated species and species with reculcitrant
seeds. Perennial species produce small amountof seeds and species with long life cycles can be also categorized
into this group.
Major tropical species of roots and tubers (i.e. sweet potato, potato, cassava, yams, taro),
banana/plantainand some export crops (i.e. rubber, sugarcane, oil palm, coffee, coconut) are
relatively
well collected
and maintained by the CGIAR centers and national programs. For thesecrop species genomic based studies need to be strengthened
to understand genetic diversity,toestablish
core collections
and to organize additional
collecting
to complement the existingcollections.
For tropical fruit and forest tree species, collecting
is much behind and systematiccollecting
should be emphasized. In addition,
land races of minor and underutilized
crops andnon-cultivatedspecies for local use should be given more attention by international
community.
Conservation methods of germplasm collectionsof clonally preserved species
Traditionally,
field genebanks has been the primary conservation
method for the type of
species.
Field genebanks
ensure the maintenance of genetic integrityof each genotypes
immediately
accessible
for characterization,
evaluation, multiplication
and utilization.
However,field collections
are labour intensive, expensive and exposed to biotic and abiotic stresses.
In vitro storage by slow growth method is an important alternativeand commonly used tocomplement field genebanks. Slow growth techniques
allow clonal plant material to be stored
under tissue culture conditions with extended subculture intervals. The techniques
are now welldeveloped and routinely applied to a range of species and across a range of genotypes within
species (Ashmore 1997). The maintenance of in vitro collections
is under reduced growth
conditions(low light intensity,
low temperature, modifications
in media by adding osmotic
inhibitors
or growth retardants etc.) but still labor intensive and only available
for midium-term
conservation.
There is always the risk of losing accessions due to microbial
contaminationorhuman error. Moreover, in vitro material of some species is subject to somaclonal variation andcareful monitoring and investigation
on stored materials are needed.
Pollen storage, true seeds populations
as gene pools, conservationof DNA and in situ or
on-farm conservation can be also used, depending on genetic feature of each crop, but only
limited extent as supplemental
methods. Therefore, development of techniquesand systems forpractical
use of cryopreservation
is keenly desired
for long-term conservationof clonallypreserved species.
Cryopreservation; Current research status and utilization
for plant germplasm
Cryopreservationis the only currently viable method for long-term storage of plant species
for which conservation in seed form is problematic.
Cryopreservation
is a method to preserve
materials
at ultra low temperature,
usually that of liquid
nitrogen(-196°C).
Under thistemperature, biochemical
and most physical processes are arrested. Therefore, the plant materials
can be stored theoreticallyin stable condition
for a unlimited period time and require low costand limited maintenance.
Orthodox seeds or dormant buds go through natural dehydration
process and show tolerance
to dehydration
or freezing. However, most of the materials for cryopreservation(cell suspension,shoot apices, embryos) are in vitro grown and contain high amount of water, thus sensitive tofreeze injuries (Engelmann 2003). Therefore, technology
development of cryopreservationforplant materials,
it has been being the key issue to developmentof artificial
dehydration
techniques
to protect materials from the damages caused by the crystallizationof intracellularwater into ice.

