Biodiversity

Integrationofand Genome Technology
for Crop Improvement

K. Oono, T. Komatsuda,

K. Kadowaki

Edited by
and D. Vaughan

Integrationof Biodiversity
for Crop Improvement

Proceedings
2000

of the Joint International

International

Science

Workshopand Technology

and Genome Technology

Workshop

Molecular Plant Breeding and Utilization

Agency (STA)

and Japan Internationalof Genetic Resources sponsoredScienceand Technology

byExchange Center(JISTEC)&
8th MAFF International
Fisheries

Workshopon Genetic

Resourcessponsoredby Ministry(MAFF)

November 28 - December 1, 2000
Tsukuba,PrintedPublishedJapan

in Japan by Sato Printing
by National

ISBN 4-93151

1-04-X

November 28, 2000

InstituteCo., Ltd , Tsukuba
of Agrobiological

Resources,

Tsukuba, Japanof Agriculture,

ForestryandPREFACE

Crop improvement using conventional varieties or wild species as genetic resources
has successfully
brought increases to agricultural
production worldwide. To respond to thedemand for increased agricultural
production research efforts have focused on development ofefficient breeding methods and finding new gene sources in conserved genetic resources.
Recent rapid developments in biotechnology
are changing both breeding methods andthe concept of genetic resources. All the functional genes of organisms are potential
genetic
resources for crop improvement by direct gene introduction.
The developments in genome technology and the accumulation of genome information
are so rapid that the complete genome sequences of 150 organisms - microorganisms, higher
plants, animals and manwill have been elucidated within two years.
Conservation and use of biodiversity
are fundamental to sustaining agriculture
and theenvironment. The applicationof genome technology to the analysis and use of biodiversity
ismost useful for the development of new breeding methods and identifying
new gene sources
for crop breeding.
Using comparative
genomics to understandvariationin biological
function,morphologicalcharacteristics
and physiologicalresponses to stresses will enable newplateaus
to crop improvement to be reached. Research into the genetic components of species andpopulations,
genetic variationand genetic differentiation
between crops and their wild
relativesand evolutionary
processes and relationships
among plants will support crop
improvement.
Enhanced linkages between plant breeding, biotechnology
and biodiversity
scientists
are to be encouraged. Research on organisms and their environments based on new
perspectives
at the gene, genome, individual,
populationlevel will require specialists
tointegrate the results for practical use. I hope this workshop will promote such integrationbybringing
together scientists
with expertise in biodiversity
and biotechnology.
I hope suchintegration
will facilitate
development of more efficient plant breeding in the 21 st century.

Kiyoharu OONO
Organizing Committee

Organizing

Committee

Kiyoharu Oono, Chairperson
Koh-ichi Kadowaki, Secretary
Duncan Vaughan, Secretary
Takao Komatsuda
Norihiko
Tomooka
Masao Iwamoto
Toshiyuki
Wako
Advisory

Board
Ronald L. Phillips,
USA
Gerhard Wenzel, Germany
Masaru Iwanaga, Japan
Shoji Miyazaki, Japan
Hiromi Kobayashi,
Japan

Ill

CONTENTS
Pre fac e
Organizing

Proceedingsi
ii

Committeesof oral presentations

Comparative genetic and physical mapping in the Oryzeae
R. Phillips,
S. A. Jackson, P. Imle, W.C. Kennard and R. A. Porter
Retrotransposons
H.Hirochika

as a tool for functional

analysisof plant genes

Genomics as a tool for an efficient utilizationof genetic resources using potato and wheat as
examplesG. Wenzel, A. Lossl, U. Frei, V. Mohler, £ L K. Hsam,X Q. Huang, F. Thummler andF. J.
ZellerTracking alien introgression
into the rice genome through molecular approaches
D. S. Brar, F. M.Abbasi, J. Talag, E. Enriquez, Z Li, M.Jones and G. S. Khush

