Proceedings ofInternational

Genetic Resources
Workshop on
the genus oryza
24-26 September 2003
Tsukuba, Japan

National

Instituteof Agrobiological

Sciences

ProceedingsofInternational

Genetic Resources Workshop on the genus Oryza

24-26 September 2003
AFFRC-Tsukub a Office
Tsukuba, Japan
Editedby
S. Nakayama, N. Tomooka, M. Hisada, D. A. Vaughan and K. Kadowaki
Organized

by
J Kunsaki,

Organizing

K. Okuno and T. Obata

Committee
Obata, Taro, Chairperson
Kunsaki, Jun-ichi
Miyazaki, Shoji (-2003.03)
Okuno, Kazutoshi (2003.06-)
Nakamoto, Yasutoshi

S ecretariatKadowaki, Ko-ichi, Head
Hisada, Masahiko
Nakayama, Shigeki
Tomooka, Norihiko
Vaughan, Duncan A.

PrintedinJapan by Asahi Printing

Publishedby
National

Institute

ISBN4-9315 ll-09-0
September

24, 2003

Co., Ltd, Tsukuba, Japanof Agrobiological

Sciences,

Tsukuba, Japan

CONTENTS
Proceedingsof oral presentations(In alphabeticalorder by author)

Distribution

and diversificationof Oryza species

in Latin

America

M.Akimoto, Y.Shimamoto and H. Monshima
QTL analysis

for several

cultivatedrice species

agronomic

1

charactersusing

four BC2 populations

between wild and

T. Ishn7Comparative

cytogenomics

and physicalmapping

S. A. Jackson,

P. SanMignel,

J G. Walling,

B. Hass Jacobus and R. M. Westerman

Genetic

potentialof wild

speciesof the genus Oryza and their

K. K. Jena, H.-g. Hwang, M.-h Lee, D. S. Brarandand its use for the study

N. Kurata, T. Miyabayashi,

M. Eiguchi,

escape and its biosafety

B.-R. Lu, S. Zhipingand
Cytologicaldiversity

related11

use in rice improvement

D. / Mackill

Wild rice core collection

Transgenein the Oryzeae

17of gene diversity

Y. Yamazaki, H. Monshima and Y. Harushima

21

concerns in rice (Oryza)

J Rong25

of FISH loci of a tandem repeat

DNA sequence in Oryza sativa

and its

speciesS. Nakayama
Phylogenetic

analysis

nucleotide
T. Nishikawa,
Originof Oryza speciessequences

sequencefrom the genomes of cytoplasmic

repeats

and its flanking

organelle
35

and wild rice with AA genome: Based on the interspersion

C Cheng, R. Motohashi,

The complete

based on simple

D. A. Vanghan and K.-i Kadowaki

of cultivated

H. Ohtsubo,

T. Sasaki,

31

Nipponbare

J Wu, N. Namiki

S. Tsuchimoto,genome sequence
and T. Matsumoto

patterns

Y. Fnkuta and E. Ohtsnbo

as a standard

of SINEs39

for Oryza genome research
45Semi-sterile
perennial
Y-I Sato, I Nakamiirawild rice {Oryza rufipogon)
and Y Kuroda

as the progenitor

oi japonica

cultivar
47

Genus, genomes and species

what do they mean in relation

D. A. Vaitghan, A. Shcherban,

N. Bauttsta,

to Oryza7

P. L. Sanchez, G. Jonson-Miranda,

T. Isemiira, A. Kaga and N.

Tomooka51

The Oryza map alignment

project

Toward a closed experimental

system for the genus Oryza

R. A. Wing, S. Jackson and L. Stein
Cytogeneticalstudies59

on alien chromosome addition

chromosomes of O. pnnctata

lines

in rice (Oryza

sativa L.), carrying

extra

Kotschy

H. Yasiu and N. Iwata
Proceedingsof poster(In alphabetical
Genetic

presentations

order by author)

relationships

N. S. Bantista,
Biased61

among the three diploid

A. Kaga, A. H. M. Jayasuriya,

distributionof pyrimidine-rice

CC genome speciesof Oryza

N. Tomooka, D. A. Vaughan

microsatelhtes

in rice and Arabidopsis

S. Fujimori, T. Washio, K. Higo, Y. Ohtomo, K. Murakami, K. Matsubara,
Hayashizaki,
S. Kikuchi, M. Tomita
Mapping
Griffof a new resistanceto cultivatedrice,gene for green rice leafhopper
Oryza sativa

K. Doi, A. Yoshimura and H. Yasui

Variationin the spacer

Y.Habu, F. Taguchi-Shiobara,

introgressed

fulllengthf Kawai, P. Carninci,

cDNAs
Y.
71

from Oryza rufipogon

L.

D. Fujita,

length67

73

of the ribosomal

RNA gene cluster

in Oryza sativa

S. Ando, S. Kamachi, T. Yamada, T. Hagio, Y. Tabet, M. Yano, T. Matsumoto

and T. Sasaki
Development

77
of DNA markers close to the wheat Wx-Bl locus using

G. Ishikazva,

M. Saito, P. Vrinten

and T. Nakamura

Rice growth

imagingsystemfor phenotypic

T. Tanabata,T. Shinomura

functional

rice genomic

sequences79

analysisfrom middle

seedling

to matureplant
T. Ishizuka,

81

Large-scale

analysisof alternative

splicing

regulation

using

full-length

H. Ito, T. Washio, K. Higo, Y. Ohtomo, K. Murakami, K. Matsubara,
S. Kikuchi
Evidence

CDNA of Oryza sativa

J Kazvai, P. Carmnci, Y. Hayashizaki,

and M. Tomita

for tissue-specific85transcription

regulated

N. Kitagawa, T. Washio, S. Kosngi, T. Yamashtta,T. Sunako, K. Murakami, K. Matsubara,by alternative

first

G. Kashima, K. Higashi,exons in Oryza sativa
K. Higo, K. Satoh, Y. Ohtomo,

J Kawai, P. Carmnci, Y. Hayashizaki,

S. Kikuchi

and M.

Tomita87

Differential

heterosis

reproductive

in a natural

strategy

populationof Asian wild

rice (Oryza

rufrpogon)

due toand edge effectY. Kuroda, H Urairong and Y-I Sato
Crossing

barrier

K. Matsubara,

within89

the primary

genepoolof rice uncoveredby means of moleculardissection

M. Ikenaga and Y. Sano

Genomic differentiationin rice using

rice and a landrace

recombinant

in the northern-mostK. Omshi, N. Ishigoh-Oka,
Identification93
inbredregionlines between

an annual type of wild

for rice cultivation

K. Takagi, Y Honuchi, W.E. Hagiwara, Y. Kishima and Y. Sanoand mappingof seed shattering

genes using

introgression

lines

95

from wild ricespeciesP. L. Sanchez, T. Kurakazu, C Hirata, Sobrizal
Identificationand mapping

P. L Sanchez, Sobrizal,
Screening

genes in rice using

gene of QTL influencing

J Yazaki, M. Ishikawa,

the quantitative

Oryza sativa

Dual origin
perennial

trait

loci responsible

L. and O. rufipogon

Y. Uga, Y. Fukuta,

Oryza glumaepatula

introgression

lines

tissue101
cultureF. Fujn, K. Shimbo,

Ohta, Y. Sato, S. Honda, K Yamamoto, K. Sakata,
Mapping

97

K. Ikeda, H. Yasui and A. Yoshimura

for a candidate

F. Shwbara-Taguchi,

of headhng

and A. Yoshimura

trait

Z. Shunatani,

T. Sasaki, N. Kishimotofor differencesusing

in floral

rice DNA microarray

Y. Nagata, A. Hashimoto,and S. Kikuchi
morphology

105
between

Griff

H. Cai, H. Iwata, R. Ohsawa, H. Monshima and T. Fujnnuraof the cultivatedrice basedon molecularstrains

rice species,

Oryza nivara and O. rufipogonof wild

T.

S. Yamanaka, I Nakamura, K N. Watanabe and Y-I Satomarkers of newly collected107
annualand
1 ll

ORAL PRESENTATIONS
Proceedings ofInternational

Genetic Resources
Workshop on
the genus oryza
24-26 September 2003
Tsukuba, Japan

Distribution

and diversification

Akimoto, Masahiro L Shimamoto,1 Obihiro

Universityof Agriculture

akimoto@obihiro.ac.jp.

Yoshiya2,

Abashin,Japan.

243-0034

Atsugi,

Keywords

O. glumaepatula,

and Veterinary

3 Tokyo University

Oryza glumaepatula,is diploid080-8555,

of Agriculture,

Facultyof Biomdustry,of Agriculture,

FacultyO. latifolia,

Japan,of Agriculture,

Latin America

O. alta, O. grandiglumis
and sub-tropicalwith CCDD genome. They are consideredhowever, their life-history

understoodsince the number of accessions

be insufficient for elucidating
diversitywide range of distribution

traitsand systematic

for wild rice species

relationshipsof respective

species

species

speciesSpecialthe rest three

genetic

have not been welland seemed to

In this study, we dealt

in Latin America using accessions

areas of respective
withinas important

are

1) 0.

rice, while,

so far examined was quite limited

entire variation

is differentiatedand O. latifolia,

Latin America (Fig.

having the same AA genome with cultivatedresources,

0. glumaepatula

ObihiroO. alta, O. grandiglumis,

in the marshy area of tropical

species are tetraploidwith genetic

Medicine,

Japan.

Four wild rice species,

glumaepatulain Latin America

Monshima, Hiroko3

2. Tokyo University099-2493

distributedof Oryza species

referencecollected

from

was paid for whether

or not and how it is geneticallyrelatedtoother AA genome wild species

in the other continents.

Fig. 1

Distributionof wild

rice in Latin America. Solidline indicates

distributionof O. glumaepatula.
line indicatesarea

Dotteddistribution

areaof CCDD genome species,
O. grandiglumis
identified

Diversification

and bio-systematics

Wegrew 57 accessions

O. barthu

togetherO. meridionalis

from Africa to examine the variability

nuclear DNA markers, and 1 0 chloroplast

Polymorphisms

at quantitative

markers were examined respectively

has not been

in Central

America.

of O. glumaepatulaof O. glumaepatularufipogon from Asia and Oceania,

butwith related

AA genome wild species,

from Oceania, and O. longistaminata
of 22 quantitatively

measurableO.
andtraits,48

DNA markers

traits,by principal

nuclear DNA markers, and chloroplastcomponent analysis

(PCA)

DNA

Subsequently,

Euclidean

distancefor each pair of accessions

by using the principal
analyses

was estimatedcomponent scores of the accessions

to constructobtainedin respective

matrices

PCA

A dendrogram using the UPGMA method was generated by each proximity
The dendrogram

accessions

were divided

for quantitativewas clusteredas perennial

rice. O. glumaepatula-IIrespectively,

and differed

be well explained

traitsanalysiswith perennial

accessions

to III, respectively)

and III were placed

overlap with each other

distributions

O. glumaepatula-I

consisted

into independent

Distribution(Fig.

tended to be separated

glumaepatulacollected
accessions

from the Pantanal

constructedon the polymorphisms

from each other forming respectivefrom Central
were
swamp msouth

at nuclear DNA, all species
clusters,

has its unique nuclear DNA type. Within the cluster

Accordingcan1)

In the dendrogramof the accessions

accessions

area of three groups did notof the accessions

from the Amazon basm, and O. glumaepatula-lll

west Brazil

clusters,of O. glumaepatula

America and north region of South America, and O. glumaepatula-ll
collected

O.

of O. rufipogon and can be regarded

from any other taxa. Subdivisionby their geographicalmatrix.

showed that O. glumaepatulainto three groups (O. glurnaepatula-l

glumaepatula-I

species

proximitythat eachof O. glumaepatula,seemed to represent

their geographicaldistributions

to the dendrogramfor chloroplast

DNA analysis,

were divided

indicating

into two groups (O. glumaepatula-Cl

accessions
and C2)

arrangement

of O.

Cl were closelyrelated

to O. barthu

C2 formed an independent

cluster,though it contained

mutations whichwere not found among the accessionsof Cl but the accessionsof O. longistaminata.

chloroplasttypes of O. glumaepatulaseem to have been derived

from different

associatedwith two African species. Accessions

quantitative

traits

While,

analysisexclusively

carriedbelonging

DNA, respectively.

of O. glumaepatula-lll

O. glumaepatulaand mtra-specificproved to be a variable

The heterogeneity

origin of O. glumaepatulain chloroplast

is not monopyletic

species

DNA found in this species

characterizedthat plants with different

in the processof their geographic

glumaepatula-l

and perennial

However, no molecular evidence

and most probably,chloroplast

expansions
accessions

such similarity

inferredwasthat theNuclear DNA of O. glumaepatulaby its own marker genotype and differentiatedindicating

differentiationand there may have been several migrations

wild rice between the New world and other continents

traitsand II mboth Cl and C2 were found among the accessions

well recognized.

species,lineages

to O. glumaepatula-l

C l and C2 type chloroplast

Two

DNA might introgressof O. rufipogon showed a similarity

is the result

wasfrom that of other AA genome

in Latin America. It is certain

was obtainedofin the present

with each otherthat O.
in quantitative

study to support

this viewof convergence but not of commonancestry.

Diversification

and bio-systematics

of CCDD genome species

O. grandiglumis

has well developedsterilelemma, which is the same length as lemma andpalea or sometimes longer than them, and by this character
species from two others
spikelet

length;characters

Key charactersdividing

O. latifolia

leaves are narrower and spikelets

show an amount of variation within

we can easily

distinguish

thisfrom O. aha are leaf width andare longer in O. latifolia
each species

than O. aha. Theseand sometimes species distinction

is obscure.
Three CCDD genome species are considered(Kiefer-Meyer

et al. 1995)

We examined variability

SSCP method using accessions
geographicaldistributions

taxonomy and systematic

relationships

In Latin America, the destruction
Introgression
1999)

of cultivarsof rice is limited
naturalIt remains

theirvagueness onmaterials

in studying

occurringas in Asia.

in Asia (Akimotoin this area. It is expectedcondition

in Asian wild rice (Morishimaof wild rice.

seemed to representof wild rice is not so serious

into wild rice, which is frequently

America is one of the valuable
phylogenetics

differentiation.

DNA byamong CCDD genome species

in Latin America growing under diverse
rice, that are vanishing

Polymorphisms

speciesof the habitats

is also rare, since cultivation

close with each otherat four regions of chloroplastof three species

rather than their

to be geneticallyconserves primitive

et althat wild ricefeatureof wild1994).

In this sense, wild rice in Latin

ecology,

population

genetics

andAcknowledgements
The authors wish to express the appreciation

to Dr. A. Ando, University

late Dr. P. S. Martins

throughout

this study.

H. (1 999)

The extinction

for helpful

suggestionsof Sao Paulo and theReferences
Akimoto,M.5Shimamoto,Y. and Morishima,

Asian wild rice, Oryza rufipogon

Griff:A case study in Thailand.

Genetof genetic

resources

Res Crop Evo. 46:

419-425
Kiefer-Meyer,
repeptive

M. C, Reddy, A. S. and Delseny,

DNA sequence associatedM. (1995)

Characterizationof a dispercedwith the CCDD genome of wild rice. Genome 38

681-688.
Monshima, H. (1 994)
Bangkok.

Tropics

Observations

3 227-233

at permanent

study-sitesof wild rice m the suburb ofofQTL analysis

for several

agronomic characters

between wild and cultivatedTakashige

Ishii

Laboratoryof Plant

Japan,

Breeding

using

four BC2 populations

rice species,

Facultyof Agriculture,

Kobe University,

Kobe 657-8501,(e-mail: tishii@kobe-u.ac.jp)

Keywords:

QTL analysis,

microsatellite

markers

the

genus

The genus Oryza consists(Tateoka

1963)

glaberrima,

respectively

reveals

were thought(Morishimaspecieswithto be ancestral

backcrossO. sativa and O.

species,O. rufipogon, O.

Of these, O. rufipogonof O. sativa

This was also confirmed

species

species,and O. barihu
speciespopulation,

and about 20 wild

five wild

O. meridionalis,

et al 1963).

species,that two cultivatedsame genome (AA)glumaepatula, O. longistaminata,and O. barthnof two cultivatedGenome analysis

share theOryza, wild

and O. glaberrima,by the phylogenetic

studiesusing molecular markers, such as nuclear and chloroplast

RFLP (Ishii

et al 1988;

Wang et al

1992)

and microsatellite

In addition,

Bautista

et al (2001)

analyzed

phylogenetic

relationships

among A-genome species

markers.

Their results

indicatethat O. glumaepatula was relatively

sativa(including

longistaminata

et al

2001)

O. rufipogori) and O. glaberrima
and O. meridionalis

Wild species
species,markers (Ishii

were highly

have wider genetic

many useful traits

have been identified(including

differentiatedvariationusing four kinds of molecularO. barthn)

whereasO.

from other A-genome species.

than cultivatedand transferredclose to the groups of O.

species.

Using wild rice

to elite breedinglines.

They

are usuallycontrolled

by single genes and can be easily identified

On the other hand, quantitative

traits,genes, and the manner of inheritancesuch as plant size and yield,
to succeeding generations

on quantitative

screeningare associatedwith many

appears to be very complicated

Therefore, limited

studiesbeen conducted

However, recently several kinds of molecular markers have been developed

for various crop species,

trait loci (QTLs)

by phenotypicusing wild rice species has so far

and their molecular linkage maps which covered almost the wholegenome have been constructed

These molecular markers have made it possible

QTL effect and identify

the chromosomal locations

In rice, Xiao et al (1996)

gave the first reportof QTLs (Tanksley

and McCouch 1997)

on wild QTL analysis.

They found that thelow-yielding

wild rice ancestor, 0. rufipogon, had alleles

productive

breeding

populations,

i.e., O. sativa (uplandvarietyThe

X O. glumaepatula (Brondani

similarresults

that can enhance yield

were also reported

2002)

These

all indicate

of highlyusingrice) X 0. rufipogon (Moncada et al 2001)
et al

to analyze

different

and O. sativa

the usefulnessof wild

germplasms improving rice varieties
In this study,a single accessionof closely-related

wild species,

O. rufipogon from

Myanmar (NIG ace. no. W630) and a single accessionof distantly-related,

O. meridionalis

fromAustralia

backcrossed(NIG ace no. W1627)

cultivars,

O. sativa Japonica

consistingof approximately

were respectively

Nipponbare
200 plants

and Indica

IR36,

each were produced

In order to identify

and distantly-related wild species,

out in the BC2 generation.

A total of llmorphologicalseed length,

seed width, grain length,

As for yield,a small scale of yield testingat two locations

Besidesyield,(days

to heading,

100-seedweight,were evaluated for four BC2 populations

was also performed in the next generation

the morphologicalthe useful

QTL analy sis was carried

characters

length, number of tillers,

grain width)rice

and four BC2 populations

QTL alleles from closely-related

photosy nthesis activity , culm length, panicle

with two typicalevaluation,

DNA was extractedof BC2F2
from BC2

plants

and their

Single-point
identify

12 chromosomal regions were surveyed with about 80 microsatellite

QTL analysis

was earned out using qGene software (Nelson

markers

1997)

in order toof P<0.01

with thethe useful QTL alleles from wild species

Wild QTL analysis

using O. rufipogon

A total of 55 and 51 QTLs
backcrossed
28 (54.9

populations

were identified

of Nipponbare%) had wild alleles

and IR36, respectively

increasing

trait values.

donor parent for two backcross populations,of two typicalrice cultivars

between two populations.

at a significanceOf these, 29 (52.7

Since a single wild plant was used as aeffect of the wild QTL alleles

was examined, and six QTL locations
Regardingon chromosome 1 was detected%) andthe wild QTL alleles

in both Indica and Japonica

in the backgroundwere found to be common

increasing

yield,

one QTL location

background populations,whereastwo (on chromosomes 1 and 2) and three (on chromosomes 5, 7 and ll) were found to be
specific

to Japonica

and Indica background,respectively.

Wild QTL analysis vising O. meridionalis
A totalNipponbareof 108 and 80 QTLs were identified

and IR36, respectively

increasing trait

values.

Of these, 47 (43.5

In the backgroundof two typicalwere found to be common between two populations.
increasingin the backcrossed

populations%) and 36 (45.0%) had wild alleles

rice cultivars,

22 QTL locations

Regardingthe wildof

QTL alleles

y ield, one QTL location on chromosome 8 was detected in both Indica and Japonica

backgroundpopulations,whereastwo (on chromosomes

chromosomes 1 and 3) were found to be specificre spectively.

1 andto Japonica

12)

and

and Indica

threebackground,(onReferences
Bautista,

N. S., Solis, R., Kamijima, O. and Ishii,of phylogenetic
Syst

relationships

and wild species

P. H. N., Brondani, R. P. V. and Ferreira,and introgressionof yield-related{Oryza sativa)

using microsatelhte

T , Terachi,
chloroplast

Ishii,

between cultivated

RAPD, RFLP and SSLP analysesof rice. Genes Genet.

76: 71-79.

Brondani, C.,-Rangel,

Ishii,T (2001)

traitsfrom Oryza glumaepatulamarkers. Theor. Appl

T. and Tsunewaki,

K. (1988)

DNA from A-genomediploid

Moncada, P., Martinez,

Morishima,

H., Hinata,endonucleaserice

analysisof

63. 523-536.

Nuclear- and chloroplast-microsatellite

C. P., Borrero, J., Chatel,variation

Quantitative

M , Gauch Jr., H., Guimaraes, E.? Tohme, J.

traitloci for yieldand yield

evaluatedcomponents

in an uplandin anenvironment

102: 41-52.
K., and Oka, H. I (1963)

cultivatedforms from two wild rice species,

Evolution

17, 170-181

Nelson, J. C. (1997)

to cultivated104: 1 192-1203.

of rice. Jap. J. Genet

Oryza sativa X Oryza rufipogon BC2F2 population
Genet

QTL mappingof rice. Genome 44: 658-666.

and McCouch, S. R. (2001)
Theor. Appl

Genet

Restriction

speciesT., Xu, Y. and McCouch, S. R. (2001)
in A-genome species

M E. (2002)

QGENE

Comparisonof modes of evolution

Oryza breviligulata

Software for marker-basedofand O. perennis.

genomic analysis

and breeding

Mol

Breed. 3: 239-245.
Tateoka,T. (1963)

Taxonomic studies

of Oryza. Ill

Key to the species

and their enumeration.

Bot Mag. Tokyo 76: 165-173.
Tanksley,

S. D. and McCouch,genetic potential
Wang, Z

among species

RFLPs. Theor. Appl
J., Li, J., Grandillo,(1996)

Seed banksfrom the wild. Science 277:

Y., Second, G. and Tanksley,

relationships

Xiao,

S. R. (1997)

Genet

and molecularmaps:

Unlocking

Polymorphismand phylogenetic

1063-1066.