Development of Cryopreservation Techniques
First successful
report on cryopreservation
was reported for winter buds of cold hardy woody
plants in 1960 (Sakai I960), and techniques based on freeze-induced
dehydration
had been main
approach to cryopreservationof plant materials. In the techniques,
freezing tolerance is conferredby the addition of cryoprotectantsand dehydrationof cells by extracellular
freezing, which is
initiatedby ice-inoculationand pursued during slow (-0.3 to -1°C/ min) cooling to -30 to -40°C(Sakai 1960, 1965, Kartha 1985).
In early 90's, some new procedures based on vitrification(Langis et ah 1990, Sakai et ah
1990, Fabre and Dereuddre
1990) were proposedand significant
progress was made incryobiologicalresearch and development of cryopreservationin the past 1 5 years. Vitrification,inthis context, refers as a phase transition
from a liquid into a non-crystalline
solid (glass)while
avoiding
crystallization.
Thus, vitrification
is a freeze-avoidancemechanism that enables
hydrated
cells and tissues to survive in LN (Sakai & Yoshida 1967). In vitrification
based
techniques,
cell dehydrationis performed before plunging
into liquid nitrogen by exposing
materials to i) a concentrated
cryoprotective
media or ii) air dessication
to induce glass transitions
during rapid cooling (Engelmann 2003). Comparing with the classicaltechniques,
these newtechniques
are less complex in operation
and require only basic equipment. Moreover, thetechniques have been successfully
applied for more complex and larger organs (i.e. shoot apices
and embryos) of both temperate and tropical species with higher post thaw survival rate (Sakai et
ah2003)
The following
different
vitrification-based
procedures
are currently
well identified;i)vitrification(osmotic
dehydration),ii) encapsulation-vitrification,i i)dehydration,iv)
encapsuration-dehydration,v) droplet method (Sakai et ah 2003,
Keller and Dreiling
2002,
Panis et ah 2005) and most suitable procedure can be selected depends on species, tissue type andlaboratory conditions
etc. Basically, those procedures are specializedbased on i) how dehydrationis performed (osmotic dehydration
by vitrification
solution or by air-drying)and ii) whether
samples are encapsulated
into alginate
beads or not. Details of these procedures
were wellreviewed in Sakai et ah (2003),
Engelmann (2003), and Engelmann and Takagi (2000).
Key points of successful cyopreservation pro tocols
Among the above mentioned different
procedures,the vitrification
method (including
encapsulation-vitrification)using osmotic dehydration
with a highly concentrated
vitrification
solution was most widely applied procedure to different species. Especially,
the protocols using aglycerol-based
vitrification
solution (7.8 M) named as PVS2 (contains 30%(w/v)
glycerol,15%(w/v) ethylene glycol and 15%(w/v) DMSO ) (Sakai et ah 1990) were well adopted to around200 species or cultivars of temperate and tropical species. Some examples of tropical species arecassava, banana , taro, yams, sweetpotato (Sakai et ah 2003).
Whatever cryogenic method is applied to cultured apices or cells, inducing higher levels of
osmo- and dehydration-tolerance
is the key for successful
cryopreservation.
Preculturewithsucrose or sorbitol enriched medium for 1 to 3 days enhanced tolerance to PVS2 for some species.
The treatment with a mixture of 2M glycerol
and 0.4M sucrose (LS solution)
followingpreculture
with sucrose enriced media enhance the substantial
increase in survival of vitrified
meristems not only in temperate species but also in various tropical species (Sakai et ah 2003).
Beside the investigationof detailed
conditionsof protocols(i.e. appropriate
time andtemperature for LS solution treatment, dehydration
by vitrification
solution etc.), it should be also
noted that optimized
in vitro culture systems are very important for successful cryopreservation.
Especially,a micropropagation
system for producing a large number of uniform and suitable invitro plants makes the production
after cryopreservation
realistic.

of a high level of osmo-tolerance

and vigorous recovery growth

Practical applications of cryopreservation at genebanks
Cryopreservation
techniques for shoot apices and embryos are now well developed. However,practical
application
to conservation at genebanks for large scale operation is still quite limited.
Cryopreservationof vegetatively
propagatedspecies currently operated at genebanks are as
follows but still limited
to temperate species;
mulberry (Japan),apple (USA), European elm(France), grape (USA) as dormant buds, and strawberry (Japan), Japanese horseraddish(Japan),garlic (Korea), potato(Germany,International
Potato Center), pear (USA), grape (USA) as shootapices.
Systematic
approach has been being carried out for Musa germplasm at the transit center,
INIBAP. Cryopreservation
protocolsin banana were developed
for seeds, zygotic embryos,
embryogenic cell suspensions,proliferating
meristem cultures and apical meristems exised fromrooted in vitro plants (Panis & Thinh 2001). Most recently, droplet-vitrification
protocol wasdeveloped for shoot apices to be applied for wider range of germplasm accessions belong toMusaspp. The optimized procedure of droplet-vitrification
protocol was applied to 56 accessions
belonging to eight different genomic groups ofMusa spp. and one Ensete spp., and an average of
52.9% post-thaw regeneration was obtained (Panis et al 2005).
Future research needs in cropreservation
Although remarkable progress in cryobiologicalstudies of plant materials
and technology
development was made in last decade, cryopreservation
as a conservation method for plant
genetic resources is still limited
and cannot be routinely adopted in genebanks in contrast withmicrobial and mammalian culture collections.
Cryopreservation
protocolsfor plant materials
were mostly developed
followinganempirical,
trial-and-error
approach. Even if a good protocol is established,
it cannot be adopted toother materials, since different species, varieties and tissue types tend to show different responses
to cryopreservation
protocol.
To further improve widly applicable
protocol for large scale utilizationof cryopreservation
for germplasm conservation, basic research should be needed to understand the biophysicalandmetabolicprocesses underlying
resistance/sensitivityof plant tissues to cryopreservation.
An
European Commission Research Project titled"Establishingcryopreservation
methods forconserving European plant germplasm (CRYMCEPT project)" was initiated in 2003. The project
involves basic researches on water thermal behaviour, analysis of proteins, sugars, membrane
lipids,polyamines,
oxidative
stress and cytoskeletal
changes. This type of approach could
provide key information to understand the mechanisms involved in cryopreservationof plant
tissues to develop cryopreservation
protocolsin a more rational,
scientificand cost-effective
manner.
Further research is also needed on i) genetic
integrityof cryopreserved
materials,
ii)
development of techniques
for recalcitrant
seeds which cryopreservation
research is much lessadvanced.
Conclusion
This paper provides
brief summary only on present status and problems
conservationfrom the technicaland research aspects for the plant germplasm
preserved.
However, plant genetic resources conservation
is not only developingof ex situ
of clonallythe most