Genetic architecture
rices
Y. Sanoand complexity

as revealed

at the molecularlevelsin wild and cultivated

The introgressionof QTL from Oryza rufipogon to IR64
MSugiono, E. M.Septiningsih
and S. R. McCouch
Evolutionary
relationships
among Oryza species inferredassociatedwith the catalase gene CatA
M.Iwamoto
The construction
B. Wang

and identificationfrom transposable

elements

of plant BAC library

Rice centromere structure with repetitive
chromosome (RAC)
N.Kurata

sequences and constructionof rice artificial

DNA marker-based estimates of coancestry and parental contributions
D. A. A. Tabanao, A, L. Carpena, R. N. Bernardo andL. S. Sebastian
Genetic analysis and breeding improvement of a single
Q. Qian, X. Li, D. Zeng, J. Ouyang, S. Teng and J. Li

tillermutant (stl)in ricein riceIV

Evaluation and utilization
K. Takeda
Sourcing, evaluating
P. Heslop-Harrisonof genetic

resources:

and utilizing
biodiversity
and T. Schwarzacher

A case study for salt tolerance

in barleyin crop genomes

Genomeanalysis in barley and its impact on the utilizationof genetic resources
A. Graner, D. Smilde, A. Drescher, K.-P.Pleissner, K Ordon and W.Michalek
Molecular diversity
T. Komatsuda

and evolution

in the genus Hordeum

Preservation,
evaluation and utilization-present and future prospects/. Atanassov and A. Atanassovof economicallyvaluableplant germplasm in Bulgaria
57

Germplasm diversity in its evolutionary
context: U.S. maize as a case study
J. Labate, S. Kresovich andK. R. Lamkey

59

Diversity of Garcinia spp. and interspecificS. Te-Chato, M.Lim and M.Masahiro

63

relationships

by DNA analyses

Population diversity analyses using different molecular techniques to determine strategies
in-situ conservation - examples from the genera Glycine, Oryza and Vigna
D. Vaughan, N. Tomooka, A. Kaga, A. Shcherban, R. Q. Xu, Y.Jinno, K. Doi andK.
Kashiwaba
Howto control biodiversityof crops by means of biotechnology?
overcoming breeding barriers
T. Adachi, L Chen, S.-H Woo,J. Alt andM. Nagano
Genomics and biodiversity
A Karpmanagement: opportunities

Genetic diversity
H Sato69

Some approaches to73

and challenges
77

RNA editing in plant mitochondria,
cytoplasmic
S. Litvak, A. Mouras andA. Araya
Mitochondrial
genome diversity
genome or nuclear genome?
K.Kadowaki

formale sterility

among plant species:

and plant breeding81

is the gene encoded by mitochondrial85

of endosperm storage mutants in rice

Problems of genetic and molecularqualitative
characters of crops
V.A. Dragavtsev

89
approaches to the hereditaryimprovement of the93Integrationof genomics and biodiversity
research at the Wheat Genetic Resources Center
B. S. Gill, G. Brown-Guedira and W.John Raupp

96

Expansion of available
K Tsujimoto

100

genetic resources for wheat improvement by super-wide hybridization

Development of novel wheat starches by the use of germplasm diversity
M.Yamamori
Rapid and simple method for purity test in radish

hybrid103

seeds using RAPD marker

S. T. Kwon andS. M. Oh

107

The molecular biology of the oil palm flowering
H.Kulaveerasingam, Z Rahmat, T. S. Hee, L C. Ching, C. M.Chooi, L Y. PingandS.
Alwee

R. S.
111

On the synteny among grass genomes
Y.Nagamura

1 15

SNP, genetic diversity
K Watanabe

1 18

and genepools for germplasm enhancement

Diversity of plant genomes revealed by analysis
R. Henryof gene sequences and microsatellites
122

Genomefusion for crop improvement, a new style of breeding
D. Shibata

Proceedingsof poster presentations

In alphabeticalorder of author

Genetic diversity and phylogenetic
relationships
on isozyme and microsatellite
polymorphisms