S. D. (1992)

in the genus Oryza as determinedby analysis

of nuclear

83. 565-581

S., Ahn, S. N., McCouch, S. R., Tanksley,

Genes from wild rice improve yield.

Nature 384: 223-224.

S. D. and Yuan, L.

Comparative Cytogenomics

and Physical

Mapping in the Oryzeae.

Authors
Scott A. Jackson1,
Westerman2

Phillip

SanMiguel2,

Jason G. Walling1,

Barbara Hass Jacobus1,

Affiliations
1 Department of Agronomy & Plant Biology
Purdue University
915W StateSt
West Lafayette,
IN 47907
siackson @purdue.edu

Program

2. Department of Horticulture
Purdue University
625 Agriculture
Mall Dr.
West Lafayette,
IN 47907
Keywords
comparative

genomics, physicalmapping,

FISH, Oryza, chromosomes

Rick M.

Text

Plant

genomes, including

those within

number and morphology,

ploidyspeciesOryzeae,and the fraction

However, even with these varying
content among plant

the tribecharacteristics,(Hulbert

differof repetitive

there is still

et al 1990)

in DNA content, chromosome
sequencesconsiderable

especiallybetween more closely

relatedmaize, wheat and millet(Devosand Gale 2000)

observations

These

evolutionary

forceshave acted

remarkably

similarbut chromosome number, ploidy,

species?

Furthermore,gene colinearity

genomes such thatof geneis the conservation

speciessuch as rice, sorghum,
beg the question:

the gene content

what

and order isand DNA content vary drastically

how or in what manner have these

of

among

genomes been remodeled

such thathas been maintained?

Physicalthe physicalon relatedconservation

Even more remarkablegene order (colmearity),in the genome.

mapping
relationshipthe molecularof related

plant

genomes gives

us the opportunity

to directlyof genetic

syntenyor colmearity

between related

speciesand physicalComparing

geneticists

to make inferences

about the mechanisms by which gene content and colinearity

were maintainedin the 50 million

years since the last common ancestor (Doebley

et al 1990)

However, the tribe

Oryzeae,which

represents

evolution,

us to look in more detailat early

10-12

millionmaps of rice and maize will permit

observe

years of independent

chromosomal

eventsWithin(megabases)

per haploid

genome, and there are both diploidOutsideduplicatedsativa.

allowsthe genus Oryza, the genome sizes vary from 343 to 1,691

the genus Oryza but within

the tribe

chromosomes (Kennard et al 2000)

Oryzeae,and autopolyploidthe genus Zizania

and a genome approximately

species(TableMbi)has three putative
twice the size of O.

To exploit

this genomic diversity

and begin to model plant

genome evolution,

especiallycereals,a toolbox

is being

made forcomparativeand evolutionary

genomics

toolboxwillof BAC (bacterial

in the(http//www.omap.org)

libraries

for several

sequenced

This

Oryza genomes (Table

and aligned

chromosome evolution(fluorescence1)

to the sequenced
by comparativein situ hybridization)

O. sativa and related

consistThese

O. sativa

physical

BAC libraries
genome.

mappingof individual

artificial
will

chromosome)be fingerprinted,

To gain further

insights

end
intoof Oryza genomes, we are using FISH

BACs or sets of overlapping

BACs (contigs)

toOryza genomes

Table 1 Oryza species being used for comparative

genomics

Oryza sativa
O. rufipogon
O. glaberrima
O. punctata
O. officinalis
O. minuta
O. australiensis
O. latifolia
O. schlecten
O. ridleyi
O. brachyantha
O. granulata

FISH can be done to either
DNA fibers(fiber-FISH)

advantages

and limitations

clonedfrom O. sativa

several Oryzeae species,(Jacksonmitotic/meiotic
et al 2000)

for genome analysis(Dong et al 1998)
including

RCS2,

the major centromericsatellite

et al 2001)

or extended

Each target,

chromosomes or fibers,

has its own(TableFISH

2)analysis

showed that some are present

Z palustris,

only in a subset of the most closely

chromosomes (Cheng

relatedrepeatof centromere repeats
at the centromeres

while others that evolve more quickly
Oryza species(Hasset al in press)in O. sativa,

was present

primarilyofwere found
For instance,in other AA-

genome species,
1998),

while

RCS1,

derivedwas found in all the Oryzeae

from a centromere specificspeciestestedretrotransposon

and m several cereal species(Dong et al(Jianget al 1996;

Hass et al 2003)
Table 2. Comparisonof FISH techniques

for plant genome analysis

We are also using FISH to analyze
across related

Oryza species

the structureof specificregionsof the rice genome

This work was begun with a BAC contig from the short arm ofchromosome 10, however, it soon became apparent

that this(Riceand that many of the sequences

Chromosome 10 Sequencing

the BACs were degenerating
regions

2003)

to fast to use heterologous

probeson chromosome 1 were chosen for 1) high gene density

BAC/PAC contigs
successful,Using a gene-richhowever, the utility

Oryza genomes is limited.
genome species
2000)

Consortium

the best

This is in stark contrast

where we have used Arabidopsis

spanning

10-35 million
In the Oryzeae,

libraries,
evolutionary

physicalthaliana

and 2) availability

are obtained

we are in the beginning

maps and cytological
the remodelingtools)

stagesthat

presentinThus, twoof a sequenced

FISH analysis

has been moreprobeson the related

in the Brassicaceae

BACs as heterologous(Lagercrantz

heterochromatic

genomes

BACs as heterologous

to our observations

years of evolution

forces effecting

results

is mostly

to related

BAC contig the comparativeof O. sativa-derived
In fact,

regionon related
AA and BB(Jackson

et alprobes on related

species1998)
of developing

a molecular

can be used to more fullyof Oryza genomes

toolbox(BAC

understandtheAcknowledgementsThis

work was supported(DBI02274

by funding

from the National

ScienceFoundation

to SAJ and PSM

14)References

Cheng,Z., Prestmg,

G., Buell,

C.R., Wing, R.A., Jiang,

J. 2001

chromosome

mappingof bacterial

artificial

chromosomes

reveals

centromere

locationand thedistribution

thechromosome 10 of rice. Genetics
Devos, K.M. and M.D. Gale. 2000.
Plant

Cell

Dong, F., Miller,
sativa)157

ofHigh-resolution

pachytene

anchoredby genetic

markers

genetic

recombination

along

in current

research.

1749-1757

Genome relationships

the grassmodel

12: 637-646.
J.T.,

Jackson,centromericS.A., Wang, G-L., Ronald,

reginsconsistof complex

P.C., Jiang,

J. 1998

Rice(OryzaSci

USA 95

8135-

Comparative

genetics

at theDNA. Proc. Natl

Acad.

8140.
Hass, B , Pires,

J.C., Porter,

R., Phillips,

gene and chromosome levels
Hulbert,

S , Richter,characterization

T.E.,

betweenAxtell,of sorghum

R.L., Jackson,

J.D.

and related

S.A. 2003

rice and wildrice.

Theor. Appl

Genet

and Bennetzen,

J.L.

Genetic

crops

1990.

in press.
mappingby means of maize DNA probes

andProc. Natl

Acad. Sci USA 87* 4251-4255
Jackson,S.A.,

fluorescence

Cheng,

Z.,Wang,

in situhybridization

M-L., Goodman,mapping

H.M., Jiang,of a 431-kb

Arabidopsis

J.

2000.
thaliana

Comparative
bacterialartificial

Jiang,

chromosome contig

reveals

the role of chromosomal

duplicationsin the expansionof the Brassica

rapa genome. Genetics

156:

J., Nasuda,

S , Dong, R, Scherrer

C.W , Woo, S-S , Wing, R.A., Gill,

1996.

A conserved

repetitive

chromosomes Proc. NatlDNA element

R., Grombacher,(Zizaniapalustris

L. 2n=2x=30)

Theor

Comparative

mapping

indicates

U. 1998
that

accompanied

Brassica

locatedintheB.S , Ward, D.C.

centromeresof cereal

Acad. Sci USA 93 14210-14213

Kennard, Ws, Porter,

Lagercransz,

833-838

A., Phillips,

genomes

R.L. 2000.

Appl

Genet

by chromosome fusions

map of wildrice

99: 793-799.

between Arabidopsis

have evolved

A comparative

throughand frequentthaliana
extensive

rearrangements

and Brassica

nigra

genome replication
Genetics

150:

1217-

view of structure,

activity,

and

1228
Rice Chromosome 10 Sequencing
evolution

Consortium.

of rice chromosome 10. Science

2003
300:

In-depth1514-1515

Genetic potential

Kshirodof wild species of the genus Oryza and their use in nee
improvement

K. Jena13*. Hung-gu Hwang2, Moon-hee Lee2, Darshan S. Brar and David J. Mackill3

1IRRI-Korea Office,National

Crop Experiment

Station,

Rural Development

Administration,

209 Seodun-Dong, Suwon 441-100, Republic of Korea
Crop Experiment Station, Rural Development Administration,
209 Seodun-Dong,

2National

Suwon 441-100,
Genetics and Biochemistry

3Plant Breeding,

Republic
Division,of KoreaInternational

Rice Research Institute,

DAPO Box 7777, Metro Manila, Philippines* E-mail kjena@rda.go.kr

Key words:

Oryza, Wild species,

Genes, Rice improvement

AB S TRACT
The genus Oryza has 22 wild species

and two cultivatedwell as the wild species

grow in a wide range of habitats

level to high altitudes

The two cultivateddomesticatedfor several thousand

Rice production

and productivity

affectedby several biotic

and abiotic

rice

againstnew biotypes

O. sativa and O. glaberrima

in tropicalstresses

distribution

and temperateof pests

as wellconditions

Some of the major biotic

stressesbacterial

(BB),

(Bl),

Tungro virus (RTV),

blast

sheathblight(ShB),et al

are severely

genes to protect

as adverse

environmental
blightashave been(Vaughancountries

due to lack of stress resistanceor pathotypes

The cultivatedworld wide ranging from below sea

years and have pantropical

2003)

plant

species,

speciesof rice are diseases

soil

and

such asdwarf virus (DV)and insects

such as brown planthopper

borer. The major abiotic
salinity.

It is essential

diversegeneticstresses(BPH),

whitebacked

are drought,

to widen the genepool

sources. The wild species

planthopper

cold, submergence,of cultivated(Wph)and stemaluminum toxicity

rice by incorporating

andgenes fromof Oryza are a rich source of beneficial

genes forrice improvement (Brar and Khush 1997)
Transfer of usefulgenes from wild species

extremely

difficultbeneficial

genes from wild Oryza species

crossability

and recombinationtransferredusingthrough production

because of crossability

advancedbarriers
techniques

into cultivatedand recombination

1)of tissueof monosomic alien addition

lines

has been

Nevertheless,severalinto rice cultivars

acrossbarriers

have been transferred(Tablerice genotypes

The genes from wild species
culturecouldbeand chromosome manipulation(Jena and Khush 1989;

1990;

Brar andKhush 1997)

Table 1 Some wild Oryza species, useful genes, associatedlocation and gene transfer mechanisms in rice.

O.nivara

O.longistaminata

O.rufipogon

DNA markers, chromosome

Transfermechanism
Homologous
recombination
Homologous
recombination
Homologous
recombination

O.rufipogon

O.officinalis
O.minuta
O.australiensis* QTL = Quantitative

Trait Loci

Recent advances
saturatedmolecular

in molecularbiologyand subsequent

map of rice have made it possible

to identify

specieson the chromosomes ofO. sativa (Jena et al 1992;

2003;

Nguen et al 2003)

of beneficial
resistance

The newly developed

genes of wild species

for the genetic

Asian countries
rice breeders(Song et al 1995,
and geneticists

McCouch et al 2002;

to BB pathotypes

Jena et al

methods have made use

O. longistaminata

introgressedof highly

genes of wild

improvement of rice cultivars

gene Xa-2\ from the wild Oryza species,

broad spectrum of resistancethe usefulmolecular breeding

gene from O. officinalis have been successfully
expressed

development

The BBand BPH resistanceinto rice cultivars

and BPH biotypes

and haverespectivelyinJena et al 2003)

These findings

have paved the way for

to make use of valuablegenetic

of wild species

resourcesof

Oryza in rice improvement

With the completionof a highly

reliable

genome sequence of

Oryza sativa by International

Rice Genome Sequencing Project(IRGSP),

it would be possible

nowto transfer

numerous beneficial

improve the yield potential,

grain quality

genes from wild Oryza species
and stress resistance.

into rice cultivars

toAcknowl edgement
We are grateful to the Rural Development Administration(RDA), South Korea andInternational
Rice Research Institute (IRRI), Philippines
for their support in preparing thismanuscript

Reference s
Brar, D. S. and G. S. Khush (1997)

Alien introgression

in rice. Plant Mol BiolJena, K. K and G. S. Khush (1989)
Monosomic alien addition
morphology, cytology and breeding behavior. Genome 32: 449-455

lines

35 35-47.

of rice:Jena, K. K and G. S. Khush (1990)
Introgressionof genes from Oryza officinalis
to cultivatedrice, O. sativa L. Theor. Appl Genet 80: 737-745
Jena, K. K., Khush G. S. and Kochert G. (1992)
introgression
lines. Theor. Appl Genet 84: 608-616.

RFLP analysis

Jena K. K. et al (2003)
Molecular tagging
rice (Oryza sativa L.) Euphytica 129: 81-88.

of a gene for resistance

McCouch S. R. et al (2002)

and mapping

Developmentof rice (Oryza

production,

Wall exWatt

sativaL.)

to brown planthopperinof 2240 new SSR markers for rice

(Oryza sativa L.) DNA Res 9: 199-207.
Vaughan D. A., Morishima H. and Kadowaki,
Opin. PI Biol 6: 139-146.

K. (2003)

Diversityin the Oryza genus. Curr.

Wild Rice Core Collection
Non Kurata. Toshie

and Its Use for The Study of Gene Diversity

Miyabayashi,

YoshiakiHarushima.

National

Institutee-mail:nkurata@iab.nig.ac.jp

of Genetics,

Key words: wild species,

Mitsugu

Mishima,collected

Shizuokacore collection,

Near 2,000 wild rice accessions
in our National

Institute

fifty years. We have selected

Eiguchi,

41 1 -8540,

gene diversity,

reproductiveof Genetics(NIG)

of 44 strains

accessions

in rank 3, as a resource for the study

from each species

for rank 1, 64 strains

habitat,place

information

are also being

gathered.

These

Oryzabaseat http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp.

of origin,performed

by several

workers using PCR technique,(Aggarwal

et al. 1999;

Ge et al. 1999;

of evolutionary

certainrelationshipkindof gene expression

and evolution

photographic

imagesis accessible

in genus

and related

to our database
Data examples

among these
specific

Chen et al. 2002;

regulatory

to search genome and species specificstart. Differencea coreandwill be introduced.

studies

attempt

diversity

information

Thoughmembers and sequences including

and preparedfor rank 2 and 171 supplemented

of genetic

characters,in the Oryzabase

have beenfrom all over the world spent more thanaccessions

consisting

future activities

barrier

9 genomes and more than 21 species

representative

Monshima,Japan

includingcollection

Oryza. Phenotype

Yukiko Yamazaki, Hiroko

elements

Oryza

sequences

specieshave beenor isozyme analysis

Cheng et al 2003 ), diversity
have not been elucidated

in geneyet. Our new

genes in a few genome species is now going toand sequence diversity,of gene members would be major attributeas well as presence or absence ofof the genetic

diversificationof1-43.

species.

Identificationof genome or speciessequence

or at the expression

microarray

analysis.

level

will

specificbe carried

diversity

genes in theout by the use of EST analysis

among species

approaches

to find them. One of the direct

sub-species,

those are in a step of evolutionary

which cause segregation

distortion

two cross combinations

barriers(Harushima

detectedin each three combinationinterested

in theirfunctionand sub-species,

approach

to detectprocesssin the crossed

almost all reproductive

identification

variableandPlans and a few examples will be shown in my talk.

As for the gene/geneticout to detect

genes and highly

othergene/genetic

barriers

betweenfor speciation,is findingof barriers

progenies.

Genome-wide survey was carried

between Nipponbare

et al. 2001

there are several

and Kasalathand in otherand 2002)

More than 30 barriers

were

and were quite different

among three crosses.

We arebarriers

are now in the processof geneproject

under the MEXT and by the riceand one of the prominent

by map-based cloning.

Acknowledgements:

This work is supported
genome project(wild

by the national

relativesbioresource

genome projectand MP1 1 1 7) in the NIAS.

References:
AggarwalRK, Brar DS, Nandi S, Huang N, Khush GS. (1999)

among Oryza species

revealed

Cheng C, Motohashi

relationshipsby AFLP markers. Theor Appl Genet 98: 1 320-1 328.

Chen X, Cho YG, McCouch SR. (2002)
and other plant species.

Phylogenic

Sequence

divergenceof rice microsatellites

in Oryza

MoL Genet. Genomics. 2002:33
R, Tsuchimoto

S, Fukuta Y, Ohtsubo H, Ohtsubo

E. (2003)

Polyphyletic

originof cultivated

rice:

based

on the interspersion

patternof SINEs.

Mol. Biol.

Evol. 20:67-75.
Ge S, Sang T, Lu B-R, Hong D-Y (1999)
origins

ofallotetraploid

species

Harushima,

Y, Nakagahra,of reproductive

barriers

Harushima,

Y, Nakagahra,

reproductive

barriers

Phylogenyof rice genomes

with emphasisonProc. NatL Acad. Sci. 96: 14400-14405.

M., Yano, M., Sasaki,in an intraspecifichybrid.

M., Yano, M., Sasaki,in three intraspecificT, Kurata, N. (2001)
Genetics.,

A genome-wide

159, 883-892.

T, Kurata, N. (2002)

rice crosses.

surveyGenetics,,160:

Diverse3 i3-322.

variationofTransgene escape and its biosafetyconcerns in rice (Otyza)

Bao-Rong Lu, Song Zhiping,and Jun Rong

Ministry of Education Key Laboratory for Biodiversityof Biodiversity
Science, Fudan University, Shanghai
Key words: Gene flow, introgression,

ecologicalbiosafety,

and EcologicalEngineering,
Institute200433, China, brlu@fudan.edu.cn
wild rice, GM crop

Rice (Oryza sativa L.) is one of the most important world's cereal crops, providing staple
food for nearly one half of the global population.
More than 90% of rice is grown andconsumed in Asia where about 55% of the world's population lives (Lu 1996), reflecting theimportance of rice in Asian people's daily life, in addition
to its significancesin cultural
aspects in this area. Rice is also one of the earliest
world's crops to which transgenic
biotechnology
has been effectively
applied for its genetic improvement (Ajisaka et al 1993;
Yahiro et al 1993). Therefore, geneticallymodified (GM) rice is not any more a novel term topublics of many societies. To date, no GM rice varieties have yet been officially approved fortheir extensive commercial cultivation
anywhere in the world, but genes conferring traits suchas high protein content, special nutritional
compounds, disease and insect resistance, virus
resistance,
herbicide
resistance,and salt tolerance, have been successfullytransferredintodifferent rice varieties through transgenic technology (Ajisaka et al , 1993; Yahiro et al , 1993;
Matsuda 1998; Messeguer et al 2001; Brooks and Barfoot 2003). It is predicted
that, as animportant world cereal crop, GM rice varieties, like many other GM crops that enhance yields,
improve human health, and make agriculture more sustainable (Snow 2003), will be released
into environment for commercial production in the near future, after their necessary food andenvironmental biosafety assessments are accomplishedby the authorized agencies (Brooks
and Bar foot 2003).
Like many other new developments, transgenic biotechnology
and the GM products haveevoked most intense debates on their biosafety concerns worldwide (Bergelson
et al 1998;
Schiermeier
1998; Crawley et al 2001; Ellstrand
2001; Prakash 2001). Biosafetyissue hasbecome a crucial constraint to the further development of transgenic biotechnology
and widerapplicationof GM products, including GM rice. Transgene escape from the GM rice varieties
and its potential
environmental
consequences is among the most controversial
biosafety
concerns across the world (Lu et al , 2003). Terminologically,transgene escape refers ingeneral to a gene or a group of genes introduced to a crop variety by a genetic engineering
method moving to its non-GM counterparts or wild relative species (including
weedy biotypes)
through gene flow (including
pollen flow and seed dissemination).
Cross-pollination
between
GM rice and non-GMrice varieties or its wild relatives is the major pathway for transgene
escape. Accordingly,
there are two types of transgene escape, i.e., crop-to-crop andcrop-to-wild transgene escape that are usually discussed by publics.
It is argued that the crop-to-crop transgene escape might contaminate non-GMrice
varieties,
affecting the purity of these rice varieties,
as well as the strategic deployment of
GM-and non-GMrice in a given agricultural
system. When normal rice varieties are mixed

with individuals
of GM rice, the exporting trade of rice, particularly
to these countries with arigid biosafetycontrol, would be considerably
influenced, and even cause some legal
difficulties.
The crop-to-wild transgene escape may lead to the persistence and disseminationof transgenes in wild or weedy rice populations through sexual reproduction and/or vegetative
propagation. If the transgenes are responsible for resistance to biotic and abiotic stresses (suchas drought and salt tolerance, and herbicide
resistance),these genes could significantly
enhance ecological fitness of wild and weedy rice species, and make the host wild plants moreinvasive, which could probably cause unpredictableenvironmental consequences in certain
ecosystems. On the other hand, when transgenes escape to wild rice populations
throughoutcrossing,
the rapid spread of the resulted hybrids and their transgene-carrying
progeny
would result in contaminationof the original wild rice populations,and even lead to theextinction of endangered wild rice populations
in local ecosystems by the so-called swarm
effect (Kiang et al 1979; Ellstrand and Elam 1993). This will jeopardize
in situ conservationof wild rice germplasm. In addition, the perennial hybrids between GM rice and their wild
relatives carrying transgenes may serve as a "bridge" to spread their transgenes to other wild
related species through outcrossing, causing even more significant ecological consequences.
Transgene escape and its ecologicalconsequences have been extensively
discussed
worldwide. Will transgene escape occur in rice through outcrossing?
Will transgene escapepose an environmental safety problem in rice ecosystems? These questions relating to thebiosafety of GM rice need to be adequately addressed for scientific and public understanding.
Normally, the occurrence oftransgene escape from GM rice needs to meet the following threeprerequisites.
Spatially,
GM rice and its non-GM counterparts/wild
relatives should have a
sympatric distribution,i.e. growing in the same vicinity and also in a close contact. In termsof crop-to-wildtransgene escape, temporally,
the flowering time (including
flowering
duration within a year and flowering time within a day) of GM rice and its wild relatives
should overlap considerably;and biologically,
GM rice and its wild relative species shouldhave a sufficiently close relationship,
also the resulted interspecific hybrids should be able toreproduce naturally.
Consequently,
the knowledge of geographic
distribution
patterns,
flowering habits, and genetic relationshipsof cultivated rice and its wild relative species willbe essential for predicting
transgene escape in rice. Similarly,
data on the actual gene flow
frequencies between different rice varieties,
and between cultivated and wild species are also
important for predicting
transgene escape and its potential
ecologicalconsequences, based on
which strategies to minimize transgene escape can be developed.
Studies have clearly shown geographicaldistribution
patterns and genetic relationshipsof cultivated rice and its wild relative species in the genus Oryza. It is known that the genus
comprises two cultivated species and over 20 wild species with ten different genome types, i.e.
the AA, BB, CC, BBCC, CCDD, EE, FF, GG, JJHH, and JJKK genomes (Vaughan 1994; Geet al 1999), widely distributedin the pan-tropics and subtropics (Vaughan 1994). Species that
contain different
genomes have significant
reproductive
barriers. Therefore, geneticallyspeaking, such species are distantly
related and spontaneous hybridization
between these
species with different genomes is extremely difficult. Cultivated rice contains the AA genome
and is relatively
easy to cross with its close relative species (including
weedy rice) that also
contain the AA genome. Theoretically,transgene escape from GM rice varieties will merely
occur to the wild rice species with the AA genome.