appropriate
and effective technology,
but also developing
strategies
and methods for proper
management systems are also critical. Joint ex situ conservation efforts for better cordinated andsynergetic
activities
between genebanks and botanical gardens at the institutional,
national andinternational
levels are essential.
References
Ashmore, S.E. (1997)
Status report on the development
and applicationof in vitro techniques
for theconservation
and use of plant genetic resources. IPGRI, Rome, Italy. 67 p.
Engelmann, F. and H. Takagi (eds.) (2000)
Cryopreservationof tropicalplant germplasm. Current research
progress and application.
JIRCAS, Tsukuba, Japan/ IPGRI, Rome, Italy. 496 p.
Engelmann, F. and J.M.M. Engels (2001)
Technologies
and strategies
for ex situ conservation.
In: J.M.M.
Engels et al(eds.)
Managing plant genetic diversities.
CAB International,
Wallingford,
UK, pp.
89-103.
Engelmann, F. (2003)
Current research status and utilizationof plant cryopreservation.
Proceedingsof theInternational
Workshop on Cryopreservationof Bio-Genetic
Resources.
National
InstituteofAgricultural
Biotechnology.
Suwon, Republic of Korea.
Fabre, J. and J. Dereuddre (1990)
Encapsulation-dehydration:
A new approach to cryopreservationofSolanum shoot tips. Cryo-Letters
1 1 :413-426.
FAO (1997)
The state of the world's plant genetic resources
for food and agriculture.
Food andAgriculture
Organizationof the United Nations, Rome, Italy.
Kartha, K.K. (ed.) (1985)
Cryopreservationof plant cells and organs. CRC Press, Boca Raton, Florida.
Keller, E.R.J. and M. Dreiling(2002)
Potato cryopreservationin Germany-Using the droplet method forthe establishmentof a new large collection.
Proceedingsof the 25th International
Horticultural
Congress, Tront, Canada.
Langis, R., B. Schnabel-Preikstas,
B.J. Earle and PL. Steponkus (1990)
Cryopreservationof carnation
shoot tips by vitrification.
Cryobiology
276: 658-659.
Panis, B., B. Piette and R. Swennen (2005)
Droplet vitrificationof apical meristems:a cryopreservation
protocol applicable
to all Musaceae. Plant Science 168: 45-55.
Panis, B. and N.T. Thinh (2001)
Cryopreservationof Musa germplasm. INIBAP Technical
Guideline
5,
J.V. Escalantand S. Sharrock(eds.).
International
Network for the Improvement of Banana andPlantain, Montpellier,France.
Sakai, A. (1960)
Survival of twig of woody plants at -196°C. Nature 185:939-394.
Sakai, A. (1965)
Determining
the degree of frost-hardinessin highly hardy plants. Nature 206: 1064-1065.
Sakai, A. (2000)
Developmentof cryopreservation
techniques.
In: Engelmann, F. and H. Takagi (eds.)
Cryopreservationof tropicalplant germplasm. Current research progress and application.
JIRCAS,
Tsukuba, Japan/ IPGRI, Rome, Italy, pp. 1-7.
Sakai, A., D. Hirai and R. Charoensub(2003)
History
and current issues of plant cryopreservation
research. Proceedingsof the International
Workshop on Cryopreservationof Bio-Genetic
Resources.
National Instituteof Agricultural
Biotechnology.
Suwon, Republic of Korea.
Sakai, A., S. Kobayashi
and I. Oiyama (1990)
Cryopreservationof nucellar cells of navel orange {Citrus
sinensis Osb. Var. brasiliensis
Tanaka). Plant Cell Reports 9:30-33.
Sakai, A. and S. Yoshida (1967)
Survivalof plant tissue at super-low temperatures.
VI. Effects ofcooling and rewarming rates on survival. Plant Physiol. 42: 1965-1701.