126

in East Asia and South Asian melons based

Y Akashi, T. Wako, M.Masuda and K. Kato
DNA sequence data and phylogeny: The genus Vigna subgenus Ceratotropis
K. Doh A. Kaga, N. Tomooka andD. A. Vaughan

129
a case study

Phylogenetic
analysis of the genus Zoysia for improvement of indigenous grazing
MEbina, A. Abe, M.Kobayashi, S. Kasugu, H. Araya andK Nakagawa
Screening of allelopathic
activity
Y. Fujii andD. R. Gealy

131
grassland
133

from rice cultivars
135

VI

Structural variation of waxy gene and differentiation
Beauv.
K.Fukunaga, M.Kawase and K. Kato

in foxtail

millet,Setaria

italica(L.) P.
137

Core collections,
DNA markers and bio-informatics:
new tools for "mining" plant genetic
resources held in gene banks - Sorghum as an example
C. GrenLr, P. J. Bramel, P. Hamon, J. Chantereau, M Deu, M.Noirot, V. G. Reddy,
S. Kresovich, K E. Prasada Rao, V.Mahalakshmi, J. H. Crouch andR. Ortiz

139

Applicationof heavy-ion bombardment to mutate wheat and barley
7. Honda, M. Wada,T. Abe, T. Asami andS. Yoshida

141

Development of DNA marker linked to fruit skin color in Japanese pear (Pyrus pyrifolia
Nakai).
E. Inoue, K. Amano, M.Kasumi, F. Sakuma, K Hara and T. Tsukihashi

143

Intra-specificgenetic differentiationin East Asian Vigna species (subgenus
revealed by AFLP markers.
A. Kaga, K. Doi, M.S. Yoon, N. TomookaandR A. Vaughan

145

Morphologicaland biochemicalvariations in radiation
H.Kajiwara, T. Kaneko and M.Nishimura

Ceratotropis)

mutants of rice147

The search for and analysis of new sources of resistance to legume storage pests
K. Kashiwaba, N. Tomooka, A. Kaga andD. A. Vaughan

149

Molecular analysis of genetic diversity
association
with adaptation to abiotic
K Kato andK Matsumoto

151

in East Asian wheat landraces
stressesand its possible

Molecular cloning and structural characterizationof a receptor-likeprotein kinase gene fromrice {Oryza sativa L.)
MKawata, T. Oikawa, Y.Matsumura, F. Fukumoto, S. Kawasaki, F. Takaiwa andS. Kuroda 153
Gene organizationof sugar beet mitochondrial
genomeT. Kubo, S. Nishizawa, A. Sugawara, K Itchoda, A. Estiati
The relationshipbetween intron splicing
under low temperature
S. Kurihara-Yonemoto and R Handa

and RNA editing

Mapping QTL's for tissue culture
Y.Manoand T. Komatsuda

in barley

traitsand T. Mikami

155

of wheat mitochondrial

cox2 gene
157

Mapping of a fertility restoration locus (Rftnl) for cytoplasmic male sterility
RAPD and STS markers
K.Matsui, Y.Mano, S. Taketa, N. Kawada and T. Komatsuda

159
in barley

using
161

Vll

Barley allele-specificamplicons useful for wheat-barley introgression
K.Murai, S. Taketa, A. K. M Rafiqul Islam andK. W.Shepherd
Geographical variation of the gene, lox-3(t),varieties
T. Nagamine, Y.Suzuki andK. he

causing lipoxygenase-3

deficiency

in Asian rice

A molecular phylogeny of genus Lilium in matK gene sequences
T. Nishikawa, K. Okazaki, K. Arakawa and T. Nagamine
Diversity in Pakistan rice genetic resources for endosperm storage
S. U. Siddiqui, T. KumamuraandH. Satoh
Isolation of microsatellites
in Brassica rapa.
K. Suwabe, H. Iketani, T. Kage, T. Tsuchiya, Y.KowyamaandM. Hirai
Origins of six-rowed cultivated barley deduced from a DNA marker tightly
vrsl locus (row type gene).
K. Tanno, S. Taketa, K Takeda and T. Komatsuda