Research data have demonstrated that apart from the two cultivatedrice species (O.
sativa and O. glaberrima),
these wild relatives,
O. ruflpogon and O. nivara from Asia, O.
longistaminata
and O. barthn from Africa, O. glumaepatula from Latin America, and O.
meridionalis
from northern Australia and New Guinea, also contain the AA genome. The
AA-genomewild Oryza species is distributed
across a significantly
wide geographic region indifferent continents, and Asian cultivated rice (O. sativa) shares sympatric distribution
withthese wild species in many areas, particularly
in Southeast and South Asia, Central Africa, andLatin America. The weedy rice is usually found in rice fields alongside cultivatedrice. Dataon geographic
distribution
evidently
indicate that spatiallytransgenes from cultivatedrice
have a great potential to escape to its wild relative species.
Floweringhabits of cultivatedrice grown in different
parts of the world varyconsiderably
depending on differencesin local cultivation
time and seasons, and in varietal
types. The floweringand pollinatingtime of different
wild rice species or different
populationsof the same species also varies significantly
across different geographic regions.
Our studies of a selected O. rufipogon population found in Hunan Province of China and two
cultivated rice varieties showed that both of the flowering period in a year and flowering timein a day had considerable
overlap between O. rufipogon and the two rice varieties.
Our
additional
experimental data also showed that pollen grains of O. rufipogon and a cultivatedrice variety could be viable in air for more than 60 minutes (Song et ah 2001). These results
suggest that outcrossing between O. rufipogon and cultivated rice will take place, if the two
species are grown near to each other.
Data on interspecificcrossability,
meiotic chromosome pairing, and fertility
in the Fi
hybrids can be used to estimate genetic relationshipsof the cultivated rice and its AA-genome
wild relatives. If cultivatedrice has relatively
high crossability
with its wild relatives, normal
meiosis, and comparatively high fertility
in the Fi hybrids, the transgenes will easily escape towild relative species through outcrossing
and persist in environment. Transgenes would also
disseminate
through reproduction
or vegetative propagation if the hybrids and their progeny
were perennial.
Results from our interspecifichybridization
showed that most of the
AA-genomewild rice species have relatively
high compatibility
with the cultivatedrice,
extremely high chromosome pairing formed in meioses of the Fi hybrids with the wild rice,and spikelet
fertilityof the Fi hybrids was relatively
high under bagged self-pollination
conditions.
These data indicate a high opportunityof transgene escape from GM rice to its
wild relatives,in terms of the close genetic relationshipsof cultivatedrice with its
AA-genomewild relatives.
In order to obtain data on the actual gene flow frequencies between different cultivatedrice varieties, and between cultivatedrice and its wild species under natural conditions,
weconducted a series of experiments involving GM-, non-GMrice varieties, weedy rice, and O.
rufipogon. The experimental data showed that gene flow frequencies between different rice
varieties were very low (less than 0.5%), although with a certain variation, comparable geneflow frequencies were observed between cultivated rice and weedy rice. However, gene flowfrom a cultivated rice variety Minghui-63 to the Hunan O. rufipogon population under four
different experimental designs were significantly
variable, with the maximumfrequency of ca.
3% under the special cultivation
conditions,indicatingclearly that gene flow from cultivatedrice to the widely distributed
O. rufipogon would occur considerably
in nature.

Our studies demonstrated that transgene escape from GM rice to non-GM rice varieties,
and to its wild relatives
is possible
to occur, although its extent might vary considerably
between different varieties
or different
species (populations).
This result is accordant withmanyother conclusions based on gene flow or introgression
studies of rice species, although
the gene flow frequencies between GM-and non-GMrice varieties were comparatively
low(e.g. Messeguer et aL 2001). It is therefore a very important biosafety strategy to establishaneffective buffering isolation
zone between GM rice and non-GM rice varieties,
particularly
between GM rice and its closely related wild species, to avoid or significantly
minimizetransgene escape, given that the spatial, temporal, and biologicalconditions for rice transgene
escape are satisfied
in many rice producing countries or regions. In addition,
our further
research activates should be focused more on ecologicalconsequences of rice transgene
escape, which is still a controversial
issue that have received an extensive attention by publics,
scientists
and government agencies. Although it is difficult
to assess and monitor potential
ecologicalconsequences caused by transgene escape within a limited period, long-term andcontinued accumulation of basic knowledge on ecologicalimpacts of transgene escape isessential
to increase our understandingof GM rice biosafety,
and reduce our biosafetyconcerns caused by GM rice. Moreover, there should be more scientificinputs allocatedforinvestigating
scientificquestions on ecologicalconsequences of rice transgene escape. This
will allow us to effectively
assess and manage the potential
ecologicalrisks resulting fromrice transgene escape, which will in return promote the development and safe use oftransgenic rice varieties.
Acknowledgements
This research project was supported by the National Natural Science Foundation of China(NSFC) for Distinguished
Young Scholars (Grant No. 30125029)
and Shanghai Commissionof Science and Technology (Grant No. 02JC14022
and 03dzl 9309).
References
Ajisaka, H., Maruta, Y. and Kumashiro, T. (1993)
Evaluationof transgenic rice carrying anantisense glutelin gene in an isolated field. Proc. 3rd Intern. Symp. on the Biosafety Results ofField Tests of Genet. Mod. Plants and Microorganisms
Jones, D. D. (ed), UniversityofCalifornia- Oakland, pp. 291 -298
Bergelson, J., Purrington,
Nature 395: 25.

C. B. and Wichmann. G. (1998)

Promiscuityin transgenic

Brookes, G, and Barfoot, P. (2003) GM Rice: Will this lead the way for global
GMcrop technology. ISAAA Briefs. httpV/www.isaaa.org/Publications/briefs/briefs
Crawley, M. J., Brown, S. L., Hails,
natural habitats. Nature 409: 682-683.
Ellstrand,
N. C. (2001)
1543-1545.

R.S., et al. (2001)

When transgenes

Biotechnology

wander, should

plants.

acceptance of
28.htm- Transgenic

crops in

we worry? Plant Ecology

125:

Ellstrand,
N. C. and Elam, D. R. (1993)
Population
genetic
consequencesof smallpopulation-size-implications
for plant conservation.
Annual Review of Ecology
andSystematics
24: 2 1 7-242.
Ge, S., Sang, T., Lu, B.R., et al. (1999)
of allotetraploid
species. Proceedings
States of America 96: 14400-14405.

Phylogenyof rice genomes with emphasis on originsof the National Academy of Sciences of the united

Kiang, Y. T., Antonvics, J. and Wu, L. (1979)
The extinction
formosa) in Taiwan. Journal of Asian Ecology 1 •E1-9.

of wild rice (Oryza perennis

Lu, B. R. (1996)
Diversityof the rice gene pool and its sustainableutilization.
In: Zhang, A.L.
and Wu, S.G. (eds) Floristic
Characteristics
And Diversityof East Asian Plants. China Higher
Education Press-Berlin:
Springer-Verlag,
Beijing, pp. 454-460.
Lu, B. R., Song, Z. P., and Chen J. K. (2003)
Can transgenic ricethrough transgene escape? Progress in Natural Science 1 3(1): 1 7-24.

cause ecologicalrisks

Matsuda, T. (1998)
Applicationof transgenic
techniques
for hypo-allergenic
rice. In:
Proceeding of International
Symposium on Novel Foods Regulationin The European Union Integrity of The Process of Safety Evaluation.
Berlin, Germany, pp. 3 1 1 -3 14.
Messeguer, J., Fogher, C, Guiderdoni,E., et al. (2001)
Filed assessment of gene flow fromtransgenic to cultivatedrices {Oryza sativa L.) using a herbicide
resistancegenes as tracer
marker. Theoreticaland Applied Genetics 103: 1 151-1 159.
Prakash, C. S. (2001)
The genetically
evolution. Plant Physiology
126: 8-5.

modified

Schiermeier,crop trails

P. (1 998)

Snow, A. (2003)

German transgenic

Unnatural

selection.

crop debate

in the context

of agricultural

face attack. Nature 394: 8 19.

Nature 424: 619.

Song, Z. P., Lu, B.-R., and Chen, J. K., (2001)
A study of pollen viability
and longevity
Oryza rufipogon, O. sativa and their hybrid. Intern Rice Res Notes 26(2): 3 1-32.
Vaughan, D.A. (1994)
Rice Research Institute,

The wild relatives of rice: a genetic
IRRI, Los Baflos, Philippines.

resources

handbook.

inInternational

Yahiro, Y., Kimura, Y, and Hayakawa, T. (1993)
Biosafety
results of transgenic rice plants
expressing rice stripe virus - coat protein gene. Proc. 3rd Intern. Symp. on the Biosafety
Results of Field Tests of Genet. Mod. Plants and Microorganisms(ed Jones, D. D.),
University of California- Oakland, pp. 23-36.

Cytologicaldiversityand its related
ShigekiofFISHloci of a tandem repeat DNA sequence in Oryza sativa

speciesNakayama

Genetic Diversity

Department,

National

Institute

Sciences,

305-8602,

Japan, E-mailKeywords:

Oryza sativa, wild nee, cytologicaldiversity,

hybridization(FISH),

DNA sequence

Genetic

diversity

in genus Oryza and domesticationand molecularanalysesgroups (A-J)

to the molecularfluorescencein situ hybridization

repetitive

biologicalby various studies

analyses(FISH)

revealed

inter-species

studieson the intra-species

diversity

diversity

in cultivatedDNA (Ishii

et al 1988),

transposableof cytology,
and genome

mapping by the

et al 2000)

and the

These cytologicalin the genus Oryza. There are few cytologicaldiversity,

intra-species

and tetraploid)

RNA genes (rDNA)

Shishido

basedfrom genome

based on physicalof ribosomal

DNA sequences (Uozu et al 1997,

in situ

From the viewpoint

based chromosome numbers (diploidstudies

2003),

fluorescenceof rice has been investigatedcharacteristics

in the genus Oryza has been revealedorganization

Tsukubaaffrc.go.jp

Os48y tandem repetitiveon their morphologicaldiversity

shignak@mas

ofAgrobiologicalhowever, there are many molecularstudiesnee, O. sativa, based on, for example, the organellar
elements(Mochizuki

RFLP and RAPD polymorphism(Suh et al 1997)

Os48 (Rc48)

tandem repeat

is a representative

et al 1993,

sequence

Cheng et alin cultivatedrice, Oryza

sativa (Wu & Wu 1987)

This 355-bp

sequence is found in only Oryza Agenome

species

In previous

FISH studies

loci were detected

in indica(Wu etal

chromosomes

1991)

Eight

found mjaponica

varietyWuyujing

8 (Cheng

Fukui 1997,

variety

was mapped on nee

Zhongxian3037 and two were

A tandem repeat sequence into Os48, was visualized

IR8 and IR36 and two mjaponica variety Nipponbare

Ohmido et al 2000)

These FISH results

the number of Os48 between nee varieties
diversity

this sequenceet al 2001)

the Agenome, TrsA, which shows high similarity
the indica varietiesonat six loci in(Ohmidoshowed that there are difference

and suggested

intra-species&
incytologicalin O. sativa.

To reveal cytologicalintra-specificdiversity

among varietiesof O. sativa, FISH of Os48

with tyramide
diversity

signalamplification(TSA,

was shown in the differences

Okada et a! 2000)

was employed

Cytologicalin the number ofFISH

signals

The number of

FISH loci was almost the same among dWjaponica varieties,

includingthe temperate(paddyBut the indica vaneties

and upland

habitats)

and tropical(javanica)

varieties

showed significant

differencesin the number of the FISH loci

These FISH results

reveal two points

about cytologicaldiversity

cytologicaldiversehas accumulated

in rice chromosomes and the other is narrow

cytologicaldiversity

is observed

Additional

FISH studies

cultivatednee from a cytologicalof rice chromosomes, one is intra-species

mjaponica

while wide diversityof O. rufipogon will shed lightis observedon the domestication

in indica.
ofviewpoint

Acknowledgements
Rice seeds were kindly
Genebank (NIAS),

provided

from Genetic Stock Research

Drs S Fukuoka,

thank Prof H. Monshima (NIG)
D. A. Vaughan (NIAS)(NIAS)

for her kind suggestions

technical

MAFF

S Yamamoto, M.Yamazaki and M. Yano (MAS)for the criticalfor her excellent

Center (NIG),

reading
support.

on selectionof the manuscript
Plantsofmatenals,

Drand Ms N. Odashima

were grown at domed green house,

NIAS, Tsukuba, Japan.
References
Cheng C, Motohashi
rice:R, Tsuchimoto

S et al (2003)

Based on the interspersion

Polyphyleticpatternof SINEs

Cheng Z, Stupar RM, Gu M, Jiang J (2001)

Atandemly

associatedIshiiwith both knob-likeheterochromatin

origin of cultivatedMol Biol Evol 20* 67-75
repeated
and a highly

DNA sequence isdecondensed

in the meiotic pachytene

chromosomes of nee. Chromosoma 110. 24-3 1

T, Terachi

K (1988)

T, Tsunewaki

DNA from A-genome diploid
Mochizuki

Restriction

speciesendonucleaseanalysis

elements

of rice strainsat nine lociOhmido N, Fukui K (1997)
genome- specific
35 963-968

of chloroplastof rice. Jpn J Genet 63 523-536.

K, Ohtsubo H, Hirano H, Sano Y, Ohtsubo E (1993)

relationships

structure

Classification

with AA genome by identification

andof transposable

Jpn J Genet 68 205-217.
Visualverification

DNA sequence (TrsA)of close disposition
and the telomere

between a nee A

sequence.

Plant Mol BiolI

Ohmido N, Kijima

K, Akiyama

genomic DNA and selecteddifferent

DNA families

Okada S, Fujisawa

Shishido

repetitivein indicasequences

andjaponica

M, Sone T et al (2000)

genomic libraries
the liverwort,

Y, de Jong JH, Fukui K (2000)

suitableR, Sano Y, Fukui K (2000)

inof male and female PAC
clones fromPlant J 24 42 1 -428
DNAs an exceptionof gene order in nee genomes Mol Gen Genet 263

sativa L.) basedchangesof Y-chromosome-specificRibosomal

Suh HS, Sato YI, Monshima H (1997)

concurrentof totalnee. Mol Gen Genet 263 388-394.

Construction

for identification

Marchantiapolymorpha.

reveals

Quantification

Genetic

to the conservation

586-591

characterizationof weedy rice (Oryza

on morpho-physiology,

isozymesand RAPD markers

H, Ohmido N et al (1997)

Repetitive

sequences

Theor Appl

Genet 94 316-321
Uozu S, Ikehashi

genome size and chromosome morphology

cause for variationin the genus Oryza. Plant Mol Biol 35

791-799.
WuHK, Chung MC, Wu TY, Ning CN, Wu R (1991)
DNA sequences
WuTY, WuR (1987)

in individual

rice chromosomes

A new nee repetitive

RNAand tRNA. Nucleic

Localization

Chromosoma 100

DNA shows sequence

Acids Res 15 5913-5923

of specificrepetitive

330-338

homology

to both 5SinPhylogenetic
analysis
its flanking nucleotide
Tomotaro Nishikawa,
Genetic Diversity
National Institute
Tsukuba, Ibaraki,Japanof Oryza species based on simple sequence repeats andsequences from the genomes of cytoplasmic organelle

Duncan A. Vaughan, Koh-ichi

Departmentof Agrobiological305-8602

Kadowaki

Sciences

E-mail : kadowaki@affrc. go.jp
Keywords:

Oryza, rice, simple

Simplethe nuclear

sequence repeats (SSRs)

genome among species

polymorphism
flanking

genome information

mitochondrion
chloroplast(Notsuback the genetic

relationships

Hence, the present

organisms.

using2002).

The complete

studies

PCR products that includes
whetherin chloroplast

SSRs

SSR plus

the size differences

sequenced(Hiratsuka

SSR informationand McCouch 2000)

inherited

Earlierinareor that of its flanking sequence. Rice is the only crop where

yet from the completely

genomes are maternallyvariation

to distinguish

both

et al

mitochondria

been used to detect allelicof amplified

is available

genome of rice (Ishii

been studied

species.

in many living

sequence. However, it was difficultof the SSR itself

chloroplast,

have widely

were based on size variation

due to the length
entiresequence repeat,

et al. 1989)
is available

whereas mitochondrial

genome. Chloroplast

for theSSRs have notand mitochondrial

and theircharacteristics

can effectively

among species,

particularly

to study the evolution

study was undertaken

andto assess the level of genetic

be used to traceof polyploidvariationin thegenomes of cytoplasmic
relationships

organelle

basedon SSRs,

and to characterize

the phylogeneticof genus Oryza based on these information.
A total of 44 accessions

tisserantiiof genus Oryza were analyzed.

were used as an outgroup. The SSR information

was obtained

from the entire sequence (Notsu et al 2002).

five chloroplast

SSR regions

sequence determination

were analyzed.

and subsequent

Two accessionsof Leersiaof the rice mitochondrial

genomeA total of seven mitochondrial

Polymorphism

phylogenetic

was identified

analysisandby nucleotide

was performed using the mostparsimony method.
In addition

to length

polymorphism

occurrence of many deletions/insertions
were also identified

in SSR sequence itself,
in the flanking

in SSRs. In conclusion,

SSR region was recognized

the length

by sequence

determination.

misledinformation

because the internal

polymorphic

obtainedboth by chloroplast

similarin principle

among species

relationshipmore detailed
theseparation

relationships

writtencomplexes

the O. officinalis

in thein theirin PCR products
flanking

substitutions

although

genus

the

analysisof theregion as well as

the deletions/insertions

alwaysof SSRs were not taken

and mitochondrial

taxonomic tree. The most parsimoniousof thepolymorphism

Previously,

into account. Results

revealedregion of SSRs. Base substitutions

from the deletions/insertions

among SSR itself

our results

SSR analysesof chloroplast

showed a

SSR showed

tree for the genus Oryza showed clearlyOryza (Fig.

1).

However,the species

complex and O. sativa complex could not be welldiscerned

due to insufficient

showed clear differences

polymorphism

among the complex. The species

with BB, whereas, it was more closely

genome. Our results

stronglysuggests

and CCDD species

analyzed.

This is the firstreportbased

on mitochondrialand chloroplastdifferent

rice

species

relatedof CC genome

to BBCC and CCDD

that CC genome is the maternal parent for the BBCC
on phylogenetic
SSRs

relationships

amongand theirflanking

sequences.

0. barthu
0. glabernma
0. glumaepatula
0. meridionahs
0. meridionahs
0. mvara
0. rufipogon
0. rufipogon
0. rufipogon
0. rufipogon
0. sativa cv Kasalath
0. sativa cv Nipponbare
0. longistaminata
0. longistaminata
0. longistaminata
O.punctata (2X)
O. punctata (2X)
O. austrahensis
O. austrahensis
O. austrahensis
O. austrahensis
O. austrahensis
O. minuta
O. minuta
O. punctata (4X)
0. punctata (4X)
0. eichingen
O. officinahs
0. officinahs
O. £aftyb/uz
O. /ar/fo/w
0. officinahs
O. punctata {AX)0. grandiglumis
0. grandiglumis
0. brachyantha
0. granulata
0. granulata
0. longiglumis
0. nW/ey/
0. longiglumis
0. riT/feyi
Leersia tisserantn
Leersia tisserantn

Fig. 1Phylogenetic

in organelle
10,000analysisof genus Oryza based on variation

genome. The numbers above the nodes represent

bootstrap

replications

W0652
Tog5923
W1169
W1625
W1635
SL10
W0120
W0157
W2078
W2099

O, sativa complex
(AA)

W1421
W1650
W1499
W1514
103887101397101410103318105267
W1639
W1213
W1337
W0043
W0015
W1525
W0614
NIAS WBC6
W0048
W1181
W0002
W1564
W0613
W1480(B)101232
W0022
W0067(B)
W1220
W0604
W1228
W0001
W1345
W1620

O. officinahs complex(BB)(BBCC)(CC)(CCDD)(EE)(FF)
O. granulata complex(GG)
O. ridleyi complex(HHJJ)of SSR loci and their flanking
bootstrap

values expressedsequencesas percentageofAcknowledgments
DNAs from four O. australiensis
providedby Dr. Ishii of Kobe University,

Wethank Ms. Nohara for her technicaldoomed greenhouse, National

Instituteaccessions
Japan.

and one O. brachyantha

We thank him for his valuableassistance.

Leaf materials

of Agrobiological

Sciences,were collected

accession

were kindlycomments on our results.
from plants

grown atTsukuba.

References
Hiratsuka,J., Shimada, H., Whittier, R. et al. (1989)
The complete sequence of the rice (Oryza
sativa) chloroplast
genome: intermolecular
recombination
between distinct tRNA
genes accounts for a major plastid DNA inversion during the evolution of the cereals
Mol Gen Genet 217: 185-94.
Ishii, T. and McCouch, S. R. (2000) Microsatellites
and microsynteny in the chloroplast
genomes of Oryza and eight other Gramineae species. Theor Appl Genet 100:
1257-1266.
Notsu, Y, Masood, S., Nishikawa, T. et al. (2002) The complete sequence of the rice {Oryza
sativa L.) mitochondrial
genome: frequent DNA sequence acquisitionand loss during
the evolution of flowering plants. Mol Genet Genomics 268: 434-445.