STRATEGY FOR CONSERVATION AND ENHANCED USE OF
CROP GENETIC RESOURCES
International

C. L. L. Gowda and H. D. Upadhyaya
Crops Research Institutefor the Semi-Arid
Patancheru-502
324, Andhra Pradesh, India

Tropics,

Introduction
The plant genetic resources (PGR) are finite and vulnerable for loss due to introductionofnewcrop cultivars in agriculture,urbanization,and natural hazards, etc. The realizationof thisfact led to the drafting of Convention on Biological
Diversity at the Earth Summit in Rio in 1992.
The PGR contribute enormously towards achieving the Millennium Development Goals of foodsecurity, poverty alleviation,
environmental
protection,and sustainable
development. PGR are
criticalcomponents of crop improvement efforts aimed at increasing
food security - both forshort-term gains as well as for long-term increase in productivity.
Over the years, genebanks havebeen establishedin a number of countries and the number of accessions conserved in genebanks
nowexceeds the six million mark (FAO 1998).
The mission of the Consultative
Group on International
Agricultural
Research (CGIAR) isto achieve sustainable
food security and reduce poverty in developing
countries through researchand developmentin the fields of agriculture,
forestry,
fisheries,
policy,and environment.
Exploration,exchange, and conservation of PGR is one of the main objectivesof the CGIAR.
The International
Crops Research Institute for the Semi-Arid Tropics (ICRISAT - one of the 15
CGIAR centers) has responded
to this need by establishinga Genetic Resources Unit forassembly,
characterization,
evaluation,
maintenance,
conservation,
documentation
anddistributionof germplasm of the sorghum, pearl millet, chickpea, pigeonpea, groundnut, finger
millet, foxtail millet, barnyard millet, kodo millet, little millet and proso millet, and their wild
relatives.
In this paper we present a brief account of (i) ex situ germplasm assembly efforts, (ii)germplasm management in ICRISAT genebank (iii) germplasm utilization
status and (iv) means
to enhance use of germplasm in research and development.
1. Germplasm assembly in the ICRISAT genebank
Since the inception of ICRISAT in 1972, efforts were initiatedto assemble the germplasmof the mandate crops from various research institutes
around the world. The Rockefeller
Foundation
donated 11961
accessionsof sorghum and 2000 accessionsof pearl millet to
ICRISAT. ICRISAT also obtained 2000 accessionsof pearl millet collectedby the Institut
Francaisde recherche
Scientifiquepour le Developmenten Cooperation(ORSTOM)
infrancophone West Africa. A chunk of chickpea and pigeonpea germplasm were the donations by
the (former) Regional Pulse Improvement Project (RPIP). We also acquired over 1,200 chickpea
accessions
from the Arid Lands Agricultural
Development(ALAJD) program in Lebanon.
Similarly,
much of the groundnut germplasm was received from the Indian groundnut research
programs and the USDA.
Newgermplasm of mandate crops was also collected from the priority areas. From 1975
to 2003, a total of213 joint missions were launched in 62 countries securing 33,194 accessions of
ICRISAT mandate crops. Besides genebank activities,ICRISAT also works on the genetic
improvement of the mandate crops. A large number of improved breeding lines, stress resistantsources or the cultivars were developed, and 3905 such lines have been registered in the genebank forfuture utilization.
At present, a collection
the ICRISAT genebank (Table 1).
2. Germplasmof 1 14,870

management in the ICRISAT

accessionsof eleven crops is conservedinGenebank

2.1. Phenotypiccharacterizationand evaluation:
Adequate characterization
for agronomic andmorphologicaltraits is necessary to facilitate
the utilizationof germplasm by researchers.
Toachieve this, germplasm accessions of all the crops were sown in batches over the years andcharacterized
for morphologicaland agronomic traits. Germplasm screening against biotic andabiotic stresses,
and the estimationsof grain food quality were conducted jointlywith various
disciplinaryscientists.
Germplasm sets were evaluated for agronomic performance over locations
jointly
with NARS scientists
in India, Nepal, Thailand, Indonesia, Ethiopia,
and Kenya and more
intensively
with the National Bureau of Plant Genetic Resources (NBPGR), India. The results ofjoint evaluations
have led to better understandingof the germplasm material.
2.2. Regeneration:
Regenerations
were carried out to meet the seed increase requirements of (i)accessions
that had reached a critical level of seed stock/viability;(ii) accessions
required for
medium-term storage (MTS) and/or long-term storage (LTS);
and (iii) germplasm repatriation.
During regeneration,
all possible
efforts
are made to grow the accessions
under healthy
conditions
and following the appropriate
pollination
control measures.
2.3. Conservation:
Germplasm conservation
requires cleaning the seed material, drying tominimal seed moisture content, storing in cool and dry conditions,and regular monitoring of seedhealth during storage. In the ICRISAT genebank, the seeds of the entire collection
are stored in
MTS (4°C, 20-30% RH) in aluminum cans. The germplasm accessions are also conserved in
LTS (-20°C) after packing in vacuum-sealed aluminum foil pouches.
2.4. Safety back up: ICRISAT's
agreement with FAO places the germplasm collections
under
the auspices of FAO, and requires safety back-up for long-term conservation in countries out side
India. We have initiatedefforts conserving
2000 chickpea
at ICARDA (Syria)
and 2006
groundnut and 4580 finger millet accessions at ICRISAT Regional Genbank at Niamey in Niger.
2.5. Documentation:
The vast germplasm data gathered on chickpea and pigeonpea germplasm
has been summarized and presented to the users in the form of catalogs (Pundir et al. 1988,
Remanandan et al. 1988). During the last 15 years, we had a very purposeful collaboration
withthe NBPGR, India, on germplasm exploration,and evaluation at a number of locations,and theoutcome were reviewed and discussed
in a workshop 'Collaboration
on Genetic Resources' heldin 1988 at ICRISAT, Patancheru (ICRISAT
1989). Core and mini-core collectionsof ICRISAT
mandate crops were establishedand the information
was publishedthrough journal articles(Grenier et al. 2001; Bhattacharjee
2000; Upadhyaya and Ortiz 2001; Upadhyaya et al. 2001a;
Upadhyaya et al. 2002; Upadhyaya et al. 2003; Upadhyaya et al. 2005; and Reddy et al. 2005) forthe benefit of fellow research workers. A 'Manual of Genebank Operations and Procedures' has
also been published(Rao and Bramel 2000) documenting
the historyof the collections,proceduresfor germplasm
acquisition,
maintenance,
documentation,conservation,anddistribution.
3. Germplasm utilization