linkedto theConstructionof a microsatellite-enrichedlibrary in azuki bean
X-W.Wang,A. Kaga, N. TomookaandD. A. Vaughan
Diversity of chloroplast
DNA SSRs in wild and cultivatedsoybeans: an evidence for themultiple origin of cultivated soybeans with different organelle genomes from wild soybeans
D. Xu, J. Abe and Y.Shimamoto
A syntenic approach for the functional
K. Yoshida, N. Imaizumi, M.Akiyoshi

genes conveying
and K Endosalinityin the family

GraminaeProceedingsof oral presentations

Comparative

Ronald L. Phillips1,

Genetic

and Physical

Mappingin the Oryzeae.

Scott A. Jackson1, Peter Imle1, Wayne C. Kennard2, Raymie A. Porter1

department of Agronomy and Plant Genetics, University of Minnesota, 4 1 1 Borlaug Hall, 199 1 Upper
Buford Circle, St. Paul, MN 55108 USA
2Monsanto Company, 3302 SE Convenience Blvd., Ankeny, IA 5002 1, USA
Keywords: comparative

mapping,

rice, wildrice,physical

mapping,

FISH

Introduction
American wildrice (Zizania aquatica) is the only grain endemic to the North
American continent. It grows in lakes and slow moving streams in the north central UnitedStates and into Canada. Wildrice was an important food and ceremonial crop for Native
Americans inhabitingthese regions of North America. It was hand-harvested by canoe andcontinues to be an integral part of Native Americans' lives. It was only in the mid-1 900fs thatwildrice began to be cultivated by farmers for mass production. The cultivated forms of wildrice
were renamed Z palustris.
From a genetic viewpoint, very little was known about wildrice until recently. As late as
the 1970's only the chromosome number was knownand it was assumed to be a diploid.
Cytogenetically,
wildrice has a haploid chromosome number of 15 and observations of meiosis
indicatedthat it regularly forms 7 rod and 8 ring bivalents (Grombacher et al. 1997). Until the
1 990's almost nothing was known about its taxonomic relationshipto other cereal crops. Similar
to maize, the male and female floral organs are anatomicallyseparated in wildrice. Thus, it wasthought by some to be more closely related to maize than to rice (Oryza sativa).
As would be expected of a newly cultivated species, there are many production problems
with wildrice.
The most obvious of which is seed shattering;
estimates range from 40% to 70%
yield losses due to shattering.
Several of the diseases affecting wildrice are the same pathogens
affecting rice. Thus, the value of research in rice holds particular value for those working on
closely related species such as wildrice.
Figure 1. Phylogram ofseveral cereal species basedon sequence data of the Adhl
genes (Ge et al. 2000). Z
palustris (American
wildrice,sequenced in thisstudy) falls within the Tribe
Oryzeae which includes O.
sativa and R. subulata. Bar
equals 10 substitutionsper
1 00 residues. Phylogram wasconstructed using the
growtree function of the
GCG package.