Originof Cultivatedand Wild Rice with AA Genome: Based on theInterspersion

Patternsof SINEs

Hisako Ohtsubo*.

Chaoyang

Cheng*, Reiko Motohashi*,

Fukuta,&JBiichiOhtsubo*.

^Instituteof MolecularTokyo 1 13-0032,

and Cellular

Japan;

Biosciences,

Suguru Tsuchimoto*,

The University

and t Hokuriku National

Agricultural

Yoshimichiof Tokyo, Bunkyo-ku,

Experimental

Station,Joetsu943-01 93 , Japan, hohtsubo@ims.u-tokyo.ac.jp
Keywords; p-SINEl , Oryza genus, AA Genome, insertion

polymorphysm,

phylogenySpecies of Oryza genus
Oryza genus comprises approximately
Australia,

and Central

and South America.

cytogenetic

analysisclassified

into six diploid(0. sativa and 0+ glaberrima)

and 0. glaberrima,

are thought

1928).

The 0. rufipogon

perennial(Morishima

strains

et al.,1992).

of 0. rufipogon, both species

Discovery

Africa,subsequentin the Oryza genus are

Among AA genome species,

and five wild species

0. meridionali).

The cultivatedspecies,morphologicallyinto indica

and japonica

have been classified

into

two ecotypes,

Although

0. sativa is thoughtshow remarkably

there are(O* rufipogon, O. BarthiU

to be derived from 0. rufipogon and 0. Barthii,

0. sativa strains have been classified

speculation

crossing,

all species

CCDD, HHJJ and HHKK).

0. longistaminata,

to various

in Asia,

genome types (AA, BB, CC, EE, FF and GG) and four tetraploid0. glumaepatula,species

distributed

Based on interspecificand genomic DNA hybridization,

genome types (BBCC,
two cultivated

22 species

high intraspecific0. sativa
respectively.
(Kato et al.,annualandto be a domesticatedvariation

leading

for the origin of 0. sativa.

of p-SINEl

p-SINElis the first

plant

SINE identified

in the Waxy gene in 0. sativa (Umeda,

et aL, 1991).

It is 122 bp in average size and contains

III,pyrimidine

1).

and an A-T rich region with a variable

lengthp-SINElin the Oryza genome in highwas estimatedto be present

3000 copies per haploidaL, 1997).

genome of 0. sativa and 0. glaberrima,

Many p-SINEl

inversePCR (IPCR)

insertionpolymorphism

members have been identified

or genomic library

screening.

among species

might have retrotransposed
withAA

genome (Mochizuki

transcripts

have been isolated

strainsofT-richa promoter for RNA polymerase

in our laboratory

Hirano et aL, 1994).

etthe divergencemembersof the rice species

Recently,

many p-SINEl

by 5f RACE-PCR from various kinds of organs of 0. sativa
et aL, unpublished).

from the internal

pol III promoter

ofp-SINEl,

activemembers.

These suggestthat the p-SINEl

duringtheSome of these transcripts
suggesting

and are derivedof AA genome species,

had athat they are the products
from some transcriptionallymembers were active
and may be stillin transposition

presently

activeinon.

RA sub family, a recentry
Wecollectedjaponica

amplified

p-SINEl

forty-seven members of p-SINEl
type,members at particularloci

as wellas indica

was determinedtype.

0. glumaepatula,

Seventeen p-SINEl

members were found polymorphic,

duringthe divergencethat these p-SINEl

populations.

Alignment

The presenceor absence of thesewith the AA genome, 0.

0. longistaminata,
indicating

and 0. barthii.

that they may have been

Among those polymorphic

members,

in between 0. sativa and 0. rufipogon. This means

members were amplified

and 0. rufipogon and therefore

loci in the genome of

0. meridionalis,of the rice species.

some showed insertion polymorphism

at different

by PCR in strains

sativa, 0. rufipogon,rufipogon

These polymorphic

et aL, 1992;

5f end of p-SINEl,

amplified(Motohashi

Some members showed interspecificloci during

at the precise

O.sativa

with 6500 andin the genome of 0. sativa by

with AA genome.

sequence startingtranspositi

copies,respectively

into the respective(Tsuchimoto

divergence

tract at the 3f end (Fig.

only recently

after the divergencehave not yet become fixedof the nucleotideof 0. sativa

in the 0. sativa

sequencesand 0.

of the polymorphic

members showed that they
sequences

share

three

commonmutations;in the pol HI promoter and one in the distal

members are transcribed,
mutationsand thus, indicate

have been amplified

RA (Recently

p-SINEl

Amplified)- insertion

end region

that p-SINEl

in recent evolutionary

subfamily

(Fig.

1).

Some of themembers with threetime.

members (Chengpolymorphism

two in the A- and B-box

We therefore

diagnostic
named themet al., 2003).

and phylogenetic

analysisof O. sativa and O. rufipogon.
Wecollected

101 strains

investigatedthe presence

polymorphic

members at particularshowed insertionof 0. sativa and 0. rufipogon.

or absence

polymorphismof 24 p-SINEl

By using PCR, we

members, including

22 RA

loci in each rice strain.

RA members of p-SINEl

among these rice strains.

To show the relationships

among those, each strain was "bar-coded" based on the presence or absence ofp-SINEl
members at the respective
neighbor-joining

loci,a phylogenetic

tree was constructed

method based on the bar codes given to all the strains

In the phylogenetic
IV (Cheng
II.

then

tree, the rice strains

et al., 2003).

The two ecotype2).

into four major groups, I

0. sativa strainsare clearly

separated

into two groups, I andof 0. sativa,

Japonica

and Indica,can be distinguishedstrains

almost exclusively

by the presence

strainsamong all four groups

are divided

are divided(Fig.

by theor absence of p-SINEl
on the tree (Fig.

members.

0. rufipogon

2),

that

indicating0.

rufipogon shows remarkably

high intraspecificvariation.

Interestingly,

all the annual

0. rufipogon

for one strain,

are present

in group II,

whereasI, III and IV (Fig.

2). supportingstrains,

perennialstrains

the

that

idea

(Morishima

exceptare in each of the other three groups,
theannualet al.,1992).

strains

an intraspecificfrom the primitive

Because annual type strainsin the 0. rufipogon populationconsidered

are derived(Fig.

variation,2), differentiation
as reported

theperennialstrainappear to form a distinct

group

into the annual type should

previously(Oka 1988).

bePolyphyletic

originof O. sativa

The phyiogenetic

tree shows the japonica

strainsof 0. sativa

perennialstrains of 0. rufipogon are in group I (Fig. 2), indicative

perennialstrainsof thisgroup

rufipogonof the perennial

type,.

that the japonica

andoriginatedfrom a common source, probably

0.

The indica

strainsof

0. rufipogon are both in group II (Fig.
from the annual strains,commonsource.
perennialplantsindicatingstrains

distinct

are essentiallyand Nepal,

are clusteredthat both types of strainsare clearly

When weconsider the fact that the O. sativa cultivars

type.

et al.,1992),

the source is most probably

However, a few indica strains,with the annual strains

may have originateddomestication.

Most indica

from another(Morishima

strains2).

of 0. sativa and annual strains

are originatedthe perennial

indicaand six

These

0. rufipogon ofincludingthose from Assam (India)(Fig.2).

This

suggeststhat thesein the annual 0. rufipogon population

by recentshow

originatedfindings

that

the

0.

sativastrains

pol yphyletically.

Evolutionary

relationships

the SINE insertion
We further

among rice species

polymorphysm
identified

new p-SINEl

polymorphismfrom representative

strains

The p-SINEl

insertion

glaberrima,

were very similar

respectively.

Phyiogenetic

the strains

to be distantly
glumaepatula.
strains
indicates

patternsof the strainsof cultivatedanalysis

relatedof 0. longistaminatathat 0. longistaminata

tree including
are related

insertion

formed a clusterwith AA genomes.
0. sativa and 0.

patterns

(Fig.

3).

to those of 0. meridionalis.

and 0. meridionalis

showed that
The strains

ancestor

to the hypotheticalare divergedofThey appear

0. rufipogon^ 0. barthii

a hypotheticalmost closely

insertion

0. rufipogon and 0. barthii,

based on the p-SINEl

to those of the other three species,

A phyiogenetic

interspecificrice species,

to those of wild rice species,

appear to be distantly
relatedmembers showingof four wild rice speciesof each of the five wild rice species

0. longistaminata

with AA genome based on

and 0.

showed that theancestor.

This

early, whereas the otherspecies

are diverged

relatively

recently,

genome might have originatedAcknowledgement

that

the Oryza genus with AA

in Africa, rather than in Asia (Chenget al.,2002).

We thank Drs. N. Kurata and Q. Xue for providing

strains.

We are grateful

ecotypesof the O. rufipogon

work was supported
Technology

and suggests

to Drs. H. Morishima
strains

and Y. Sano for the informationand for their criticalby grants from the Ministryof Japan and from the Ministry

the rice

readingof Education,of Agriculture,on theof the manuscript.

Culture,Forestry

Sports,This

Scienceand Fisheries

andof Japa

References
Cheng, C, Motohashi,( 2003)

R., Tsuchimoto,

Polyphyletic

SINEs.Mol.

Biol.

originof cultivated

Y and Ohtsubo,

rice: Based on the interspersion

E.
patternof

Evol.20:67-75

Cheng, C, Tsuchimoto,relationships

S., Fukuta, Y., Ohtsubo,

S., Ohtsubo,

among rice species

H. and E. Ohtsubo,

E. (2002)

Evolutionary

with AA genome based on SINE insertion

analysis.

Genes Genet. Syst. 77:
Hirano, H.-Y, Mochizuki,(1994)

K., Umeda, M., Ohtsubo,

Retrotransposition

H., Ohtsubo,

E. and Sano, Yof a plant SINE into the wx locus during evolutionof rice.

J. Mol. Evol. 38:132-137.
Morishima,

H., Sano, Y and Oka. H. I. (1992)

rice and its wild
Motohashi,

relatives.

R., Mochizuki,

and distributionofp-SINEl
Umeda, M., Ohtsubo,
insertionof mobile

Oxford Surveys
K. Ohtsubo,

Evolutionary

studies

Evol. Biol.

8: 135-184.

H. and E. Ohtsubo.

in cultivated1997. Structures

members in rice genomes. Theor. Appl. Genet. 95:359-368.

H. and Ohtsubo.

E. 1991. Diversification

DNA elements

into introns.

of the rice Waxy gene by

Jpn. J. Genet. 66:569-586.

III

Fig 1 Nucleotide
sequences of the p-SINEl members, most, except rl , showing insertion polymorphism among the rice species A concensus sequence(CON)of p-SINEl (Motohashi et al 1997) is shown at the top A-box and B-box of the pol
promotor are shown in baldface letters
RA subfamily diagnostic
mutations are boxed.

RA subfamily

members may be further divided

into four groups as indicatedby the square bracket

Fig 2 A phylogenetic
tree showing relationships
among the O. sativa and O. rufipogon strains The tree was constructed by the NJ method based on the pattern
the presence or absence of p-SINEl members The four groups ( I-IV) identified
are shown by four deep internal branches indicated by thick lines Each majorgroups are circled. The strains with hatched box belong to O. sativa mdica group The large star indicate a hypotheticalancestor with no p-SINEl insertionofFig 3 A phylogenetic
tree showing relationships
among the 72 strains with AA genome The tree was constructed by the NJ method based on the pattern of thepresence or absence of p-SINEl members All strains were clustered into five groups, corresponding to O. longistaminata,
O. meridionalis, O. glwnaepatula, O.
barthii/O. slaberima and O. rufipogonlO. sativa Note that the strains of O. barthii and O. glaberrima are in the same cluter so as those of O. rufipogon/O. sattva

a

The Complete Nipponbare
genome research
Takuji Sasaki,
Rice

Jianzhong

Wu, Nobukazu Namiki, and Takashi

Genome ResearchInstituteof the Societytsasaki@nias.affrc.go.j
Keywords

genome sequence as a standard

Program,National

for Techno-innovationp

genome sequence, in silico

Institutemapping,

Matsumotoof Agrobiologicalof Agriculture,

Nipponbare,for Oryza

Sciences

Forestryand Fisheries

Kasalath,

Oryza sativa/

The genome sequence of Nipponbare,japonica type variety of Oryza sativa
has been almost completed by the internationalsequencing consortium, IRGSP. All
sequence data for BAC/PAC clones correctly aligned along the 12 rice chromosomes
using DNA markers and fingerprint
data has been read with at least 99.99% accuracy.
As of August 2003, IRGSP has constructeda physicalmap covering 96% of the
Nipponbare
genome which has a total size of 400 Mb The genome sequence provides
the most basic information necessary to understand phenotypicvariations and to clanfy
how these differences
are geneticallycontrolled.
Rice is one of the major cereal crops
and an important food source for more than half of the word' s population.
Improvementof rice production has always been a major concern to guarantee a stable food supply
particularly
in nee consuming regions in Asia, Africa and Latin America where a drastic
population
increase is predictedin the next 20 years The accurate genome sequence ofNipponbare
is expected to now widely used for gene identification
corresponding
tophenotypeby genetic and reverse-genetic
methods For example, precise tagging oftarget phenotype
using DNA markers with sequence information facilitatesefficientapplicationof map-based cloning method in rice. So far, several important disease
resistanceand morphologicaltrait-related
genes have been identified.
The sequence
information is also indispensable
for rapid identificationof corresponding
candidategene within the specific genomic region tagged with DNA markers Reverse-genetic
method is now commonly used to identify the function of a gene with known sequenceby phenotypic
analysisof plants in which the function of the gene is impaired. Thisapproach is greatly facilitatedwith the utilizationof the nee retrotransposon,
Tosl7
which moderately transposes
during tissue culture and serves as an efficient insertionalmutagen. The accurate genome sequence of Nipponbare can be useful for corresponding
gene identification
and analysis of the functions of more than 40-60,000 predictedgenes

in the rice genome.
Rice is produced in a wide range of environmental and climatic conditions,andca. 120,000
varieties
are cultivatedaround the world. Each variety has uniquecharacteristics
adaptedto each cultivating
conditionand differencesin phenotype
account for enormous genomic diversity
This indicatesthat there are at least 120,000
differencesin the genome sequences of cultivatednee, O. sativa. These differences
range from subtle changes involving
one or a few nucleotides
among closely related
varieties to more conspicuous changes such as insertions
or deletions commonly foundbetween japonica and indica ecotypes
More defined differences
can be expected
between O.sativa and its wild relatives
such as O.glaberrima and O.rufipogon, whichcarry favorable
characteristics
not found in cultivatedspecies At present, map-based
cloning following
a detailed
genetic analysis using chromosomal substitutionlines hasmade it possible
to identify genes involved in complex traits controlled
by more than
two loci Analysis of flowering time in nee clearly has shown that subtle differencesincorresponding
genes between Nipponbare
and an indica ecotype Kasalath conceitedly
contributeto the differencein phenotype.
This suggeststhat similar variationinnucleotide
sequences in genes responsible
for the same phenotype exists in other nee
varieties
The Nipponbare
genome sequence with an accurate positional
assignment as
may serve as a standard for clanfying
variation across a wide taxonomical range.
We tried to construct
an accurate physicalmap of Kasalathby in silico
mapping ofBAC-end sequences using the Nipponbare
genome as reference sequence. A
BAC-based physicalmap of an indica ecotype is expected to facilitate
genome-widepractical
applications
such as map-based cloning or position-directed
detailed
genomesequencing. Using ca. 78,000 Kasalath BAC-end sequences, BAC clones were assigned
to chromosome 1 resulting
in 22 contigs with a total length of38 7 Mb corresponding
to89% of the chromosome. This strategy can be applied to other varieties of O.sativa andmight be promising for other Oryza species by introducing
less stringent
conditionforin silico

mapping.
Our knowledge on the diversityof Oryza species at the genome level is very
limited,but we must re-evaluate the gene resources to discover many unknown genes
which may have been activated during the domesticationof wild relatives to cultivatedrice. It may also provide an accurate estimate of the evolutionary
relationships
which
may elucidate the genetic mechanisms of species diversification.
Also information on
relation of variation of nucleotide
sequence such as SNPs with variation of phenotype
may provide an effective
tool for improvement of nee plants based on breeding
strategies

Semi-sterile
perennial
japonlca cultivar
Yo-Ichiro

wild

rice (Oryza rufipogori) as the progenitorof

SATO1 , Ikuo Nakamera2 and Yosuke KURODA3

1. Shkuoka Univ*9 2. Chiba Univ. 3. Kyoto Univ.
We have been collecting
wild rice (Oryza rufipogon and O. nivara) inSoutheast Asia since 1983.
After 1990s, we collected
stem nodes in stead ofseed-samples, in case that the population shows extremely low seed productivity*Here
we report genetic traits among 85 samples.
To classify those samplers into indiciaand japoirica4iomologues,
coda types (deletionor non-deletion
types at PRF 100
regions; Chen et al. 19939 and sequence at the PS-ID region; Nakamura et al. 1997)
were determined.
The results obtained are as follows.
1. Samples used were likely classified
as O. rufipogon (or, perennial type of O«
rufipogon in a broad sense), but scarcely be done as O. nivara.
2. Of the samples, 50 ones showed low seed productivity
and were classified into two
types. First type shows extremely strong seed sterility,
while they bore a lot of
panicles. Second type shows poor panicle bearing throughout a year (Fig. 1).

Fig. 1

A perennial wild rice population
but no panicle was born)in Mekong delta (Phototaken in November,

Wesawthe second type only in Mekong delta with a few exceptions.
first type was seen morewidely.

On the other hand,3.

We compared the proportion of plants having japonica-homologous
CPDNA (eg.
Non deletion at ORF 100 region and/or 6C7A or 7C6A PS-ID types) and indica-homologous cpDNAs (eg. Deletion at ORF 100 region and/or 6C8A or 7C7A
PS-ID types, see Nakamura et al. 1997) in both of the stem-sampled and theseed-sampled populations. In the stem-sampled populations, japonica-homo\ogo\xs
CPDNAwas significantly
frequent compared to homologue of indica (data will beshown at the conference site).

From these facts, and also Yamanaka et al. (2002),
homologous rufipogon was originally vegetative propagator

we consider(Fig. 2).

that japonica-

Fig.2 Schematic
figure showing evolution of rice based on CPDNA types. ND:
Non-deletion at ORF100 region, 6C7A etc. are PS-ID types (see Nakamura et al. 1997).
S: Sterile, F: Fertile.

The experimental results obtained by using collected
seed samples may cause
mislead, because the seeds of such sterile populations
are likely resultantsofoutcrossing by pollen grains derived fromoutside of the population.

References
Chen, B. W.,Nakamura, L, Sato, Y. I. and Nakai, H. 1993. Distributionof deletion typein CPDNA of cultivated and wild rice, Japan. J. Genet. 68: 597-603.
Nakamura, I., Kameya, Y., Kato, N., Yamanaka, S., Jomori, H. and Sato, Y. I. 1997. A
proposal for identifying
the short ID sequences which addresses the plastidofhigher plants.
Breed. Sci. 47: 385-388.
Yamanaka, S., Nakamura, I., Nakai, H. and Sato, Y. I. 2002.
Dual origin of thecultivatedrice based on molecular markers of newly collected annual and perennial
strains of wild rice species, Oryza nivara and O. rufipogon.
Genetic Res. & Crop
Evol. (in press)

Genus, genomes and species:
D A. Vaughan1, A. Shcherban2,

what do they mean in relation

N. Bautista1,

to Oryza?

P.L. Sanchez1, G. Jonson-Miranda1,

T.

Isemura1, A. Kaga1 and N. Tomooka1
National

Instituteof Agrobiologicaland Genetics, Novosibirsk,

Sciences,

Japan; 2 Currently

Instituteof Cytology

Russia.

1. What is the genus Oryza?
Genera are delimited

based

species

includeleavesThe genus Oryza is most closely

(Fig.

1)

presence

on key morphological

In 1965

transferredand shape

threespeciesof sterile

thatto the genus Leersia.

traitsTraitsdistinguishlemma and morphology

relatedto the widely

were previously

Two species,

thatand anatomy of

distributed

consideredO. brachyantha

Oryzagenus Leersia

Oryza species

and O. schlechten,

wereremainin the genus Oryza but are on the boundary between Oryza and Leersia. O. brachyanthaiscloser to the genus Oryza than Leersia although

it shares with Leersia anatomical featuresof the leaf midrib

featuressmallest

ratherthan Oryza. Distinct

genome size of Oryza species(iron)

rock pools

closely

relatedOryza species

and its narrow ecologicalwhere it can be sympatnc

due to the presence

palea

habit

of coriaceousand morphology
Therefore

are that it has theadaptation

with Oryza barthnof the spikelets

that are also polyploid.

very different

speciesto Leersia but scanning microscopy

However, overall
speciesof thisto laterite

O. schlechten
confirmed

is alsothat it is an

and lemma and sterilelemma.

of O. schlecten

are more similar

to Leersia

O. brachyantha

and O. schlechten,

while bothfrom each other, could be considered

speciesthat link to the genus Oryza to

Leersia.
One much studied
Oryza but detailed
species

species,taxonomic studies

to have been placed

Porteresia

was establishedcaryopsis
2. Where are speciesof Fi hybrids

was previously

in the genus

showed it is sufficiently

different

from other Oryza

in its own genus The morphologicaltraitson which the genus

are morphologicalfeaturesin the genus

betweenmeasure of the divergence
determining

coarctata,and anatomicalof leavesandon chromosome pairingatand genomes of Oryza distributed?

Genome designationmeiosisPorteresia

different

Oryza is based
taxa.

This

cytologicalof the whole genome. Recently

genome designation

have been

employedsuch

approach

other

rapid

as total

providesaapproaches

togenomicDNAhybridization(Aggarawal

et al , 1997)

genomic DNA hybridization
seem reasonable.
genome levelapproachand gene phylogenies

is a whole genome approach

However, use of gene phylogenies
for genome designations

to understanding(Ge et al , 1999)

and results

requiresto be accepted.