3.1. Germplasm supply to users: ICRISAT Genebank supplies healthy, viable, and geneticallypure seeds of genetic resources to the research workers. Scientists
at ICRISAT are the largest
users of the germplasm followed by the scientists
in India. During 1973 to 2004, we havesupplied over 660,000 seed samples to the scientists
outside ICRISAT representing
143 countries.
3.2. Germplasm repatriation:
The global collections
held at ICRISAT serve an important
purpose in restoration of germplasm to the source countries when national collections
are lost due
to some reasons. For example, we repatriated362 sorghum accessions to Botswana, 1827
sorghum and 922 pearl millet to Cameroon, 1723 sorghum and 931 chickpea to Ethiopia,
838
sorghum, and 332 pigeonpea to Kenya, 1436 and 445 sorghum accessions respectively
to Nigeria
and Somalia and 71 pigeonpea
accessions to Sri Lanka. The ICRISAT germplasm collections
include 44,822 accessions received from or jointly collected with the Indian NARS. On request,
these accessions have been restored to the NBPGR Genebank, New Delhi, India.
3.3. Impact of germplasm supplied
to the NARS, worldwide:
Besides the utilizationofgermplasm in ongoing research at other institutes,
66 germplasm accessions of seven crops fromthe ICRISAT supplied
samples have been directly released as cultivars in 44 countries. These
cultivars have greatly benefited countries where released.
4. Strategies

to enhance germplasm

utilization

ICRISAT genebank has supplied more than 660,000 germplasm samples to users outside
the ICRISAT from 1975 to 2004. This figure could be considered
as satisfactoryuse ofgermplasm. However, the use of basic germplasm in various breeding programs is scanty. We
have adopted the following strategies to enhance utilizationof the germplasm in research:
4.1. Germplasm evaluation for specific traits: Germplasm accessions are evaluated for traits ofagronomic importance such as yellow endosperm trait in sorghum and pearl millet and largeseeded trait in chickpea and groundnut. Based on the emergence of new priorities,
future plans
will be worked out.
4.2. Developing
core collections:
One of the reasons that plant breeders are using less of basic
germplasm in research is the lack of information on traits of economic importance, which often
shows high genotype
x environment interactions
and requires replicatedmultilocational
evaluations.
This is a very costly and resource-demanding
task owing to the large size of the
germplasm collection.
To overcome this, our research now focuses on studying the diversityofgermplasm collection
and developing"core collections,"
which are about 10% of the entire
collection,
but represent almost full diversity of the species. From the germplasm collection in the
ICRISAT genebank, we have already developed core collectionof seven of the ll ICRISAT
related crops.
4.3. Developingmini-core collection:
When the size of entire collection
is large, even a corecollection
size becomes unwieldy for evaluation by breeders. To overcome this, we developed atwo-stage strategy to develop a mini-core collectionthat would consist of 10% accessions of thecore collection(or 1% of entire collection).
(Upadhyaya
and Ortiz 2001). At ICRISAT, we havealready developedmini-core collectionsof chickpea(21 1 accessions)(Upadhyaya
and Ortiz2001),
groundnut (184 accessions)finger millet (65 accessions).