Taxonomic relationshipof wildrice to rice When the molecular investigation
ofwildrice
wasinitiatedin our lab the relationshipof wildrice to rice was unclear. Chloroplaststudies had
suggested that this species was in the Tribe Oryzeae (Duvall et al. 1 993), although,morphologically,
wildrice did not appear to be a close relative of rice. Southern blot analysis of
genomic hybridizationsof wildrice genomic DNAto rice, oat and maize DNA revealed that the
DNAof wildrice was most similar to that of rice thus confirming the relatedness of wildrice torice (Kennard et al. 2000). Furthermore, sequencing of the Adhl and Adh2 genes showed inmoredetail the relationshipof wildrice to other cereals and confirmed the inclusion of wildrice in
the Tribe Oryzeae (Fig. 1).
Molecular mapping of wildrice
Recently, a project was initiated to apply modern molecular
genetics to wildrice. Chloroplastevidence suggested that wildrice may be closely related to rice
(Duvall et al. 1 993) and Southern analysis of genomic DNA from wildrice and rice confirmed
this relationship(Kennard et al. 2000). Therefore, RFLP probes mapped in rice were used toconstruct a molecular map of wildrice. Rice RFLP probes were used to exploit the potential ofcomparative genetics to determine the underlying genetics of agronomic traits in wildrice.
The molecular genetic map of wildrice showed extensive colinearity of RFLP markers
with the genetic map of rice (Fig. 2). Also, the map indicated that three of the wildrice
chromosomes were duplicates of rice chromosomes (wildrice chromosomes 1, 4 and 9). Wildrice
is the only species in the Tribe Oryzeae that has a chromosome complement that is not a multipleof 12. There are several allopolyploid
species within this tribe, but wildrice appears to be theonly one that is an aneuploid. We are investigating
the nature of this aneuploidy by comparative
physical studies and examining the superchromosomal structure of the duplicatedchromosomes
by looking for hypermethylation.
Comparative genomics within the Tribe Oryzeae The value of comparative studies has
increased due to the availabilityof the sequence of the rice genome (http://www.riceresearch.org; http://rgp.dna.affrc.go.jp/Seqcollab.html).
The molecular genetic map of wildrice
constructed in our lab is >80% colinear to the rice genetic map making it the most colinear
comparison of genetic maps to date. Since very little is known about the underlying genetics ofagronomic traits in wildrice, rice has become a very important model for our studies. Several
lines of investigation
are being pursued to exploit the similarity of wildrice to rice.
Conservation of genetic information between rice and wildrice can be seen in thecomparative analysis of QTLs between the two species. QTLs for traits such as seed shattering,height, maturity, panicle length, and stem color have all been mapped in wildrice (Fig.2)(Kennard et al. in preparation).
Many of the colinear regions in rice contain QTLs forcomparable traits such as the dwarf gene sd-1 on linkage group 1 (Kinoshita1997) and qSHT-4(Sh-3, Causse et al. 1993; Cai and Morishima, 2000). This information highlights
the value ofcomparative genetic mapping and can be utilized to find analogous QTLs between related
species. Several of these QTLs have been, or are being cloned in rice providing candidate genes
for the related wildrice QTLs and, in the short term, are providing more RFLP markers to further
refine the genetic positions of these QTLs.
Another line of investigationthat is being pursued is comparative physical mapping ofseveral cereal genomes including rice, wildrice, maize and sorghum. The value of fluorescencein situ hybridization
on chromosomes and extended DNA fibers (fiber-FISH)
has been very
useful for comparative studies in both plants and animals. Fiber-FISH is a tool that allows one to
examine genomes at a resolution approaching the limits of the Watson-Crick DNAmodelwithout the expense of sequencing across several genomes. For instance, in mammals thechromosomal evolution of primates can be visualized
by using entire chromosomes as probes topaint chromosomes from related species (Archidiacono
et al. 1 998).
In plants, fiber-FISH
hasbeen used to examine the evolution of several hundred kilobases
ofDNA from chromosome 2 ofArabidopsis
in the Brassicas by hybridizing
Arabidopsis
bacterial
artificial
chromosomes (BACs)
to genomic fibers from Brassica rapa (Jackson et al. 2000).
In the cereals, we are pursuing thisline of investigationby using rice (BACs - each containing
up to -150 kb of rice DNA) as FISH
probes to examine chromosomal evolution by hybridizing
rice BACs to chromosomes and thengenome evolution/architecture
by hybridizing
to genomic fibers from related species using fiberFISH. By hybridizing
to chromosomes we first determine whether rice regions have been
duplicatedin related cereal species.
Once this is done, analyses using fiber-FISH
permit us todetermine what has happened to genes within regions delimitedby the rice BACs.
SupportThis research was funded in part by Grant No. USDA 95-34340-1605(Purduesubcontract 593-0220-02),
Minnesota CultivatedWild Rice Council, and the U.S. Department
Agriculture- Agriculture Research Service Cooperative
Agreement.