Totalfrom this method

confirmation

at the whole

Genome diversity

is a better

Oryza evolution

than species

diversitydifficult to distinguishfrom one another,intermediate

accessions

between species

known and different

nee workers have different

regardingthe number of species

opinions

since species

are rather
arein the genus Oryza.
The distributionof genomes of the genus Oryza reveals that more genomes are to

be found in Australia-New

Guinea than anywhere else. Latin America has Oryza species

with 3 genomes, Africa with 4, Asia with 7 and Australia-New Guinea with 8. Comparisonof the main islandsof the Malay Archipelago

NewGuinea (Table

1)

Fig. 1. Relationships

genome diversity

onamong genera within the tribe Oryzeae. Size of box approximatestothe number of species (from Vaughan, 2003)

also reveals

highestTable 1. Oryza species
Archipelago.

and their

Borneo (O. meyeriana,

genomes on the largest

O. officinalis,

meyeriana, O. minuta, O. officinalis,ridleyi,O. minuta, O. officinalis,(0. granulata,

O. rufipogori),

O. rufipogori),that a member of the O. granulata

O. ridleyi,(O.

New Guinea (O. longiglumis,O.

to the west and southeast

O. schlechteri),O. rufipogori)complex is present

in the Malay

Mindanao

O. rufipogon, O. meridionalis,O. meyeriana, O. officinalis,

this complex are present

O. ridleyi,of the islands

SumatraIt might be expected

in New Guinea since species

fromof New Guinea. However, to date noreports of species from this complex in New Guinea have been reported.
2 In this table it is assumed based on distribution
and morphology that the two genomes ofO. schlecten

are different

from others in the genus Oryza.

3. Ecologicalfactors,

speciation

and Oryza species

the species

complexes in the genus Oryza requires

Understanding
the their characteristic
Only species
wet habitats

in the O. granulata

in both the O. granulata
Southeast

complex do not occur in seasonal

complex species
and O. ridleyi

complex species

maybe in lowland

complexes

or upland

occur in forests,

are always in lowlandsofparticularlythus,

or permanently
habitats

Speciesshadedhabitats

along rivers

banks ofAsia and New Guinea.
O. sativa complex and (9. officinalis

taxonomic section,sectioncomplex species

Oryza. However, thesedivergedand ecologicallypressures

The O. sativa complex has evolved

only)consideration

habitats

O. granulata

The O. ridleyi

complexes

selectionhydrologicalpressure
regimeresultingevolvedin diversity

Regional

variation

two complexes

in responseof species

are geneticallyto different

in aquatic- thus annual and perennial

water on an annual basis
is sometimes

have

have been put in the same

habitats

primary

reflect

selectionand the main (but notin this complex is responselife cycle

wellto thelack or abundanceofwithin some species of the O. sativa complex

very clear. However, in some locations

a single

population

may consistofplants

exhibiting

annual, perennial

hydrologicalregime,domesticationof rice must have targeted

does not strictlyforand intermediate

example,correspondprimarilylakesidedue to local variationin thehabitatHuman selection

duringwild nee ecotypes

and thishigh seed producing

to life history.

The Oryza officinalis
diversityalife cycles

complex in contrast

to theO. sativa

complex exhibits

disturbance,a characteristic

in response

to light

A measure of habitat

associatedwith weedy plants

appears

commonto most species

populationsof species

in this complex today tend to be small and fragmented.

size maybe one reason, in contrast

to species

complex have not been domesticated.

A full understanding

were not domesticatedprovideneeds investigation

Populationin the O. sativa complex, species

in thisof why species in this complex

and advances in Oryza genome studies

may

to the 0. sativa complex includes

severalthis
The O. officinalis

allotetraploid

species

workshop(see abstract

genome species

complex in contrast
The three

Among diploideichingen

(CC),

three

withinfrom results

et al ), to have theirallotetraploid

species

is found in shaded habitats
severalthis complex (Fig.

2)

are of shadedor partiallyshadedone species,

complex and speciesof diversity

to the ancestralin thetype for thecomplex.

There are scientificquestionsof relevanceto cultivated

may be sought in the O. officinalis complex. Among these, reflecting
Do species in the O. officinalis complex have the capacity

rice for whichanswers

comments above are;for domestication(seedsize andproductionof some of the 0. officinalis complex species

are not much different

presumedprogenitors

not successfully

Oryza species?
complex?

O.

Vaughan et al 2003c)

show the high levelthat may be closest

at thisgenome from CC

onlyof the O. officinalis

The resultspresented

maternal(see Vaughan 2003b;

studiesCC genome species O. eichingen

O. officinalis

appearBB and CC genome Oryza species

We have undertakenradiation

CCBB species

by Nishikawa

These

habitats

diploidin the complex and thusof rice)?

Why has autopolyploidy

Why is genome size markedly

Why is genome radiation

largera marked characteristic

but not O. sativa complex even though distribution

in species

fromoccurredinof the O. officinalisof the O. officinalis complex

is similar and genetic

studiesthat these two complexes may have evolved over a similar time span (cf Second,suggest1984)

Fig. 2. Diversity

in O. officinalis

codingof a gypsy

domain2001)*

lettersof their specificcomplex based on sequence diversity
likeretrotransposon

BB genome; •E CC genome; T(modifiedof cloned

integrasefrom Shcherban

DD genome; species indicatedet al ,

by first two

name.

Asian group

4. Future directions
1 Despite intensive collecting
to be explored.
Americaof Oryza genetic resources

there are importantTwo vast river systems not yet explored

where wild

Oryza species

are (a) the Orinoco

are known to exist

Pegunungan Foja river system, Irian Jaya, Indonesia

locationsand (b)thewhere information

yetof SouthMamberamo-on Oryza species

is lacking.
i Thereis much that is not known about Australian

O. australiensis
distributedthat has the largest

acrossnorthern

Oryza genetic resources,

particularly

genome in the genus Oryza. O. australiensis

is widely

Australia

and its habitat

is distinct

with AA genome species in Australia.

O. australiensis

drier

It appears

areas than AA genome species

rhizomatousand non-rhizomatous

also reported

for O. australiensis.

naturalhabitatforms

occurs in more elevatedto be a highly

by its afternoon

and thus

variable

specieswithand perennial

strategies

arebut reproductive

isolationin itsAnnual, biennial

It is high seed producing

seems to be ensured

from but often sympatricpollenshed (Oka,

1978,

Second,1987)The diversity

determiningof thisspecies

if within this species

has been little

studiedthere is much variationand it may be worthwhilein genome size,in There has been much speculation

regardingregarding

DD Oryza genome species,

the yet undiscovereda. It is present
currently

diploidin an Oryza accession

no credible

evidence

the DD genome. The are three explanations

alreadyin germplasm

collections

There

is

that this is the case.

b. It occurs in a species that is now extinct
c. The DD genome species
America not yet exploredexplanation.

is yet to be found.

Given the large

for Oryza genetic resources

this later

areas of Latin

seems most plausible

For example, Oryza germplasm of the O. officinalis

complex from the

Pacific coast of Ecuador has not been studied.
iv. Variation within species
As indicatedand standard

at the startother genera. Similarly
We have identified,intermediateof this paper some species
some accessions

based

identified

representativeof two speciesof O. rhizomatis

et al , this conference)

based on morphologicalThus all accessions

that isTateokacharactersof O. eichingenof a species

are not equallyof a species

Speciesoften show clear genetic

grow. Thus an accessionaccession

markers, one population

with 0. punctata.

of the genus withat the boundaryin Sri Lanka (Bautista

two populations

that were intermediate

are at the boundary

can be consideredon genetic

with O. eichingen(1965a,b)

accessionsof O. rufipogondifferentiationin relation

to locationof O. rufipogon for Indonesia

means somethingfrom Sumatra, Indonesia

and O. rufipogon

where theydifferent

for anfrom Irian Jaya,

Indonesia.
These

comments explain

germplasm or accessions

the need for what we have called

representativeof species diversity.

repeatedlyused, for a wide range of studies,collection

best represent

the species

diversityinternational

agreement

to study

species

understandingof the genus Certainly

the genus Oryza would logicallystandard

germplasm set and core collection

more restricted

in number and accessionsof theThisspeciesnecessarily

requiresthat from ourof Asian Vigna. A similar approachin the genus

set ofOur Asian Vigna research has

the same set of accessions

the accessions

be included

a standard

Oryza would help

based onfurther

ourused to create the BAC libraries

forin the standardof germplasm

set Our distinctionis that the standard

betweenset is much

represent

as near as possible

typicalaccessions

analysisof intra-specificgenetic

diversity.

Nipponbare

and Kasalathresearch,which accessions

have become two of the main standard
should be the standard

Some of the characteristics

of a standard

accessions

set of accessions

for neefor Oryza research?

should include:a. Accessions that produce sufficient seeds for research purposes
to high seed producing

accessions(thiswill mean some bias

population

still exist and areaccessions),b. To the extent possiblechoose accessions

where the original

accessible;c. Accessions for which herbarium

specimens have been widely distributed;

d. Coordination

Genetic

Cooperative
originatedby theand Genetic

Rice

Resources

Stock

Committee

coordinatorsof theof countries

International

where theseRicepopulations

and exists

v. What does Oryza research offer to rice research?
The usual merits given for researchsource of genes for resistanceresistanceto pests

on the wild relatives

to pathogens,and diseases

might

pests

of rice are theirand eco-edaphic

most successfully

be sought

usefulness

stressesas a

Genes forin accessions

areas where wild rice grows in areas where rice has been grown since ancient

from

times

The

zone south of the Asian mountain chains into which winds and rain are driven during themonsoon are likely

placesto find pest and disease

is not necessarily

an approach

to finding

resistanceare know in rice germplasm.

resistancemight be more widely found in the genus
Rice is a genome model for the monocots
have widely

cereals to understanddiverged.

cereals

A full understanding

domesticatedprovideanswers in relation

complex and O. officinalis

allopatnc

Oryza is a rather primitive,

stresssmall genus
offeredcomplex may furnish answers not available

questionsandsources of eco-edaphic

syndrome means The contrastof the genetics

are key research

Usefulsince both

Oryza may offer the best example among major

what the domestication

O. sativa complex and O. officinalis

in wild rice. However, logic

sources of resistancesympatric

in which species

resistanceof domestication

Contrasting

to animal domestication.
complex can provide

and inabilityby thewith otherto becomeAsian and African elephants
Perhapscontrastingsome answers to thesecanthe O. sativa
questionsforplantsReferences
Aggarawal,

R.K., D.S. Brar, and G.S. Khush. 1997

Mol Gen Genet 254 1-12;

Ge, S.,

T. Sang, B.R. Lu, D.Y. Hong. 1999. Proc. Nat Acad. Sci USA 96 14400-14405,

Oka,

H.L 1978.

an observationof wild rice species

NIG, Japan 24 pages,

Second,G. 1984.

notesof wild

rice

on a collection

Tateoka,T. 1965a.

76.366-373,
154;

species

D.A. 2003a.

The genepools

Vaughan,

D. A. 2003b

contribution

27:44-48;

Second,Tateoka,on genus Oryza. Oxford(ed.)

Peradeniya,

of Agrobiological
Sri Lanka;

Fieldlibrary),

Mag. Tokyo

Genetica

108 145-

to Nanda, J.S

D. A., N. Tomooka and A. Kaga, 2003c

in

Japan

historyNew
rice,

Department

Sciences,

G. 1987.

of wild

A.H.M and D.A. Vaughan (eds ) Conservationof the joint

from& DBH Publishing,

The natural

Jayasuriya,

Institute

Vaughan,Botof genus Oryza. Pages 1 13-138

University

Proceedings

T. 1965b.

and N. Tomooka. 2001

in H. Morishima

1216

May, (mimeo- IRRI,

Hokkaido

crops

Press,

Australia,

in Australia,

A., D.A. Vaughan

and S.D. Sharma (eds ) Monograph
Dehli,The Nucleus

Bot Mag. Tokyo 78 198-201,

Shcherban,

Vaughan,in tropicaland use of wild relativesof Agriculture,

Sri

Lanka

workshop.

Departmentofand Nationalof Agriculture,

The Oryza Map Alignment
Oryza.

Project

Toward a closed

experimental

system for the genus

Rod A. Wing 1, Scott Jackson 2, Lincoln Stein 3
1 Arizona Genomics Institute,
Department of Plant Sciences, The Universityof Arizona,
Tucson, AZ, 2 Purdue University,
West Lafayette, IN, 3 Cold Spring Harbor Laboratory,
NY.

With the approachingcompletion of the International
Rice Genome Sequencing Project's
penultimate goal of assembling a base-perfect finished nee genome sequence by
December 2004, science must now set its sights on a complete functional
characterizationof rice. Such analyses will include functional annotation of the nee genome using fulllength cDNAs and mutant knockout lines, proteomic and metabolic pro filing andcomparative genomics Ultimately,
the knowledge gained from this work will lead tosuperior rice cultivars that are essential to feed our ever expanding world population.
One important area in the functional characterizationof rice is in the analysis of
the wild relativesof nee. Wild rice species offer an enormous potential to expand thegene pool for cultivatedrice as well as help in the identificationof conserved regulatory
elements To this end our consortia was just funded by the US National Science
Foundation to develop and exploit a set of tools designed to physicallyalign the genomesof representativesof 12 wild rice species (Table 1) to the sequenced Oryza sativa ssp.
japonica cv. Nipponbare genome - The Oryza Map Alignment Project (OMAP
www.
omap.org)
The long term objective of OMAP is to create a genome-level closed
experimental
system for the genus Oryza that can be used as a research platform to study
evolution, development,
genome organization,
polyploidy,
domestication,comparative
genomics, gene regulatory networks, positional
cloning, and crop improvement
The specific objectives
are to:•E Construct fingerprint/BAC
end sequence physicalcomprising the 10 genome types•E Align the 12 physicalmaps with the sequenced
Oryza sativa ssp. japonica cv. Nipponbare•E Reconstruct

rice chromosomes

maps of 12 wild rice species
reference

AA genome diploid1, 3 and 10 across all 12 wild species•E Data mine BAC end sequence/positional
elements etc.

informationfor SNPs, repetitive

In my talk I will discuss OMAP, recent progress in BAC library construction of the wild
species, and a proposal to determine if the wild species accessions that we are aligning to
the sequencedjaponica
genome can be used as the standard reference species forexperimental work throughout the world.

Table1

Genus species
Oryza rufipogon
Oryza rufipogon
O. glaberrima
O. punctata
O. ojficinalis
O. minuta
O. australiensis
O. latifolia
O. schlechten
O. ridleyi
O. brachyantha
O. oranulata

Cytogenetical

Studieson Alien Chromosome Addition

sativaL.), Carrying

Yasui and Nobuo Iwata

Plant

Breeding

Laboratory,

Fukuoka, 812-8581

Faculty

Japan;

e-mailKotschyof Agriculture,

Graduate

School,KyushuUniversity,

hyasui@agr.kyushu-u.ac.jp

Key words: Oryza sativa L., Oryza punctata,mtrogression,(OryzaExtra Chromosomes ofO. punctata

HideshiLines in RiceKotschy,monosomic alien additionline (MAAL),

insect resistance,
Abstract

Genome analysis
been carried
includesof the genus Oryza based on chromosome pairingout and A, B, C, D, E, and F genomes have been identified.

22 species,

two of which are cultivatedO. sativa

and O. glaberrima

remaining

20 species

which

includinggenes for resistancewellstresses

as to abiotic

and wild rice accessions

pests

resistantshowed resistanceand pathogen

be easily

obtained(Fujita

et al 2003)

are A genome species

to major diseases(Swammathan

are useful1986)

to various

usefulconsistare wild relatives

A comparison
pests

revealed

to insectpests

germplasm

for rice breeding.

The wild relatives

genes from more distantly

Hybrids

for instance,

related(Vaughan1994)

rice and theWild relatives(Heinrichs

the highare

et al 1985)of the percentages
that

has

The genus Oryzaof cultivatedand insect pests

and alien gene from wild relatives,

To transfer

hybrids

and the others are wild relatives

five A genome species

sources of usefulaccessions

in interspecificasof cultivatedproportionof wild

with such resistanceto insect

withinA genome species

transferred

species

to cultivated

can
rice

is much more difficult

inbecauseof theirpoor crossability

and such wild relatives
step in transferringwhichwith cultivatedhas been carriedrice.

out (Jena

Jena and Khush (1989)
genome)and Khush 1984,

alien genes is the development

have an alien chromosome in addition

Wide hybridization

O. sativa

Brar et al 1991)

The firstof monosomic alien addition
to the diploid

complement

lines

a complete

set of MAALs for O. officinalis

The second step involves

the screening

transferring

alien

genes to cultivatedrice(2n=24,CCof alien introgression

lines with intactalien

genetic

Another approachis throughchromosome fragments

derivedresearch

was conductedto study the cytogenetical

additionlines

each carrying

about transferringfrom wild relatives

an O. punctata

background.

the integrationbasis of various kinds

chromosome(s)Fifty-three

from the three

original

interspecificbetweenautotetraploidO. sativa

L. of Japonica

diploidstrain

12 typeson the basis of their morphological

Kotschy,

Nipponbare(Iwatacorrespondto any of the primary

the 12 morphologicaland Omura 1984)

types,

hybrids

W1514(2n=2x=BB)

Morphological

10 were fertilealien traits(2n=3x=AAB)

cultivarresemblancetrisomics,

basic

specificknowledge

Nipponbare

to O. punctata

plantsobtained(2n=25)to the 12 primaryfeaturesof the other

MAAL 3 and MAAL 12, however, were completely
at meiosis

from O. punctata

into O. sativa cv. Nipponbare

sterile.

in all the MAALs suggesting
throughand aAmong them, 39 can be grouped
trisomics

recombination

toof cultivar

14 MAALs did notunder any groups

and the alien chromosome can be transmitted

12UH-1x association

were

from a cross(2n=4x=AAAA)

hence could not be classified

showed mainly

This

rice.

monosomic alien addition

isolatedof smallof alien chromosome

and to advanceA complete set of MAALs was produced to determineof O. punctata

or substitution

to the normal chromosome complement

alien genes of wild rice to cultivatedchromosomes (Yasui and Iwata 1 991)

(MAALs),of the rice cultivar

first developed

genes or alien chromosome segments in the recipient

progenies,

between

Cytological
that

Of

to eachanalysisgene transfer

is very low in frequency.

Severaltraitsderivedfrom a diploidstrainof O. punctata

were assigned

chromosomes by comparing each primary tnsomics with the respective
same genetic

backgroundof japonica

chromosome 12 to the genetic
cytoplasmcausedcultivarbackgroundsevere male sterility.

Nipponbare.

successfully
purplealien

to eliminate

leafhopper

pigmentation,

to chromosomes
resistanceon O. punctata

to Japonica
spottedalien

with Nipponbare
for F{ sterility

to chromosome 12 transferredcultivarof O. punctata,
was also

chromosome could

from O. punctata.

chromosome 12 to transfer
Nipponbare.

leaves at ripening

from O. punctata

chromosome of a MAAL in which

Nipponbarethat one of the gene(s)3, 7, 8 and lltransferredof O. punctata

suggestsgene(s)genes from O. punctata

stigma, red pericarp

also assigned

these

The addition

cultivar

between O. sativa and O. punctata was assigned
Thus it is necessary

MAALs, which have theof japonicaThis

to the specific

Major genes forstage

and late heading

were

respectively.

In addition,

green

assigned

to a specificalien

not be identified

by karyotype

analysis.
More fertilealien chromosome addition

the gametes

carrying

These plants

were designatedand 9S (short(MaAAL)acrocentric

at diakinesis

than

the

chromosome fragment

chromosome (Yasui

alien addition

lines

transmissionofand Iwata i998a)(MtAALs:

chromosome 9) and monoacrosomic

and first

metaphase.

This

from a misdivisionby chromosome breakage
respectivewith stablealien

2n=2x+lt)

2, 7

additionline

Most of the PMCs in the MtAALs and the MaAAL showed \2xl+\x

chromosomes originatedanaphase II followed

or acrocentric

as monotelosomic

arm of O. punctata

4S4L, respectively.

configuration

higheran alien telocentriclines were developedthator acrocentic

that

the extra

telocentnc

orat anaphase

I or

MAALs Their seed fertility

wasof an alien univalent

in the respective

MAALs, suggestingsuch as telocentricsuggests

theadditionlines

carrying

a smallchromosomes can producefunctional

gametes

Ditelocentric

telocentricchromosomes of O. punctata

7, ll

alien additionand one unidentified

chromosome of the three

separated

to each daughter

telocentricchromosome at hightransmission

high(DtAALscellchromosome are functional

completely

at anaphaseI, givingfrequencies

The transmission
progeny

chromosome(s),in both female

is a small

chromosometransmittedprogenies

and stable

chromosomes may be transferred

appliedto detectspecific
introgressed
carrying

The DtAALs showed stablewithan extrafunctional

telocentricthat a telocentric

centromere,

can begenetic

backgroundthroughhybridization.

using

total

using O. punctata

O. punctata

from O. punctata
total

rice. This

chromosomes

DNA as a probe wasto the Japonica

DNA allowed

methodcultivar,

the detection

may be usefulin the progenies

telocentricof theof ato identify

MAALs eachan alien chromosome of wild relatives
Introgression

singlechromosome

alien

1997)

or translocatedwith an alien

These

et al

segments

gametesand usually

generation.

transferred

into cultivatedthe alien

next

chromosomes

chromosome transferred

the meiosis,

This suggests

with

alien

GISH

MtAALsrates of the alienof DtAALsa pair ofof respectiveat the pachytene

rise to viablefragment

to any cultivated(GISH)(Yasuipairedwhere gametes

in theGenomic in situ hybridization

Nipponbare

Duringand male germ cells

chromosome, thatto thefrom the progenies

DtAALstelocentricof rice each carrying

and Iwata 1998b)in the self-pollinatedof alien

2n=2x+2t)

were isolated

MtAAL (Yasui

telocentricwere considerably

lines

homoeologous

has been extremely

limitedin the disomic

chromosomes from O. punctata

MAALs Thus, it may be impossibleof MAALs To transfer

alien

derivatives

in the BC2 progeny

proposed.

The F, plants

usuallyprogeny

form univalentof MAALs because
duringmeiosisthat homoeologous

recombination

occurs during the meiosis

chromosome segments

to cultivatedrice,

derivedbetweenfrom the hybrids

have a complete

set of chromosomes

monitoring

wild and cultivatedof cultivatedofdisomic
rice will be

and wild species

inwhere the homoeologous
for favorable
disomic
alien

traitschromosomes may rarely

had been successfullyprogenies

ofBC2 plants

introgression(Jenapair and exchange

introgressedchromosome segment(s)conferringfrom wild relatives

and Khush 1990,

will be gamma-ray irradiation
favorable

each other

Ishii

Severalto cultivatedet al 1994)of MAALs carrying

rice in the

Another
limitedgenes

approachforamount of alienagronomic traits

Acknowledgments
I am greatly

indebted

to Dr G. S. Khush, Plant(PBGB),

International

acceptingme to conduct my thesis

National

Agriculture

Center,for their

guidancein the insect

T. Omura, Faculty

Rice ResearchInstitute(IRRI),

work in IRRI

Ministry
resistanceof Agriculture,

Breeding,

research.
Kyushuand Biochemistry

Divisionfor his kind

supportand forare also due to Dr R. Ikeda,

formerPhilippines,
Thanksof Agriculture,

Genetics

Dr E. Angeles,
Special

University,

PBGB, IRRI, Philippines,

appreciation
for

his

is to Professor
continuous

Emeritus

guidanceandencouragement.