(Upadhyaya

et al. 2002),

pigeonpea(146

accessions)and4.4. Identificationof sources for traits of economic importanceusing core and mini-corecollections:Due to reduced size, the core collection
can be evaluated extensively
to identify
useful parents for crop improvement. By evaluatingcore collectionof chickpea, we identified
new sources of early maturity (28 accessions),
and large seeded kabuli (16 accessions)types. By
evaluating groundnut core collection
we found new sources of early maturity in 21 accessions,and tolerance to low temperature at germinationin 158 accessions(Upadhyaya
et al. 2001b).
Also were found 15 Valencia, 20 Spanish, and 25 Virginia type germplasm lines in groundnut
with high-yield,
good shelling
percentage
and 100-seedweight throughmultilocational
evaluationof the 'Asia region core collection'(Upadhyaya
et al. 2005). These new sources
performed better than or similar to the best control cultivars
for particular
trait (s) but were
diverse from them. We have also identified
10 drought- and five salinity-tolerant
accessions fromthe chickpea
mini-core and 18 diverse accessions
with high water use efficiency
from thegroundnut mini-core collection(Upadhyaya2005) (Figure 1).
4.5. Multilocational
evalutionsof mini-core collections:
The chickpea mini-core was evaluatedat the Indian Instituteof Pulses Research (IIPR), Kanpur, India during the 2002/03
and 2003/04.
IIPR scientists
were particularly
excited with the large-seededkabuli accessions. They selected
12 accessions
for subsequent large plot evaluationand their use in the breeding
program.
Similarly,
scientists
in China, Vietnam, and Thailand have identified
8, 10 and 12 germplasm
lines, respectively
from evaluation of groundnut mini-core in their country.
5. Future outlook•E In future, we will have increased focus on assessment of the germplasm for theirusefulness
for crop improvement. The core and mini-core subsets of the germplasm will
be evaluated at diverse locations to identify trait specific diverse parents.
•E We have initiateddeveloping
composite sets of ICRISAT mandate crops under the
Generation Challenge Program. Phenotypicand genotypic characterizationof these sets
will provide
vast scope of identifying
useful and unique germplasm resources forutilizationin the crop improvement.
•E Search for new and useful crop germplasm and try to secure and conserve them in thegenebank.
•E Weneed to secure new germplasm of wild Arachis from Peru, wild Cicer from Iran andpigeonpea from Myanmar and Uganda to fill the genetic gaps in the collections.
References
Bhattacharjee
R (2000)
Studieson the establishmentof a core collectionof pearl millet(Pennisetum glaucum). Ph D Thesis, CCS Haryana Agricultural
University,
Hisar - 125 004,
India 162 pp.
Convention on Biological
Diversity(CBD)(1992)
Convention
signed by the International
Community during the Earth Summit at Rio de Janeiro, Brazil, 1992.
FAO (1998)
The state of ex-situ conservation. Page 90. in The state of the world's plant genetic
resources for food and agriculture.
FAO, Rome.

Grenier CPJ, Bramel PJ and Hamon P (2001)
Core collectionof the genetic resources ofsorghum: 1. Stratification
based on eco-geographicaldata. Crop Science 41 : 234-240.
ICRISAT(International
Crops ResearchInstitutefor the Semi-Arid
Tropics)(1989)
Collaboration
on Genetic Resources: summary proceedingsof a joint ICRISAT/NBPGR(ICAR)
workshop on germplasm explorationand evaluationin India, 14-15 Nov 1988,
ICRISAT, Patancheru, India. Patancheru 502 324, Andhra Pradesh, India: International
Crops
Research Institute for the Semi-Arid Tropics.
Pundir RPS, Reddy KN and Mengesha MH (1988)
ICRISAT Chickpea
Germplasm Catalog:
Evaluation
and Analysis. Patancheru, A. P. 503 324, India: International
Crops ResearchInstitutefor the Semi-Arid Tropics. 94pp.
Rao NK and Bramel PJ (2000)
Manual of Genebank Operations
and Procedures. Technical
Manual no. 6. Patancheru, A. P. 502 324, India: International
Crops Research Institutefor theSemi-Arid Tropics. 190 pp.
Reddy LJ, Upadhyaya HD, Gowda CLL, and Sube Singh (2005) Development of core collectionin pigeonpea (Cajanus cajan (L) Millsp. Genetic Resources and Crop Evolution (in press).
Remanandan P, Sastry DVSSR and Mengesha MH (1988)
ICRISAT
Pigeonpea
Germplasm
Catalog:
Evaluation
and analysis.
Patancheru,
A. P. 503 324, India:
International
Crops
Research Institutefor the Semi-Arid Tropics. 89 pp.
Upadhyaya HD (2005) Variability
for drought resistance related traits in the mini-core collectionof peanut. Crop Science 45: 1432-1480.
Upadhyaya HD, Bramel PJ, Ortiz R and Sube Singh (2002) Developinga mini core of peanut for
utilizationof genetic resources. Crop Science 42: 2150-2156.
Upadhyaya HD, Bramel PJ and Sube Singh (2001a)
Development of a chickpea core subset using
geographic
distribution
and quantitative
traits. Crop Science 41 : 206-2 10.
Upadhyaya HD, Gowda CLL, Pundir RPS, Reddy V Gopal, and Sube Singh (2005) Developmentof core subset of finger millet germplasm using geographicalorigin and data on 14 morphoagronomic traits. Genetic Resources and Crop Evolution (in press).
Upadhyaya HD, Mallikarjuna
Swamy BP, Kenchana Goudar PV, Kullaiswamy BY and Sube
Singh (2005)
Identificationof diverse
groundnut
germplasm through multienvironment
evaluation of a core collection
for Asia. Field Crops Research 93:293-299.
Upadhyaya HD, Nigam SN and Sube Singh (2001b)
Evaluationof groundnut core collections
toidentify
sources of tolerance
to low temperature
at germination.
Indian J. Plant Genet.
Resources 14:165-167.
Upadhyaya HD and Ortiz R (2001) A mini core subset for capturing
diversityand promoting
utilizationof chickpea genetic resources. Theoreticaland Applied Genetics 102: 1292-1298.
Upadhyaya HD, Ortiz R, Bramel PJ, and Sube Singh (2003)
Developmentof a groundnut corecollection
using taxonomical, geographical,
and morphologicaldescriptors.
Genetic Resources
and Crop Evolution 50: 139-148.