References
Brar, D. S , R. Elloran and G. S. Khush (1991)
rescue between cultivatedand eight wild species

Interspecifichybrids
produced throughof rice. Rice Genet. Newsl 8 91-93

embryoFujita, D., K. Doi, A. Yoshimura and H. Yasui (2003)
Mapping of a new resistance gene forgreen rice leafhopper
introgressedfrom Oryza rufipogon Griff, to cultivatedrice, Oryza sativa L.
Rice Genet Newsl 20 (in press)
Hemnchs, E. A., F. D. Medrano and H. R. Rapusas
resistance in rice. IRRI, Philippines,
p356.

(1985)

Genetic

evaluation

for insect

Iwata, N. and T. Omura (1984)
Studieson the tnsomics in rice plants (Oryza sativa L.)
accomplishmentof a tnsomic seriesjaponica rice plants Japan. J. Genet 59 i99-204.

VI An

Ishii, T., D. S. Brar, D. S. Multani and G. S. Khush (1994)
Molecular
tagging of genes for brownplanthopper
resistanceand earlmess introgressedfrom Oryza australiensis
into cultivatedrice, O.
sativa. Genome 37 217-221.
Jena, K. K. and G. S. Khush (1984)
Embryoimprovement Rice Genet Newsl 1 133-134.

rescue ofinterspecifichybrids

Jena, K. K. and G. S. Khush (1989)
Monosomic alien addition
morphology,
cytology,
and breeding behavior
Genome 32 449-455
Jena, K. K. and G. S. Khush (1990)
Introgression
cultivatedrice, O. sativa L. Theor Appl Genet
Swaminathan,

M. S. (1986)

Plant

research

80

lines

and its scopeof rice:of genes from Oryza officinalis
737-745

and world agriculture.

Plant

in riceproduction,

Well

ex Watt to

Mol BiolRep. 4: 1-17.

Vaughan, D. A. (1994)
The relationshipbetween the genus Oryza and other grasses
relativesof rice" International
Rice Research Institute,
Philippines,
3-5

In "The wild

Yasui, H. and N. Iwata (1991)
Productionof monosomic alien additionlines of Oryza sativa
having a single O. punctata chromosome. Rice Genetics II, International
Rice Research Institute,
Philippines,
147-155
Yasui, H. and N. Iwata (1998a)
Development
additionlines in rice {Oryza sativa L.) carrying
Breeding
Science 48 181-186.

of monotelosomic
and monoacrosomic aliena single chromosome of O. punctata Kotchy.

Yasui, H. and N. Iwata (1998b)
Cytogenetics
of ditelosomic
alien addition
extra pair of telocentricchromosomes of Oryza punctata Kotschy.
J. Fac. Agr., Kyushu Umv.43(1-2)
1-9.

lines

Yasui, H., K. I Nonomura and N. Iwata (1997)
Detectionof alien Oryzapunctata
chromosomes in rice, Oryza sativa L., by genomic in situ hybridization.
J. Fac. Agr., Kyushu Umv. 42(1-2)
63-68

each carrying

Kotschyan

POSTER PRESENTATIONS
Proceedings ofInternational
Genetic Resources
Workshop on
the genus oryza
24-26 September 2003
Tsukuba, Japan

Genetic relationships
N. S Bautista1,^neticamong the three diploid

CC genome species of Oryza

A. Kaga1, A. H. M Jayasunya2, N. Tomooka1 and D. A. Vaughan1

Diversity

Department,

National

Instituteof Agrobiological

Sciences(NIAS),

Japan.2 Plant Genetic Resources Center, Department of Agriculture,

Sri Lanka.

Tsukuba,

Email nonnie@affrc.go.jp
Keywords:

Oryza; CC genome species,

molecular

markers

There are three diploid

CC genome species,

distribution

CC genome species is from West Africa to Papua New Guinea. However,of diploid

only in southern

South Asia can all three

analyze the genetic
Materials

O. eichingeri,

diversity

species

Sri Lanka),of 19 accessionsof thisstudy

was tofrom South Asia.

of O. rhizomatis,

7 of O. eichingeri

4 of O. officinalis (from Kerala and Assam, India and China)
were consistingof 8 accessions

from southern South Asia) and 1 accession
Three moleculartechniques,

polymorphism(AFLP)and simple

SSR primers from Cornell
polymorphism

polymorphic(SSR)

O. rhizomatis, O. eichingeri

University
in the diploid
diploid

accessions

used only in SSR analysis)

DNA (RAPD),

sequence repeats

SSR primers derived

Outgroup(all fromof O. nivara and 5 of O. rufipogon (allofO. sativa (Nipponbare-random amplified

CC genome species,

there are no available

detecting

The objectiveof the three CC genome species

used consistedof AA genome speciesof diploid

be found.

The

and Methods
Materials

lengthO. officinalis and 0. rhizomatis.

amplifiedwere employedfragment

in the analysis

and O. officinalis accessions

from CC genome species,

Since

the AA genome derived

were surveyed to find out which primers can be usefulCC genome species

polymorphism

among the threeCC genome accessions

the 12 linkage

groups of rice, but relatively

Of these primers,in17.2% revealed

These primers are distributedonfewer primers were found in chromosomes 4, 9 and

10.

Results
1 All three molecular
to O. eichingeri
eichingeri

techniquesrevealed

than O. officinalis (Table

accessions(Fig.

rhizomatis but was collected

1)

This
withinthat O. rhizomatis

is genetically1)of O. rhizomatis

accession

One accession

has all the morphological

5 km of populationsof O. eichingeri

more closely

relatedclusters

with O.

characteristicsof O.

in Sri Lanka.

Both O.

rhizomatis

and O. eichingeri

occur in Sri Lanka whereas O. officinalis is found in other parts of

Asia.

2. AFLP and RAPD analyses revealed
analysisgroupedtheaccessions

eco-geographic

differentiationin O. rhizomatis.

of O. rhizomatis

from northernand southern

by SSR analysis

Cluster

Sri Lanka

separately.

This was not revealed

and that may be due to the larger

number ofpolymorphic

bands revealed by the dominant markers RAPD and AFLP compared to those of theco-dominant SSR markers
3

AFLP and RAPD analyses

genome species

revealed

that inter-population

was greater than AA genome species

same region (Table

1)

This suggests

genetic

diversity

genetic

diversity

in diploid(O. nivara and O. rufipogon)CCfrom thethat the CC genome species may be a rich gene source forrice improvement
4. Intra species

among the CC genome species

officinalis

and least

eichingeri

compared with the other two CC genome species

Table

for O. eichingeri

1. Average genetic distanceThis

analysed

may be a reflection

between pairs of the 3 diploidwas greatestof habitat

stability

for O.
of 0.

CC genome species and among

AA and CC genome species of rice based on RAPD, AFLP and SSR analyses
C o m p a riso n

R A P DN

A FL PSSR

G D A veN

G D A veN

G D A vee tw e e n O . r h iz o m a tis a n d O . e ic h in g e r i 1 0 2

0 .1 9 0 1

13 3

0 .1 3 6 9

13 3

0 .0 9 0 8

e tw e e n O . r h iz o m a tis a n d O . offi c in a lis

68

0 .1 9 3 076

0 .1 6 3 376

0 .1 1 4 6

e tw e e n O . e ic h in g e r i a n d O . offi c in a lis

24

0 .1 9 5 4

28

0 .1 7 0 1

28

0 .1 1 0 2A m o n g C C g e n o m e a c c e s sio n s

3 51

0 .1 4 9 3

43 5

0 .1 0 6 3435

0 .0 8 0 7

A m o n g A A g e n o m e a c c e ss io n s

78

0 .1 0 9 678

0 .0 6 3 991

0 .0 8 9 2

Acknowled gements
The authors are gratefulstudy and to colleagues
with usto IRRI, the Philippines
for supplying some of the materials used in thisin the Department of Agriculture that have cooperated in Oryza studies

Fig. 1. Dendrogram of diploid
AFLP analysis.

CC genome species

Rh - O.rhizomatis;

diversity

Ei - O. eichingeri;

based on polymorphism

Of- O. officinalis.

revealed byBiaseddistribution
Arabidopsis
Full-lengthofpyrimidine-rich
cDNAs

ShigeoFujimori1'3,

Takanori

Washio1 , Kenichi

KenichiMatsubara5,

Jun Kawai6, Piero Carninci6,

Higo4,

microsatellites

inYasuhiroKazuo Murakami5,

Yoshihide

Ohtomo5,
Hayashizaki6,

Rice

Shoshiand

Kikuchi4,

and Masaru Tomita1'2
institutefor Advanced Biosciences,

2Department of Environmental

Keio University,

Tsuruoka, Yamagata 997-003 5, Japan

Information

3Graduate School of Media and Governance, Keio University,
National

Instituteof AgrobiologicalFujisawa,

Sciences, Tsukuba, Ibaraki 305-8602,

foundation

for Advancement of International

laboratory

for Genome Exploration

Science (FAIS),

Kanagawa 252-8520,
Japan

Tsukuba, Ibaraki 305-0062,

Research Group, RIKEN Genomic Sciences

Tsurumi-ku, Yokohama, Kanagawa 230-0045,

Japan

Japan

Center (GSC),

Japan.

Email: washy@sfc.keio.ac.jp

Keywords: microsatellite,

tandem repeat, full-length

CDNA

Summary:
Repeats, such as microsatellites,
are ubiquitously
present in eukaryotic
genomes. Microsatellites
located within or near transcribed regions can potentiallyaffect thetranscriptionalor translational
activities.
In this research, we conducted a computational
analysis to assess the variations of microsatellites
in transcribedregions using foil-length
cDNAs of rice (Oryza sativa ssp. japonica c.v. Nipponbare)(Kikuchi S. et ah 2003) andArabidopsis
thaliana.
In addition,
human and mouse (Okazaki Y. et ah 2002) were
simultaneously
analyzed for a comparative analysis. Our analyses confirmed that thedensities of microsatellites
near transcription
start sites (TSSs) in plant genes were higher
than those in mammals (Fig. 1). It was also confirmed that microsatellites
containing CpTdinucleotides,
such as (CT)n, and (CCTT)n are found intensively near the TSSs, specificallyin the two plants, but not in the mammals. Our additional
finding is that microsatellites
containing CpG dinucleotides(e.g., (CG)n, (CGCT)n, and (CGTCT)n)are preferentiallylocated in TSS-flanking regions only in rice. Our results suggest that microsatellites
locatedwith high frequency in 5'-flanking regions can potentiallyact as factors in transcriptional
and/or translational
regulation, possibly repressing transcription
in response to methylation,
controllingtranscription
efficiency through forming unusual DNA structures, or being
cis-regulatory element to control translation efficiency.

Figure 1. Relative frequencies of microsatellites
in full-length
cDNAs. Histograms
illustrate
percentages of repeats on each position, in cDNAs. The relative position of the
microsatellitein each CDNAwas calculated as the sequence length of the upstream of the
microsatellite
divided by the length of each CDNAexcluding the microsatellite.

Acknowledgement
Wewould also like to thank TMRI for offering Oryza sativa genomic sequence data. This
work was supported by the Ministry of Agriculture,
Forestry, and Fisheries of Japan (RiceGenomeProject SY-1 104).

References
Kikuchi S. et al ; Rice Full-Length
CDNA Consortium; National Institute of Agrobiological
Sciences Rice Full-Length
CDNA Project Team; Foundationof Advancement ofInternational
Science Genome Sequencing & Analysis Group; RIKEN. (2003) Collection,
mapping, and annotation of over 28,000 CDNA clones from japonica rice. Science 301,
376-9.

OkazakiY. et al. ; FANTOM Consortium;

RIKEN Genome Exploration

I & II Team. (2002) Analysis of the mouse transcriptome
60,770 full-length
cDNAs. Nature 420, 563-73.

Research Group Phase

based on functionalannotationofMappingof a new resistancegene for green rice leafhopperfrom Oryza rufipogon Griff, to cultivated

Daisuke Fujita.

KazuyukiDoi, Atushi

Plant

Breeding

Laboratory,

Faculty

Fukuoka, 8 12-8581

Japan

Keywords,

Oryza sativa,

introgressed

rice, Oryza sativa L.

Yoshimura and Hideshi

of Agriculture,

Yasui

Graduate

School,KyushuUniversity,

E-mail* dfujita@agr.kyushu-u.ac.jp

Green rice leafhopper(GRH),

resistancegene, introgression,Oryza

rufipogon

Wild relatives

are sources of useful

The Nephotettix
pests

cincticepsof rice in temperate

was found to be highly
present

study,

resistant

The hybrids

rice leafhopper

to GRH by testing
lines

GRH)

antibiosis

of rice each carrying

the GRH resistanceof O. rufipogon (W1962)
parent(Greento major diseases

Asia. An accession of wild relative,

mtrogression

were used to analyze

Uhler

genes for resistanceintroduced

and continuously

pests

is one of the serious

insectOryza rufipogon(Kishino

O. rufipogonfrom O. rufipogon.

as female was crossed with a Japonica

was obtained

and insect

backcrossed

variety

Griff.

(W1962)and Ando 1978)

In thechromosome segments
A resistantaccession

Taichung

65 as maleby Taichung65 to BC4F1!

introgressed

To evaluate

nymph mortality

as GRH resistance,

excisedand infested

with 7-10

first-

collected

in Fukuoka

Prefecture

in 1991,

dark condition.
69 resistantand 29 susceptible

indicating
Grh5

The nymph mortality(Greenlocationriceof Grh5,

or second-mstar

The segregation
was controlled

resistance

5),

BQFj

were analyzed

by a single

tentatively.

analysisone SSR marker, MRG3154

was detected.

Further

SSR analysis

Grh5 was located

MRG0615

GRH resistancegene locatedgenes located

Grh5

distribution,

segregating

into

to a 3 1 ratio( % 2=1.35),
asthe chromosomal

Sequence

Repeats

between(SSR)

Grh5 andusing BC4F2 revealed

that

linkedandand tightly

to MRG2754

in long arm of chromosome 8 (Fig.

1) Thereon chromosome 8. We finallya new GRH resistancein BC4F2 population.

than that of variety

8h

The linkage

identified

on chromosome 8 using O. rufipogon introgression

for Grh5 was selected
higherregionand 16h light

To determine

segreganton the distal

at 25°C

dominant gene designatedby bulked

MRG2761

wasusing 767 Simple

and MRG5845

werepopulation

ratio fitted

markers (McCouch et al 2002)

betweenof 98 BC4F2 plants
The insect

and have been rearing

leafhopper
plantsnymphsin BC4F2 showed a discrete

plantsthat the GRH resistanceleaf blades

carryingfrom O. rufipogon

singleThe nymph mortality
GRH resistanceis a favorable

lines

Nearlyhave been no

isogenicline

was more than 90% that is

genes reported

previously.

gene resource for rice improvement

Thus,

Linkage map of Grh5, a gene forresistance to green rice leafhopper,
on chromosome 8. Frameworkmap on the upper was quoted from
Harushima et (1998)

al

References
Kishino,K. I. and Y. Ando (1978)leafhopper

Nephotettix

J.ApplEnt Zoolcincticeps

Cartinhour,

Q. Zhang,

D. Schneider,mapping of2240

Uhler

resistance

1 Laboratoryof thetechnique

rice

plant

for testing

to thegreen

the antibiosis

rice
Jpn.

22: 169-177.

McCouch, S. R., L. Teytelman,
Z. Li, Y. Xing,

InsectY. Xu, K. B. Lobos, K. Clare,

I Kono,M. Walton,

B. Fu, R.Maghirang,

G. DeClerck,

D. Schneider,

M. Yano, R. Fjellstrom,

S. Cartinhour,

D. Ware, L. Steinnew SSR markers for rice (Oryza sativa

L.)(2002)Development

DNA research

9: 199-207

S.
andVariation
sativa.

in the spacer lengthof the ribosomal

RNA gene cluster

in Oryza

Yoshiki Habu, Fumio Taguchi-Shiobara,
Sugihiro Ando, Shinichiro
Kamachi, Tomohiro
Yamada, Takashi Hagio, Yutaka Tabei, Masahiro Yano, Takashi Matsumoto and Takuji
SasakiNational Instituteof Agrobiological
Ibaraki, Japan (habu@affrc.go.ip)
Keywords

ribosomal

Sciences,

RNA gene, non-transcribed

Kannondai

2-1-2,

spacer, heterogeneity,

Tsukuba

305-8602,

Oryza sativa

Genes encoding 17S, 5.8S and 25S ribosomal RNA (rRNA) are clustered in tandem at
one or few loci in the genome and form nucleolus, that is a structurally
distinct entity innucleus and the site for biogenesisof ribosome. RNA polymerase I transcribes
theprecursor RNA that contains above three rRNA sequences and the transcriptisprocessed to generate each rRNA species Transcription
start and termination
sites
locate within the non-transcribedspacer region that separates 25S and 1 8S rRNA coding
sequences In general, the non-transcribedspacer region consists of a set of complex
repeats and, in some organisms, additional
promoters that generate short transcripts
withunknown function have been identified
in the non-transcribedspacer region. DNA
methylationin the non-transcribedspacer region has been shown to be involved in theepigenetic
regulationof the rRNA gene activity in inter-specifichybridsof Brassica,
known as nucleolar dominance.
Previous studies have shown that there is a considerable
variation in the lengthof the non-transcribedspacer in the genus Oryza. The length heterogeneity
has often
been observed even within individuals
in a cultivar and the length variants were
inherited independently,
indicatingthat the rRNA gene clusters carrying different
spacervariants locate at unliked loci in the genome.
Towards understanding
the epigenetic
regulationof rRNA genes in Oryza
sativa, we re-examined the length heterogeneity
in the non-transcribedspacer length intwelve cultivars of Oryza sativa, including
three japonica,
two javanica and sevenindica cultivars. The length heterogeneity
within individuals
was observed all of thecultivars ofjavanica
and indica, but not in those of japonica. The differencesin thelengthcasesof the variants are often slight and intensity of one of the signals is weak in many
Since it has been shown that Kasalath has two rRNA loci each on chromosome 9and 10, respectively,
we analyzed the Nipponbare-Kasalathchromosome segment
substitutionlines carrying the Kasalath sequence of the short arm of chromosome 9 in
the Nipponbare background and the results indicate that the major variant of the nontranscribedspacer in Kasalath locates in the short arm of the chromosome 9.
The results presented here show that the length heterogeneity
in the nontranscribedspacer of rRNA gene cluster is common in javanica and indica cultivars
The presence of the length heterogeneityin javanica and indica, but not japonica,
cultivars coincides with the previous data of in situ hybridization
showing that javanica
and indica, but not japonica, cultivars tend to have two rRNA loci in the genome. The
representative
data of the Nipponbare-Kasalathchromosome segment substitutionlines
suggest that most of the javanica and indica cultivars possibly
carry a distinct but
homogeneous sequence of the non-transcribedspacer in each cluster of the rRNA genes

Development of DNA markers close to the wheat Wx-Bl locus using ricegenomic sequences
Goro Ishikawa,
National

Mika Saito, Patricia

Agricultural

Japan (e-mailVrinten and Toshiki Nakamura

Research Center for Tohoku Region, Morioka, Iwate 020-0198,

tnaka@affrc.go.jp)

Keywords: Triticum aestivum, Wx-Bllocus, DNA marker, genetic colinearity
In hexaploid wheat (Triticum aestivum L.), there are three granule-bound starchsynthase genes (GBSS) genes, also known as waxygenes, which synthesize amylose inendosperm tissue. Recently,
wheat varieties

null Wxalleles

for each Wx protein have been identified

from around the world by several groups. Each null allelein

has an effecton the amylose content in starch, thereby greatly influencing
the quality of the Japanese
noodle, Udon. Zhao et al. (1998)
reported that all of the Australian cultivars preferred
for making white salted
superior

noodle carried the null Wx-Bl {Wx-Blb)

noodle making qualityof these cultivars

still

but the deletion

unknown. Thus, there is a possibility

However, thecould not be explained

decrease in amylose content caused by the null Wx-Bl allele.
The molecular analysis of Wx-Blb indicateda deletion
coding unit (Vrinten et al. 1999),

allele.

breakpoints

that gene(s)simplyof the entire

by a
Wx-Bl

of the Wx-Blb allele

relatedareto noodle making quality

were lost along with the Wx-Bl gene when the deletion event occurred. Development ofDNAmarkers around the Wx-Bl locus is one way to discover the deletion breakpointsof Wx-Blb, but there is little
any reported

sequence information

DNA makers reported

are distantdevelop new DNA markers tightly
linkedbetween the rice and wheat genome.

around the waxy loci of wheat, andfrom the loci.

we tried toto the Wx-Bl locus using the colinearity

The Wx-Al and Wx-Dl genes of wheat are locatedarms of respective

Therefore,

on distalparts of the shortgroup 7 chromosomes, while the Wx-Bl gene is locatedon the longarm of 4AL because ofa translocationof the 7B short arm (Gale and Devos 1998). The
positionsof commonDNA markers for cereals, including the waxy gene, indicate abroad level of colinearity

between wheat group 7 chromosomes and rice chromosome 6.

We are now able to access all rice genome sequence data provided
Instituteof Agrobiological

Sciencesby the National

DNA bank. Weused wheat EST clone sequences asqueries and searched for rice genomic sequences showing high homology using BLAST.
Several wheat EST clones showing high homology scores to sequences near the ricewaxygene were identified.
A number of primer sets were constructed using sequence
information fromthe EST clones.
From these primer sets, we were able to select two sets of primers which only
generated the expected PCR fragment from the 4A chromosome. Subsequent analyses
with deletion lines of 4AL (Yamamori et al. 1 994) showed that the amplified fragments
could be used as markers. These are the closest markers to the Wx-Bl locus that havebeen identified
to date. Based on a rice physical map, one marker locates on a position
about 6.5 kbp distal to the centromere from the waxy gene and the other one locates
about 40 kbp proximal tothe centromere. However,isthe marker fragments were
amplifiedby the PCRprimer sets when DNA
samples from Australiancultivars
carryingtheWx-Blb allele were used as
Fig. 1. Breakpoints of deletion lines are indicatedby arrows beside a 4A chromosome ideogram
templates.
This indicatedL(Yamamori et aL, 1994) Presence (+) or absence
that

that

breakpoints
allelethe(-) of Wx-Bl gene and amplified products withthe generated DNAmarkers are shown in tabularform. Based on a rice physical map, the marker(A) locates on a position about 6 5 kbp distal tothe centromere fromthe waxy gene and the (B)locates about 40 kbp proximal to the centromere

deletionof the Wx-Blb

are insideof thesemarkers.
Acknowledgments
The deletionlines were provided

from Laboratoryof Plant

Kyoto University,

which supportedby the National

Schoolof Agriculture,

Projectfrom the Ministryof Education,

Science

Referenc es
Batey I. L., Gras P. W.and Curtin B. M. (1997)
Gale, M. D. and Devos, K. M (1998)

and Culture,

Graduate

Bio Resource

Japan.

J Sci FoodAgric

Proc NatlAcad

Genetics,

Sci USA95:

74:503-508.
1971-1974.

Vrinten, P., Nakamura, T., Yamamori, M (1999) Mol Gen Genet 261 : 463-471.
Yamamori, M.,Nakamura, T., Endo, T. R. et al. (1994) TheorAppl
Genet 89: 179-184.
Zhao, X. C, Batey, I. L., Sharp, P. J. et al. (1998)

J Cereal

Sci 27: 7-13.

Rice Growth Imaging
Seedling

System for Phenotypic

Functional

Takanan Tanabata, and Tomoko Shinomura

HitachiResearch

CentralLaboratory,e-mail* torui @hat1.hitachi.co

Key words: phenotype,jp

System;HI-RIS)

phenotypes

350-0395,Japan

URL: http //www gs.dna.atfrc

go jp/SY-

for assigning

an automaticfor analyzing

digitalrice gene functions

in mutants and transgenic"Type junior"

Hatoyama, Saitama,

plants(1).

imagingsystem (Hitachi

For the purposethe growth of rice plants and tillering.

at 60-mmute intervals.

The image analyzer

measures forms of organs continuously(14-hvisualizes

and comprehensively

we measured the growth of Oryza sativa
hght/10-h

Rice Imaging

by measurement of detailedof an image capturer and an image analyzer. The image capturer

Firstly,

1 108/

image, measurement, leaf growth, long-day

Wehave been developing

conditionfrom Middle

to Mature Plant

TomIshizuka,

plantsAnalysis

discussed

visiblehere, we madeThis system is composed

acquiresgrowth images of 24

the growthusing sequentialof plants

andimages.

L. cv. Nourin-8 under a long-daydark) from the 14th day to the 50th day after seeding

using HI-RIS

system. The temperatureacquired

approximately

possible

to identify

was 28°C in the light

4000 images from 8 different

was measured as the distanceleaves of main culm. Similarly,

tillers

withinelongated

tenth leaves
theirangles. These sequential

each leaf of main culm and tillers

growth. Plant heightappearedand 23°C in the dark. From each plant wethe heights

for measuring

subsequently

growth rate decreasedof two primarytillers

with the corresponding

and the long-day

controls(light28°C/dark

23°C and lightunder long-day

condition

in higher

condition

Under

the

long-day/higher

appearance

was shortest(3.1-3.8days),and the growing

30°C/dark

25°C)

The fifth totheperiodwith two different(10-htemperature

We found that the plants

grown

condition,

theperiodicity

maximum leaf

growthrate

was greatest(3.2-4.3

days).

of each leaf was shortest
affect their

growth and development

condition

grew faster than those grown under the othertemperatureand temperature

under a short-day

in combination

temperature

conditions.

covered these visible

Each leaf of the primary

within 4 days

dark)that photoperiodofgrew at a maximum growth rate (3-5mm an hour) for 2 days, thenlight/14-hsuggests

point

were examined. New leaves

leaf of the main culm (2)Next, we compared the growth of the same material(9.6-12.9cm/day)the rate of plant heightfrom the ground to the highest

a cycle of 3 to 5 days in both main culm and tillers.
synchronously

images made it

phenotypes.

proceduresof leafThis

HI-RIS images effectively

quantitatively.

This work was partly
and Fisheries

1)

2)

supportedby grants from the Ministryof Japan (Rice Genome Project

TakananT., Ishizuka

analysis

The Journal

Engineers(in press).

Katayama T. (1951)

InstituteStudiesT. (2003)

of Electronics,

on tillering

Forestry

SY-1 108 and DP-1006).

T. and Shinomura
ofof Agriculture,

Imagingsystem in gene functional

Informationof rice and barley.

andCommunication

Yokendo. Tokyo.

Large-scale Analysis of Alternative
CDNAof Oryza sativa

Splicing

Hitomi

Itoh1'2,

Higo3,

KenichiMatsubara4,

Takanori

Washio1,

Kenichi

Jun Kawai5, Piero

Carninci5,

Regulation
Yasuhiro

Yoshihide

Ohtomo4,
Hayashizaki5,

using

Full-length

Kazuo Murakami4,
Shoshi

Kikuchi3,

and Masaru Tomita1 '2
institutefor Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0035, Japan
2Department of Environmental Information, Keio University, Fujisawa, Kanagawa 252-8520, Japan
3National Institute ofAgrobiological
Sciences, Tsukuba, Ibaraki 305-0062, Japan
foundation
for Advancement of International
Science (FAIS), Tsukuba, Ibaraki 305-0062, Japanlaboratory
for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC),
Tsurumi-ku, Yokohama, Kanagawa 230-0045 , Japan.
Email : washy@sfc.keio.ac.jp

Keywords: alternative1

splicing,:

exonic splicing

enhancer (ESE),

full-length

CDNA.

Introduction

Although an extensive number of genes have been reported to be alternatively
spliced, mostof the information obtained by this process has not allowed for general understandingofalternative splicing regulation, especiallyin plants. The basic requirements for plant splicing
is similar to those for vertebrate splicing;the consensus sequences of the 5'-splice sites and3'-splice sites and the branch point region at the -30 nt distance from 3'-splice sites are
similar in plants and vertebrates (Lurhrsen, 1 994). On the other hand, there are plant-specificrequirements for pre-mRNA splicing; plant introns have a strong compositional bias for UAor U-rich sequences and plant exons contains more GC-rich sequences than vertebrates(Lorkovic, 2000). Prior studies have indicated that the 3 '-splice sites and 5'-splice site locatedat transition regions from UA-to GC-rich sequence is prefentiallyselected for splicing (Lou,1 993). Also, the mechanisms of splice-site recognition may differ between dicot and monocot
plants; it has been indicated that the sequence composition of both introns and exons wasclearly different
between dicot and monocot plants (White, 1992). Here we comparedalternative splicing regulation in four eukaryotes, O. sativa, A, thaliana, K sapiens, and M
musculus, and constructed models for alternative splicing regulation in O. sativa.

2

Results

First, we computationallydetected putative splice variants by mapping full-length
CDNA
clones to complete genomic sequences; clones were then grouped into clusters if all theinternal regions were correctly mapped. Putative splice variants were then classified
according to alternative
splicing
patterns. We then compared the distributionof known
splicing regulatory sequences, which tended to be exonic in all four species, suggesting thatthe basic mechanism of pre-mRNA splicing
is similar in all eukaryotes.
Higher O/E(observed/expected)
values of regulatory sequences in plants indicatedthat there may beplant-specificregulatory features other than known regulatory elements in metazoans. Basedon these results, we constructed models for alternative splicing regulation in Oryza sativa.
Figure 1 shows one of the constructed models; all of the constructed models are available athttp ://www. bioinfo. sfc. keio. ac jp/research/intron/.

Figure 1. Cross-species comparison of putative alternate exon in transcripts homologous to A.
thaliana unknown protein. Shaded box represent conserved regions across rice, barley, andwheat.

Acknowledgement
Wewould also like to thank TMRI for offering Oryza sativa genomic sequence data. This
work was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan (RiceGenomeProject SY-1 104).

Referen ces
Lorkovic,
Z.J., Wieczorek Kirk, D.A., Lambermon, 3VLH. and Filipowicz,
W. (2000)
Pre-mRNA splicing in higher plants. Trends Plant ScL 5: 1 60-167.
Luehrsen, K.R., Taha, S., and Walbot, V. (1994)
Nuclear pre-mRNA processing in higher
plants. Prog Nucleic Acid Res Mol Biol. 47: 149-193.
White, O., Soderlund, C, Shanmugan, P., and Fields, C. (1992)
Information
contents anddinucleotide
compositionsof plant intron sequences vary with evolutionary
origin. Plant Mol
Biol 19: 1057-1064,
all of the constructed

models for Alternative

Splicing.

Evidence for Tissue-specificExons in Oryza sativa
Noriyuki

Kitagawa1'3,

Kashima1'2,

Kenji

Takanori

Higashi1'2

Kazuo Murakami6, Kenichi
Shoshi

Kikuchi5,

KenichiTranscription
Washio1,
Higo5,

Matsubara6,

ShunichiKoji

Satoh5,regulatedby AlternativeFirst

Kosugi1,

Tomoya Yamashita1'4,

Goki

Yasuhiro

Jun Kawai7, Piero Carninci7,

Ohtomo6, Tomomi Sunako6,
Yoshihide

Hayashizaki7,

and Masaru Tomita1'2

institutefor Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-003 5, Japandepartment of Environmental Information, Keio University, Fujisawa, Kanagawa 252-8520, Japandepartment of Policy Management, Keio University, Fuj isawa, Kanagawa 252-8520, Japan
4Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa 252-8520, Japan
5National Institute ofAgrobiological
Sciences, Tsukuba, Ibaraki 305-0062, Japan
foundation
for Advancement of International
Science (FAIS), Tsukuba, Ibaraki 305-0062, Japanlaboratory
for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC),
Tsurumi-ku, Yokohama, Kanagawa23 0-0045 , Japan.
Email: washy@sfc.keio.acjp:

Keywords: alternative
first exons, transcription
chain reaction (RT-PCR), 5 '-end EST.

start site, reverse transcription-polymerase

Abstract
The diversity of transcription
start-site and alternative splicing greatly contribute to expansionof protein diversity, but their regulations
are still not clear, especiallyin plant. Although
recent studies have revealed the existence of an alternative first exon (Hugnot et al 1992;
Kelner et al 2000; Zavolan et al 2002% most of them have been carried out in individual
genes; as such, the mechanism regulating
the alternative
first exon has not yet to beunderstood. In the present study we detected a putative alternative first exon in Oryza sativa,
verified the candidates using reverse transcription-polymerase
chain reaction (RT-PCR), andsearched for core promoters that might regulate the alternative first exon. As a result, we havepredicted a number of unreported alternative first exons, some of which are regulated in atissue-specificmanner.

Figure 1. RT-PCR results for each cluster, indicating tissue-specificexpression. Tissue names
are given above each lane (Seedling,
Leaf blade, Leaf sheath, Flowerl just after heading,Flower2 two weeks after flowering, and Callus). The transcript from exon la is shown in a,and that from exon lb in b. Figures on the right are structures of 5'-end EST. Filled triangles
indicate predicted start codons on full-length
CDNA, corresponding to 5'-end EST of eachcluster.

Acknowledgement
Wewould also like to thank TMRI for offering Oryza sativa genomic sequence data. This
work was supported by the Ministry of Agriculture,
Forestry, and Fisheries of Japan (RiceGenomeProject SY-1 1 04).

References
Hugnot, J.P., Gilgenkrantz,
H., Vincent, N., Chafey, P., Morris, G.E., Monaco, A.P.,
Berwald-Netter, Y, Koulakoff, A., Kaplan, J.C., Kahn, A., et al (1 992) Distal transcript of thedystrophin gene initiatedfrom an alternative first exon and encoding a 75-kDa protein widelydistributed
in nonmuscle tissues. Proc. NatJAcad Sci USA, 89: 7506-10.
Kelner, M.J., Bagnell, R.D., Montoya, M.A., Estes, L.A., Forsberg, L., and Morgenstern, R.
(2000) Structural organizationof the microsomal glutathione
S-transferase gene (MGST 1 ) on
chromosome 12p l 3. 1 - 1 3.2. Identificationof the correct promoter region and demonstration oftranscriptional
regulation in response to oxidative stress. JBiol Chem, 275: 1 3000-6.
Zavolan, M., van Nimwegen, E., and Gaasterland,
T. (2002)
Splice variation in mousefull-length
cDNAs identified by mapping to the mouse genome. Genome Res. 9 12: 1377-85.

Differential

heterosis

rujipogori)in a naturaldue to reproductive

strategy

Yosuke Kuroda1, Hathairat

Urairong2

Graduate

Schoolof Asianand African

606-8501,

Japan(kuroda@asafas.kyoto-u.ac.jp),

Pathum-Thani422-8259,

12110,

Thailand;

populationof Asianrice(Oryzaand edge effect& Yo-Ichiro

Sato3

Area Studies,^Faculty

wild

Kyoto

University,

Sakyo-ku,

Kyoto

Rice

ResearchCenter,Universty,

Ohya,

Shizuokasystems,

2Pathum-Thaniof Agriculture,

ShizuokaJapan

Key words: heterosis,
sampling

SSR (Sinple

Sequence

Repeat),Oryza rufipogon,

reproductive

rufipogori)is a polycarpic

perennial

locations

Abstract
Asian commonwild rice (Oryza
mixed inbreeder/outbreeder
the population.

strategy.

Seeds and clones

However, when collected

to whether

they have experienced

evaluating

mechanisms

in the flowering

naturalfor the survival

selectionof a population

plantwith aare both reproductive

componentsofstage, the two differ

substantiallyasdirectly

or not This study

by comparing

genetic

is aimed atstructureamongsubpopulations

that

also

(fringe2) location(SimpleSequence

outcrossing

are classified
or inside;

Repeat)t = 35.5%)

alleles(A) and expected

0.692

in clones

likelyto survive

outcrossing

althoughhigherof 1) reproductive
1)

First,

thethat parametersin clones

respectively;

with highin the naturalof both the fringe

be due mainly to the restriction

areas (Table

anddeterminedat seven

SSR

(Ho)

and

of observed

heterozygosity

Table

indicates

1)

Secondly,Thisof wind strength

resultresultin fringe

for the number ofand seeds, HE = 0.709,
thatloci and outcrossing

parameters1). Thisthan in seeds (Ho =the same values
clonesarea (Ho = 0.443, t = 60.7%),

and insideor seeds)(HE) (A = 4.43 in bothrates were lower for seeds from the fringe

to seeds from the inside(clones(Ho = 0.609, t = 82.4%)

numbers of heterozygous
habitatsystems

genotypes

the two had approximately

heterozygosity

and seeds,"heterosis",Fig.

loci showed

rate (t) were clearly

0.346,

showingin termsof observed

the individuals
rates,are moreheterozygosity

andarea (Ho = 0.238, t = 24.6%)

compared

althoughvaluesin clones

suggeststhat the "edge effect"

were similararea of the forested

swamp.

might

Fig.

1.

Sampling

areas along the pier in the in-situ

four subpopulations

S-fringe,

Table

and observed

1.

Expected

subpopulationsof O. rufipogoncomponent (clones^lone1973);or seeds)= C-fringebObserved

C-fringe,+ C-inside;

heterozygosity;

conservation

S-inside,and C-inside.

heterozygosity,

fixationin the conservationand location(fringearea, classified

rufipogon

and outcrossing
in termsof therate

forof reproductive

or inside).

"Seed = S-fringe
cOutcrossing

index

area ofO.

+ S-inside;

rate (Weir,

1990).

aExpected

heterozygosity(Nei,References
Nei, M. (1973)

Analysisof Gene Diversity

in Subdivided

Populations.

Proc. Nat Acad. Sci

USA 70: 3321-3323.
Weir, B.S. (1990)
Sunderland,

Genetic

data analysis

methods

for discrete

population

genetic

data. Sinauer,

Massachusetts

Acknowledgements
Thanksare due to researchersat the National

Rice ResearchCenterin Thailand,

K. Tanaka of Kyoto University

for providing

the Pathum-Thani
Professor

for this research.

Agricultural

ResearchCenterin Laos,

Dr Y Kono of Kyoto University
equipment

and valuablesuggestions

and

cim

Crossing barrier withinmolecular dissection

the primary

genepoolof rice uncoveredby means of

Kazuki Matsubara, Mitsunobu Ikenaga, and Yoshio Sano
Plant Breed. Lab., Dept Appliede-mail rysano@abs
Keywords:

Biotech,Hokkaido

Univ., Sapporo, 060-8589

Japan

agr.hokudai.ac.jp

Post-fertilization

barrier,

Hiddenvariation,

Near-isogenic

is one of the most effective

isolatingbarriers

line, Sex-specificreaction,

Primary genepool
Crossing

barrierdiverged

populations.

Sexual affinity

or cross-compatibility

which restrict

gene flow between

has been widely

surveyed in crops andtheir wild relatives

since knowledge about the primary gene pool is a prerequisite

breeding

1975),(Harlanhowever, our present

phenomena and their distribution

understandingfor hybridizationof the genes involved

within the primary genepool is limitedin theseplant species including

The present study was earned out to examine the genetic basis of the unidirectional
incompatibility

observed

in hybrid

derivatives

rufipogori) rice strains

A domesticatedbiologicalconsists

species,correspondingwhich

cultivatedcomplex (Harlan
strainsof backcrossing

The near-isogenic
This provides

line ofT65wx

generallyto the same

(Japonica

interbreeding

recombination,

no distinct

crossing

Sitch etal

1989)

1969;

The genetic basis
by testcrosses

cross-incompatibilitytype) carrying

isolatingbarrierof this cross-incompatibility
using recombinant

the introgressedsegments of wild and cultivatedcross-incompatibility

reaction was controlled

populations,and theto be the O. sativa - O. rufipogon
was detected

and cultivatedin(O. sativa) ricean alien segment of chromosome

a unique example in which genes for crossing barriers

althoughbelongonly when crossed with T65wx as the male (Sanoand a distinct

investigatedand its progenitor

between wild {Oryza rufipogon)

primary genepool as hidden variation,complex (Chu etal

and wild (O.

The unidirectional

6 from a wild strain gave a reduced seed setting
1 992)(Oryza sativa)of groups of potentiallyOka 1988)

cross-

between cultivatedand wild forms of rice are regarded

1975;

advanced generations

plant

rice.

barrier resulted

has been reported
reactionswere present within thefrom hybridization
withintype)

strains

the rice species

in the female and male wereinbred lines that were established(Indicaandthroughdissecting

The results revealed that theby Cif 'mthe female and by dm in the male.

When

the female plant with Cif was crossed with the male plant with dm, a failure of early endosperm
development

was observedin the hybrid

zygotesAmong cultivarsof O. sativa,

waspredominantly
suppressor,

distributedin the Japonica

type but not in Indica

Su-Cif, which changes the reaction

type.

In addition,in the female from incompatible

proposed to present near the centromere of chromosome 6 of the Indica type.
for the genie interactions

responsible

as shown in Figure

The gene block detected

the established1

for the cross-incompatibility

sexual affinitieswas

We propose a model

on chromosome 6 predicts

actually

to compatible

reactions in the female and malesexual reactions against a breakdown due to recombination,

might generate the diversified

a dominantthat it might maintainor the recombined genes

observed in nature.

Figure 1 Putative genetic modelby which the three genes Cif,cim and Su-Ci/are involved inunidirectionalcrossincompatibility(Matsubara et al
2003)
Cross-incompatibility
occurs when gametes with thecross-incompatibility
reactionsin the female (Cif) and the male(cim) are used for fertilization.
+ indicates the crosscompatibility
reaction.

References
Chu, Y.-E., H. Morishima, and H.-I Oka.
rice species and their wildrelatives
Harlan, J. R.
Matsubara,(1975)(1969)

Reproductive

Japan. J. Genetin wild and cultivatedY. Sano.
rice.

Genetics

A gene blockcausing cross-incompatibility

OriginSano, Y.

(1992)

Gnentic comparisons of chromosome 6 between wild and cultivated

Rice.

JSSP, Tokyo / Elsevier,potential

6:58-60.

Amsterdamrice.

42: 561 - 572.

L. A., R. D. Dalmacio, and G.O. Romero. (1989)
theirhidden(in press)(1988)

Sitch.

in cultivatedof Agronomy, Madison, WI

Oka, H. I

Japan. J. Breed.

of Cultivated(2003)

distributed

44: 207 - 223

Crops and Man. American Society

K, Khin-Thidar,

barriers

use for improvementof cultivatedCrossabilityof wild Oryza species

rice (Oryza sativa L.)

Rice Genet

and

Newsl

Genomic differentiationin rice using recombinant
inbred lines between an
annual type of wild rice and a landrace in the northern-most region for ricecultivation
Kazumitsu Onishi, Noriko Ishigoh-Oka,

Kyoko Takagi, Yuki Horiuchi, Wilhelm Eigo Hagiwara,

Yuji Kishima, and Yoshio Sano
Plant Breed. Lab., Dept Appliede-mail rysano@abs

Hokkaido Univ., Sapporo, 060-8589

Japan

agr.hokudai.ac.jp

Keywords: Adaptive traits,
The objective
adaptive

Biotech.,MITEs, QTL cluster, Rice, RIL
of the present study is to investigatechanges reflecting

the domestication

genetic differentiation

as well as varying environments

associatedwithin the primarygene pool of Asian rice.

The wild progenitor

of the cultivatedmostly habits

areas while cultivatedforms of O. sativa are widely distributed

in tropicalto temperate regions
to be successfully
involved,

In Hokkaido,
cultivatedrice species is O. rufipogon which isthe northern area of Japan, intensive

after the beginning

we compared a landrace (A58)of the last century

from Hokkaido

between A58 and Wl 07.

In addition

inbred lines (RILs)

To examine the genetic basesin latitudinal
invertedof wild rice
distribution.

from the F5 population

to PCR-based markers, miniaturefrom tropic

improvements made rice

and an annual form (W107)

from India by means of QTL mapping since the two strains are different
We raised 79 recombinant

withfrom the crossrepeat transposable

Figure 1 A constructed
map showing the effectivenessfor discrimination
between Indica and Taponicu types and positions
ol QTL s
Positionof Mil b and PCR-based markers are indicatedby thin and thick lines along chromosomes
æf shows effective
MITE markers lor discrimination
between Indica and laponica types
Putative QTL is represented
as one-LOD support interval
A LOD thresholdol 2 4 was selected lor declaring
thesignificanceol a putative
QTL 2LBL 2LSL
Leaf blade and leaf sheath length of2nd leaf LS Leaf sheath length at vegetative
stage TN Tiller numberFI
Illlertype,
LA Leal angle CL Culmlength
PN Paniclenumber
PL Paniclelength
SPP
Spikelets
perpanicle
HD Headingdate
SH Shatteringol fertile seed stSH
Shatteringof sterile spikelet
SPR Spreading
ofpanicle
SW Seed width AHL Apiculus
hair length
F% Seed fertility

elements(MITEs)were used to constructmolecular

map. MITEs are a major componentof interspersed

repetitiveof polymorphic

changes are effectivelyasequences and a number
detected

among rice taxa using

the MITE transposon

display

et al.

methodconstructed(155(Takagi

map included

markers)which

over 12 chromosomesThe

total length of the maDwas 1376.8

average distanceThe

a total of 268 markers

MITEs and 1 13 PCR-based

were distributed

2003)

cM and the

between two markers was 5.4 cM5 showing that the constructedanalyze a whole region in rice genome (Figure
Genomic differentiation
cultivatedFigure 2 Comparison of plant type at the vegetative stage among
T65/te(W107)
and its parental lines
Bar= 20 cm

and 7 wild forms

map enables us to1)

was examined based on polymorphic

patterns of MITEs in 9

In each MITE marker, the degree of discrimination

show to what extent it discriminates

different

taxa (Figure

0 to 1 , and the value of 1 indicatesthat it perfectly1)

discriminates

was estimated to

The value of the degree ranges fromdifferent taxa.