Table 1. Germplasm holdings
Patancheru, December 2004.

in theRajendra

S Paroda

Genebank,

Figure. 1. Dendogram of 1 8 selected drought tolerant germplasm accessions
groundnut based on scores of first 12 principal
components

ICRISAT,and control cultivarsofCOMPLIANCE, ACCOUNTABILITY AND ETHICS FOR
TRUST-BUILDING PARTNERSHIPS IN INTERNATIONALCOLLABORATIVE RESEARCH ON PLANT GENETIC RESOURCES
Plant

Genetic

Makoto Kawase
Resources Laboratory, Genebank, National InstituteofAgrobiological
1-2, Kan'nondai 2, Tsukuba, Ibaraki 305-8602,
JAPAN
kawase@afxrc.go.jp(Tel: +81 29 838 7458)

Sciences

Introduction
Along with the technicaldevelopmenton molecular genetics
and genomics, many new
methods have been applied to evaluation,
genetic analysis and use of plant genetic resources forfood and agriculture(PGRFA).
Different kinds of internaitonal
research collaboration
have
been done and will be done on PGRFA in Asia. In this article, I would like to focus on fieldresearch.
Still, field research is an important one in international
collaboration
on PGRFA:
exploration for ex situ conservation, monitoring for in situ conservation, wild-weed-crop complex
studies, and so on. PGRFA researchers are requested to have trust-building
partnerships
withcompliance, accountability
and ethics in international
collaboration.
Although some scientists
have given warning on public awareness and intellectual
property rights (IRP), there are many
problems to solve, for example, a limitted
number of legal specialists
or patent attorneys on
PGRFA, and shortage of recognitionof the ownership of and benefit sharing from the PGRFA(Watanabe et al., 1998).

Present situationand prospect of cooperation
on PGRFA
Generally speaking, we have a considerable
amount of ex situ collection
in CG centers nad
national gene banks concerning major crops for which the major concerns are how to access andhow to use them developing
new strategies
on allele mining, genomics-based
germplasm
enhancement, utilizationof core collections,and so on rather than obtaining
much moreaccessions as suggested by many researchers.
With regard to minor crops or underutilized
crops,
still we have to consider how to cooperate to collect and conserve them systematically.
We haveto pay much attention to the traditional
knowledge considering
IPR. About wild relativesofcultivatedplants, even in major crops, we have to study to dtermine what should be conserved insitu or on-farm, how to monitor genetic dynamics of populations,and wild-weed-crop complex.
A broad range of studies on PGRFA including academic studies and pragmatic studies will haveto be done, to which international
trust-building
partnerships
will make good contribution.
PGRFA are attractive to various scientists
working in different disciplines
who will promote
cooperation certainly.
There are different types of access to PGRFA (Fig. 1). I dare to focus
on collaborative
field research, an long-establishedactivity on PGRFA for the discussiononmutually beneficial
international
cooperation.
Although exploration is unlikely to be needed for
major crops, it is importantfor minor crops, for wild relatives,
for activities
for in situ
conservation and for studies on wild-weed-crop complex. Field studies on PGRFA based on
taxonomy, genetics, ecology, and ethonobotany provide ex situ conservation, extend knowledge
and idea on plant-human relationships,
and may provide some information on biosafety.
Paradigm