For discrimination

between Indica and Japonica types, the high value was observed in 35 MITE markers among the
155 examined and they were frequently

distributedon chromosomes 1, 2, 3 and 6.

However, fordiscrimination

between wild and cultivatedforms, most of the markers showed an intermediate

value althou^ithe reason is uncertain.

Thissuggeststhat genomic differentiation

may haveoccurred in different regions of chromosomes depending on different taxa.
To survey the genomic region associatedperformed in 24 traits
QILs

detected

plant height,

tiller1)

Thiscluster

forming

included

forms

was

Among 33

were found on the short arm ofvarious traits

associated07) showed a rosette-like

into T65 from W107

with plant type such as

for understandingby backcrosses

habit at the early stage of development

the region might carry various genes responsible
progressa cluster

QTL analysis

and leaf angles and so on. To confirm the presence of the QIL duster, the shortarm of chromosome 7 was introduced
T65J?c(W1changes,which were different between the wild and cultivatedover the genome, 7 QILs

chromosome 7 (Figure

with adaptive(Figurefor weedy or wild forms

the genetic bases of adaptive changes Including

The NIL of2), showing that

Further studies
cold tolerances

are inat various

stages of development
References
Takagi,detects

K., Nagano, H., Kishima,polymorphisms

Y and Saeo9 Y (2003)

among the OryzaAA-genome species

MITE-transposon

display

Breed. Sci 53: 125-132.

efficiently

Identificationand mapping of seed shatteringlines from wild rice species

genes using introgression

PaulinoSobnzalL. Sanchez.1 Toshio

Kurakazu, Chiharu

Hirata,and Atsushi

Yoshimura

Plant Breeding Laboratory, Faculty of Agriculture,
Graduate School, Kyushu University,
Fukuoka, 8 12-858 1 Japan, l Currently
Crop Evolutionary
Dynamics Laboratory,
National Instituteof Agrobiological
Sciences (MAS), Tsukuba, 305-8602 Japan,
psanchez@affrc.gojpKeywords : Seed shattering,

Seed shattering
Two species

is an importantof wild

Oryza meridionalis

Oryza glumaepatula,

Ng (W1625),

Taichung

65 and O. glumaepatula

65

O. meridionalis

variation
each(N=65)

forpolymorphism

and BC4F2

et al
108

were planted.

it affects

yield.

Ace. No. 105668)

andand map genes controlling

seed

(IRGC

were derived

etal1999),

2001)

from a crossbetweenand a cross between

Taichung

BC4F2

from O. meridionalis
After heading,

One hundredlines

24 population

O.

exhibiting

wide

the leaves

were collected

from

six RFLP

markersand(N=71)

fromshowingin the two crosses were used. Whole genome surveys were performed

(Fig.

1)

The retained

heterozygous

Shattering

genes were mapped using phenotyping

data, and were analyzed

GrainsSegregation(Sobnzal

DNA extraction.

BC3F1 generation.
generation

Steud.

(BC4F2*s)(Kurakazuin seed shattenng

plantshattenngpopulations

Introgression

trait in nee because

were used to identify

Backcrossed

glumaepatulaagronomic

nee, Oryzaglumaepatula

shattenng.

andOryza meridionalis,

were further

evaluated

in BC4F1

and genotyping

using MAPMAKER/EXP ver 3.0.

of O. glumaepatula
was observed

andregionsin

16 normal

and O. meridionalis

in the population
plantsderivedwere observed

shatterfrom bothin BC4F2

at ripeningstage.

crosses

Forty

nine

population;37

24-glumshatteringandpopulation.

Segregation

shattering

was controlled

segregation
observed

34 normal

plantsratio

in BC4F2

108

the 3 1 ratioin BC4F2

24-glum,fittedby a dominantof BC4F2 108-merin thiswere observed

population.

O. glumaepatuladid

not fit the 3 1 ratio

Shattering

was controlled

allelein BC4F2

108-mersuggesting(Tablethat

1)

In contrast,

due to segregation

distortion

by a dominantfrom O.

allelemeridionalis.

Linkagepopulations

analyses

were locatedshowedlinkedgene in BC4F2 108-mer was tightly
shattering

linkedgenes were designatedgenetic

mechanisms of seed shatteringin rice.

study

was supported,

Advancement Institution

controllingto RFLP marker C726S,

genes could be used for further

This

genes

seed

shatteringon chromosome 5 The gene controlling

BC4F2 24-glum was tightly

detected

that(BRAIN),

seed

in

both

shatteringinwhereas the seed shatteringto RFLP marker R2232(Fig.

2) Theseas Sh5-glum and Sh6-mer, respectively.
studies

to fullyin part,

by

newly
These

analyzethe biologicalnature andBio-oriented

Technology

Research

A high-density
Genetics 148

rice genetic
479-494.

linkage

Japan.

References
Harushima, Y., Yano, M., Shomura, A. et al (1998)
map with 2275 markers using a single F2 population.

Kurakazu, T., Sobnzal, Sanchez, P. L. et al (2001)
Developmentof Oryza meridionalis
introgression
lines in rice, Oryza sativa L. Rice Genet Newsl 18 81-82.
Sobnzal, Ikeda, K., Sanchez, P. L. et al (1999)
Developmentof Oryza glumaepatula
introgression
lines in rice, O. sativa L. Rice Genet Newsl 16 107-108.

Fig.
1 Graphicalgenotypes
heterozygous
regions in BC4F1(B).

showing
the retained
24 (A) and BC4F1 108

Table 1. Segregationof shattering
plants in introgression
lines with O. glumaepatula and O. meridionalis cytoplasm.

Fig. 2. Linkage

map showing the locationsof Sh5-glumand Sh6-mer on chromosome 5.

Identification
glumaepatulaPaulinoand mapping of heading
introgression
lines

L. Sanchez,1Sobnzal,genes in rice using

Kosuke Ikeda, Hideshi

Yasui and Atsushi

Oryza
Yoshimura

Plant Breeding Laboratory,
Faculty of Agriculture,
Graduate School, Kyushu University,
Fukuoka, 8 12-858 1 Japan, l Currently
Crop Evolutionary
Dynamics Laboratory,
National Instituteof Agrobiological
Sciences (NIAS),
Tsukuba, 305-8602 Japan,
psanchez@affrc.
go.jp
Keywords:
Introgression

Days to heading,lines

Days to heading

Early heading,is an important

identify

and map genes controllingagronomic

65 (T65)

used.

BC4F2 254 (N=72),

exhibitedwide variation

conditions

glumaepatularetained
(Fig.

1A)

IB)

it determines

maturityin rice.

populations(BC4F2)from a cross

DTH, backcrossed

222(N=78),
in late204 (N=74)

and early

Steud.

(Sobrizal

and 204 (N=72)

DTH were planted

for DTH. After heading,regionswere

populations

which

under natural

daylength

leaves were collected

six RFLP markers showing polymorphism

were further

evaluatedTo

et al 1999)

forbetweenand T65 were used for the whole genome survey in BC3F1 generation.

O.
The

in BC4F1 and BC4F2 generations

DTH genes were mapped using phenotyping

and genotyping

data, and wereusing MAPMAKER/EXP ver 3.0 and MAPMAKER/QTL v. l 1

The frequency

distribution

O. glumaepatula

did not flower under natural

BC4F2 254 plants(late)

becauseand Oryza glumaepatula

One hundred

heterozygous

analyzed

trait

Each plant was monitored

DNA extraction.

Oryza glumaepatula,(DTH, the number of days from sowing to emergence of the firstpanicle)between Taichung

Late heading,

ratio,for DTH of selected

(20

early

and 52 late heading

indicating

that

late

heading

BC4F2 populations
daylength

conditionsindividuals)

agreedwas controlled

by a single

are shown (Fig.
Segregationin

1 (early)

3

dominant

gene

with

Fig. 1. (A) The graphicalgenotypes showing the retained heterozygous
regions in BC4F1 generation;(B) The frequency distributionsof days to heading of BC4F2 254, 222, 204 and 271 populations;and(C) The linkage maps showing the locations of Lhdl (t)9 Lhd2(t)9
Ihd3(t)9and Ehdl (t)on rice chromosomes
6, 7, 2 and 10. Each left vertical bar shows the RFLP linkage map (Harushima et ah 1998).

Taichung 65 homozygous for C560
Heterozygous for C560
O. slumaepatula homozvgous for C560

Taichung 65 homozygous for C234
Heterozygous for C234
O. glumaepatula homozygous for C234

(Lhd](t))

from O. glumaepatula.

BC4F2 222 agreed

withpartially

gene (Lhd2(t))

dominant

into 27 lines
segregation
headingearlier

for early
distortion.

A segregation

1 3 ratio,

indicatesand 55 late heading

plantslate heading

was governedby a single

BC4F2 204 plants,

classified

thatfrom O. glumaepatula.

and 47 lines
Variation

for lateheadingdid

not fit thelfor DTH in BC4F2 204 was controlled

gene from O. glumaepatula,than T65

of 23 early

designated

The gene controllingearly

as Ihd3{\)
heading

due toby a recessive

BC4F2 271

in these

3 ratioplantsinplantslateheadedwas designatedasEhdl (t)Chromosomal
was tightly

linkedlocationsof the identified

DTH genes are shown (Fig.

to RFLP marker XNpb27 on chromosome 6. Lhd2(i)RFLP markers C383 and C145 on chromosome 7. Ihd3(t)markers C560 and R759 on chromosome 2 (LOD 18.0)
marker C234 on chromosome 10. Ehdl(t)naturaldaylength
This

study

conditions

Lhdl(\)

was linked(BRAIN),

in

part,

by

Ehdl{t)gene was linked

to RFLP

early heading

by about 10 days underBio-oriented

Technology

Research

A high-density
Genetics 148

rice genetic
479-494.

linkage

Japan.

References
Harushima, Y., Yano, M., Shomura, A. et al (1998)
map with 2275 markers using a single F2 population.

towas mapped between RFLP

compared to T65

was supported,

Advancement Institution

promotes

1C)

Sobnzal, Ikeda, K., Sanchez, P.L. et al (1999)
Development of Oryza glumaepatula
introgression
lines in rice, O. sativa L. Rice Genet Newsl 16. 107-108

Screening

for a candidate

gene of QTL influencing

tissueculture trait using rice

DNAmicroarray

Fumio Taguchi-Shiobara

1 , Junshi

Shimbo 2, Zempei Shimatani
Sato 1, Sachiko
Kishimoto1, Shoshi

Kikuchi

1 National

InstituteofAgrobiological

0854,

Ibaraki,

1 , Fumiko

Fujn 1 , Kanako

2, Tomoya Ohta 1, Yuki
1, Takuji

Sasaki1, Naoki

1

Sciences,

2 STAFF-Institute,Kannondai 2- 1 -2, Tsukuba 305-8602,
446- 1 , Ippaizuka,

Kamiyokoba,

QTL, candidate

gene, Oryza sativa

Ibaraki,

Tsukuba 305-

Japan

DNA microarray,

Candidategenes for quantitative

screenedusing DNA microarray analysis

from two varieties,

tissue culture

Koshihikan

chromosome 1 , Kasalath

alleleregeneration.

Four near-isogenic

Koshihikan

background,and tissue culture

abilitycontrolling

Throughand Kasalath,

QTL analysis

culture traits

abilityin subculture

andmostlyregion of the QTL had homozygous

with aKasalathallele,mRNAs from these four lines wereusing rice 8987 ESTs arrayed on slide(1) one week after subculture
medium containing

glassesmRNA wasin medium containing
cytokinins

ratios were more than three fold were screened. Throughin the htgs and nr database

derivedIn two QTL onlines for these two QTL were developed,

to regenerationin rice wereusing a population

six QTL were detected.

was improved over Koshihikari

2,4-D, and (2) 1 hr after transfer

tissuegave improved tissue culture

from callus at two time-points

which expression

trait,

trait loci (QTL)

and the putative

compared to those of Koshihikari

homology

Ishikawa

Yamamoto 2, Katsumi Sakata

Keywords

prepared, Masahiro

2, Yuko Nagata 2, Akiko Hashimoto

Honda 1, Kimiko

Japan (fstagu@affrc.go.ip\

Yazakil

ESTs ina search forusing the NCBI BLAST program, ESTs were locatedonthe linkage
locatedmap. In (1),54 ESTs were screened, out of which four clones were found to bein the region for QTL on chromosome 1 In (2),153 ESTs were screened, among

which there wereeleven ESTs also found m (1) Nine of 153 ESTs were located
ESTs were screened by Northernblot analysis

in the region for QTL. Twoof ESTs and locatedin the region for QTL.

Mapping the quantitative
morphology

trait loci responsible

Fukuta2, Hongwei Cai3, Hiroyoshi

Hiroko Morishima6of Molecular

Genetics,

Kannondai, Tsukuba, Ibaraki
2 International

305-8602,

Nishinasuno,

4 Department of Information

Ibaraki

305-8666,

6 Tokyo University
7 Institute

Keywords: floral

ofAgrobiological

Sciences.

2- 1 -2Japan
DAPO Box 7777, Metro Manila, Philippines

Tochigi

Forage Crop Research Institute,Japan

Science and Technology,

National

Research Organization,

Agricultural

Research

3- 1 - 1 Kannondai,

Tsukuba,and Forestry,

Universityof Tsukuba, 1 - 1- 1 Tennodai,

Tsukuba,Japanof Agriculture,of Agricultural

Tsukuba, Ibaraki

Fuj imura7

Japan

of Agriculture
305-8572,

Iwata4, Ryo Ohsawa5,329-2742,

Center, National Agricultural

5 Institute

Institute

Fanning and Forage Seed Association

3 88-5 Higashiakada,

Ibarakiand Tatsuhito

National

Rice Research Institute,

3 Japan Grasslandin floral

between Oryza sativa L. and O. rufipogon Griff.

Yusaku Uga1, Yoshimichi

1 Department

for differencesand Forest Engineering,305-8572,

morphology,

loci, differentiation

1 737 Funako, Atsugi 243-0034,
University

Japan

ofTsukuba,

1 - 1 -1 Tennodai,Japan

Oryza sativa L., Oryza rufipogon Griff., quantitative

trait

Asian cultivatedrice (Oryza

rufipogon Griff.).

sativa L.) was domesticatedThis domestication

morphologicaland physiologicalmorphologyof Asian rice varies

Measurements of eleven traits
and 53 wild accessions

process resulted

characteristics
widelyrelatedfrom its wild progenitor
in substantialof Asianchangesrice.

showed that cultivatedin theIn fact, the floral

between that of cultivatedto the pistil,(0.

and wild

rice.

stamen, and glume of 128 cultivatedrice had a less exserted

stigma,shorterstigma, shorter anther, and thicker

and wider glume than wild rice. Moreover, the degreeof stigma exsertion,

and anther length were negatively

stigma length,

thickness

and width of the glume.

variations,

we analyzed 16 traitslines,To clarify the genetic

relatedbasis

correlatedwith theof these interspecificto the three organs using recombinant inbred

derived from a cross between 0. sativa, Pei-kuh, and O. rufipogon, W1944.

quantitative

trait

composite intervalloci(QTLs)

responsible

for floral

mapping using a linkage map constructed

RFLPs. A total of7, 4, 14, and 6 QTLs were detected
and glume size and shape, respectively.
revealedmorphology

10 QTLs thatwere detectedbyusing 147 markers, mostly

for traits related

to the pistil,stamen,

A comparison of 3 1 QTLs affecting these organs
regionsonchromosomes 2, 4, 5, and 10, but most of the QTLs (68%)

were located separatelyonvarious chromosomes. Although

4 QTLs for stigma breadth,anther length,of the lemma and palea explained

more than 25% of the total phenotypicof the QTLs (87%)
importantaffectedThethe different

organs in four adjacent

had smaller effects. Our data indicate

variance, mostthat such genes play a morerole than genes with large effects in the differences

between cultivatedand thickness

and wild rice. For example, the anther length

in floral morphologyof W1944 was fourtimes that of Pei-kuh, and 4 QTLs that had small effects on anther length were detected.

Acknowledgments
Wethank Dr. E. L. Javier,providing

Ms. M. S. Almazan, and Ms. A. Alcantara

the plant materials.

We also thank the staff of the research

Double Cropping,

PBGB Division,

was performed

under an IRRJ-Japansupportedby both the Ministry

Forestry,and Fisheries

IRRI for theirexperimental

Corroborativeof Foreign

Affairsof IRRI

group at the Ricesupport.

Research

forThis research

Project(PhaseIV)and the Ministryof Agriculture,

Variations

traitsin Asian(O. rufipogon Griff.).

Breedingof Japan.

Referen c es
Uga, Y., Y. Fukuta, R. Ohsawa, and T. Fujimura.
cultivate

rice (Oryza

Science(in press).

sativaL.) and its wild

relativesof floral

Uga, Y, Y. Fukuta, H. W. Cai, H. Iwata, R. Ohsawa, H. Morishima,(2003)

Mapping QTLs influencing

derivedfrom a cross between Oryza sativa L. and O. rufipogon

AppliedGenetics

107: 21 8-226.

rice floral morphology

and T. Fujimura

using recombinant
Griff.

inbred lines

TheoreticalandDual origin of the cultivatedannual and perennial
Shinsuke

rice based on molecularstrains of wild rice species,

markers of newly collected

Oryza nivara and O. rufipogon

Yamanaka1'2, Ikuo Nakamura3, Kazuo N. Watanabe1 and Yo-Ichiro

1 Gene ResearchCenter,Japanyamanaka@gene.tsukuba.ac.jp),(E-mail

University,

Shizuoka,Matsudo,Japan.Key words
sampling

Instituteof Biological

Sciences,
2:

University

Faculty

Japan, 3 Graduate

Schoolof Scienceand perennial

types,

indica-japonica

annualof

Agriculture,

Shizuoka

Chiba

University,

p-SINEl-r2,

methodrice (O. sativa),

into annual (O. nivara)tends

sensu srticto)

types

to be differentiatedPreviousof O. rufipogon

has a monophyleticmonophyletic
perennialstudies

supposed(e.g.

Sano et al , 1980),

origin

(Oka,

1974;

the wild progenitor

that O. sativa

Chang,

1976)

ongin(s),and if diphyleticareancestorsofthep-SINEl-r2,

Umeda et al 1991)
relativesa short interspersed
was analyzedof rice. Results

p-SINEl-r2,

while

p-SINEl-r2

polymorphism

indicain 46 strains

indicatedmost perennial
corresponds

types

that

of Asian cultivatedand perennial

diverged

To elucidate
origins,

acceptedthat

whetherO. sativa

has

the

annualanddistribution

of

the

whetherand japonica,

thenuclear(SINE)

element(O. rufipogonfrom the intermediate

and it has been widelyor diphyletic
types

retrotransposonof wild

Tsukuba,differentiation,

sensu lato),(e.g.

of Tsukuba,

and Technology,

Asian commonwild rice {Oryza rufipogon

type

Sato2

O. sativa

at the waxy locus(23 each ofO.

nivara and O. rufipogon)

most annual typesof O. nivara possessedof 0. rufipogon
to annual-perennial

did

not have,differentiationsuggestingin wild

that

relatives

To clarify

the relationshipalso appliedbothbetween annual-perennial

two DNA markers discriminating

chloroplast

non-deletionand nuclear

indicaDNAs (Yamanaka(ND) in the ORF100

and indica-japonica

et al 2003)

region of chloroplast

DNA; alternately,

most of O. nivara showed deletion

annual-perennial

habit

and nuclear

In this(P, -, ND, J) and typical
study,

perennial
indicathe wildat

Most of O. rufipogon

DNA andjaponica(D) and indica

showed(J) type of nuclear
From the results,

polymorphism(+, -), chloroplast

DNA (D, ND)

associatedwith each other indicating

typicalO.

O. nivara (A, +, D, I)
of O. sativa

had clearly

differentiatedintoannualandgroups by p-SINEl-r2

polymorphisms,

and these

two groups

are corresponding

toof O. nivara and japonica

studies

intermediate

We consideredtypes
severalrathertype of O. rufipogon, contradictingsome of the materials

than typicalperennial

types,

seed propagation

for multiplication.

were collectedin the form of living

clonesprobablethatcollection

genetic

structure

was strongly
japonica^of O. sativa effectively

relatives

type

previousthrough(A, P), p-SINEl-r2

we(I) type.

DNA (I, J) were nonrandomly

rufipogon

andjaponica

differentiations,of the materials,

suggested

respectively

Acknowledgements
Vaughan (NIAS)

and multiplicationthatparticularly

used in the previous

becausearecollection

and

types

used in thisstudy

by vegetative

propagation.

It is

could

affecttheperennialstrainsItof indicaandof seed-derived(1 e. seedin the partiallyto thestudies

The perennial

and maintained
methods

resultsor clone)outbreeding

O. nivara and O. rufipogon are wild

ancestors

and the origin of O. sativa seems to be diphyletic.
The authors

for valuable

thank

Drs

H. Morishimacomments of in this study(TokyoU. of Agr)and D.A.

References
Chang,

T.T. (1976)

The origin,evolution,

Asian and African rices Euphytica
Oka, H.-I (1974)

Oryza perennis

dissemination,and diversificationof

25 425-441

Experimental

Sano, Y, Morishima,cultivation,

studieson the origin of cultivatedH. and Oka, H.-I (1980)

found in Thailand

and their

Intermediate
evolutionary

rice. Genetics

78 475-486.

perennial-annual
significance.

populations

Bot

of

Mag. Tokyo 93

291-305
Umeda, M., Ohtsubo,
insertionof mobile

H and Ohtsubo,

E (1991)

DNA elements into introns

Yamanaka S., Nakamura I Nakai
markers

Jpn. J. Genet

H and Sato Y-I

based

on molecularof newly

species,Oryza nivara and O. rufipogoncollected

Diversificationof the rice Waxy gene by

66 569-586.

(2003)

Dual originof the cultivatedriceannualand perennial

strainsrice

Genet Resour

Crop. Evol

50 529-538

of wild