shiftand collaboration

Long before the Convention on Biological
Diversity(CBD), collaborative
field works on
PGRFA have been done on researcher-researcherbases, institute-institutebases, by means ofbilateral
relations
and/or through coordination
initiativesof internationalorganizations.
The
International
Board for Plant Genetic Resources (IBPGR)
was establishedin 1974 to play animportant role to promote and assist the worldwide efforts on PGRFA and reformed to be theInternational
Plant Genetic Resources Institute(IPGRI)in 1991.
The FAO International
Undertaking
on Plant Genetic Resources for Food and Agriculture(IU) was adopted as an
instrument to promote international
harmony in matters regarding
access to PGRFA in 1983,
when PGRFA had been generally considered
as common property of human beings and to be
freely accessible
until effectuationof CBD in 1993.
We have experienced a drastic paradigm
shift on PGRFA, since the CBD assigns sovereign rights to the country of origin and requires that
Prior Informed Consent (PIC) is received
from the country in which access to organisms isrequested.
Cartagena Protocol effectuatedin 2004 is important as a biosafety protocol which is
closely related with PGRFA.
Because of the difficulty
in determining
origins of PGRFA and for their secured accessibility
for crop improvement, amendments of IU had been discussed
in order to be well harmonizedwith CBD, and then, International
Treaty on Plant Genetic Resources for Food and Agriculture(ITPGR)was settled and effectuatedin June 29, 2004 to ensure access to and equitablebenefitsharing for PGRFA.
At present, we understand that Material
Transfer Agreement (MTA) should be settled
whenever PGRFA is provided.
It was a good change that many countries have begun to pay
attention to PGRFA as national heritage.
But, at the same time, it has become more difficult toplan and organize collaborative
field studies internationally.
Without considerationof IPR, we
are not able to access PGRFA.
International
cooperationof Japan on PGRFA and the NIAS Genebank Project
Roughly speaking,overseas field studies of Japan on PGRFA might be divided into two
categories.
One has academic purposes and is done mainly by universitiies
scientists
supportedby MEXT Grant-in-Aid or else, and another has pragmatic purposes, for example, collecting
materials
for crop improvement and is done by agriculturalresearch institutes
like National
InstituteofAgrobiological
Sciences (NIAS).
NIAS operates the Genebank Project as the central
coordinating
institute(center bank) for plant, animal and microorganism genetic resources incooperation with different research institutes(sub-banks)in Japan (Okuno et al. 2004).
Amongvarious kinds of activities
in the Genebank Project, overseas collaborative
exploration
with material transfer and field research cooperation
without material transfer are essentialasinternational
collaboration.
More than 70 missions have been dispatchedoverseas since 1975,of which two thirds are in cooperation with Asian countries (Kawase 2004).
Recently we areenhancing field research cooperation
without material transfer.
The NIAS Genebank Project
has made good collaboration
with IPGRI (IBPGR) on exploration and field research for possible
in situ (on-farm) conservation.
Other activities
on PGRFA as international
cooperationarecollaboration
for gene bank establishment
projects
implemented
through Japan International
Cooperation
Agency (JICA) in Sri Lanka, Chile, Pakistan and Myanmar. Unfortunately
thiskind of cooperation
is not implemented at present.
During the JICA project term or even after
that, NIAS Genebank Project has been positively
engaged in collaborative
explorations
togetherwith other cooperation on PGRFA activities.

Fig. 1. A schematic

drawing of possible

typesof access to PGRFA under CBD.

Trust-building
relationships
in collaboration
on PGRFA
Considering
the values ofPGRFA not only for breeding purpose but also their multiple values
for people and huge related research fields, all scientists,
gene bank staff, relevant administrative
officials
and farmers should pay much attention on compliance, accountability,
ethics as well as
serious considerationof intellectual
property right for trust-building
relationships
for fruitfulinternational
cooperation.
For example, I would like to take up collaborative
field studies for discussion
among various
kinds of access of PGRFA. Based on the understandingof potentialof, the ownership of andbenefit sharing from PGRFA, certain documentation
should be prepared
for internationaltransaction in the movement of PGRFA. In prior to collaborative
field study, we have to reachmutual agreement such as the Memorandum of Agreement (MOA), Memorandum ofUnderstanding(MOU), and Germplasm Acquisition
Agreement (GAA), although it often takes
time to reach the agreement.
When the collectedmaterials are to be transferredoverseas, a material transfer agreement(MTA) should be settled following
the rules of the country. Export permit and quarantine
procedures are also needed.
After the field study, all passport data, characterization
data, andevaluation data on the PGRFA should be shared by both parties.
It is cardinal to point out thatfurther considerationof the harmonizationand governance of the international
discussion
as thereare new developments also by the involvement of Trade-Related
Aspects of Intellectual
Property
Rights (TRIPS)
on WTO and the World Intellectual
Property Organization(WIPO) on thematerials and knowledge derived from PGRFA.
Conclusion
I would like to propose more close international
collaboration
on PGRFA particularlyin
Asian countries.
We will have to start considerationand discussions
about feasibilityofresponsibility
sharing of ex situ conservationin Asia although it may be difficult
to realize
immediately
I believe that fruitful collaborative
field study between countries is a good firststep, and we will have to make sure of compliance,accountability,and ethics through thecollaboration.
particularly

The NIAS Genebank
with other countries.

Projectof Japanwishescontinuously

to cooperate

References
Kawase M (2004) International
cooperation
by Japan on conservation
and utilization
agricultural
plant genetic resources. Farming Japan 38: 24-3 1.
Okuno K, K Shirata, T Niino, M Kawase (2005) Plant genetic resources in Japan: platforms
destinations
to conserve and utilize plant genetic diversity.
JARQ 39: 23 1-237.
Watanabe K, V Ramanatha Rao, M Iwanaga (1998) International
trends on the conservation
use of plant genetic resources. Plant Biotechnology
15: 1 15-122.

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