ofReferences
ArchidiaconoN., C. Storlazzi,
C. Spalluto,
A. Ricco, R. Marzella and Rocchi. 1998. Evolutionofchromosome Y in primates. Chromosoma 107:241-6.
Cai, H.W., H. Morishima. 2000. Genomic regions affecting
seed shatteringand seed dormancy
in rice. Theor Appl Genet 100:840-846.
Causse, M.A., T. Fulton, Y. Cho, S. Ahn, J. Chunwongse, K. Wu, J. Xiao, A. Yu, P.C. Ronald, S.
Harrington, G. Second, S. McCouch, S.D. Tanksley. 1994. Saturated molecular map of the ricegenome based on an interspecificbackcross population.
Genetics 138: 125 1-1274.
Duvall, M. R., P.M. Peterson, E.E. Terrel and A.H. Christensen.
1993. Phylogenyof NorthAmerican Oryzoid grasses as construed from maps ofplastid
DNA restriction
sites. Amer. J.
Bot. 80:83-88.
Ge, S., T. Sang, B-R Lu and D-Y Hong. 1999. Phylogenyof rice genomes with emphasis on
origins ofallotetraploid
species. Proc. Natl. Acad. Sci. USA 96:14400-14405.
Grombacher, A.W., R.A. Porter, L.A. Everett. 1997. Breeding wild rice. Plant Breed. Rev.
14:237-255.
Jackson, S.A., Z. Cheng, M-L. Wang, H.M. Goodman and J. Jiang. 2000. Comparative FISH
mapping of a 43 1-kb Arabidopsis
thaliana BAC contig reveals the role of chromosomal
duplicationsin the expansion of the Brassica rapa genome. Genetics 1 56:833-838.
Kennard W. , R.L. Phillips, R. Porter, A. Grombacher. 1999. A comparative map of wild rice
(Zizaniapalustris
L. 2n=2x=30). Theor. and Appl. Genet. 99:793-799.
Kinoshita,T., 1997. In Science of the Rice Plant. Vol III. Genetics.
Matsuo, T., Yuzo, F., Fumio,
K,, Hikoyuki,
Y. eds. Food and Agriculture
Research Center, Tokyo.

Figure 2. RFLP map of Zizania palustris(American wildrice)
showing mapped
QTLs for height, maturity, shattering,
panicle length and stem color. Gray shadedboxes along the linkage groups indicate regions of colinearity
with Oryza saliva(numbers are rice linkage groups).

Retrotransposons
Hirohiko

as a tool for functionalof Molecular

Tsukuba City, Ibaraki
Screeningcausative

Genetics,

305-602,

National

Instituteby insertion

important

plant-specificrapidbiological

functionsfor plant

processes

progressanalysis

Insertion

molecular

to sequenced

biology.

projects,

gene tagging,of maize,

has been developed,

are being developedin another important

insertional
insertion

are summarized

are stablebecausetarget sites are unlinked

to generatea large collection

transposition

can be easily

inactivatedby regeneration,transpose

into gene-rich regions,to identify

tagging

activeas follows.
they

(1)

transpose

regulated,(4) they are highly(5) the original

the transposed
retrotransposons

copy

activesystem available

and reverse genetic

tosystemsover traditional
genetic

via a replicativemode, (2)copy, so that it is easy

for saturation
activatedmutagenic

tool forRetrotransposon-mediatedwith the site of the originaland strictly

Basedmodel plant, rice, whose genomic sequencingof random insertions

The rice retrotransposon7oyi

and thereforeof active

clonesthat make them an idealtransposition

method.

thatelements and their unique features
mutationsendogenous

such as Ac,

have other advantages

in plants

and maize.

elements,

Plant retrotransposons

gene tagging

reverseof tagged insertional

which can make the tagging

Indeed, retrotransposon-based

has been initiated.

For thisthe PCR-based method for cloning

many plant species.

thefor assigning

such as Arabidopsis
Providedin

Reflecting

those elements can also be used for gene tagging.

on unique features of plant retrotransposons,retrotransposons

is one of themethodlarge populations

and T-DNA.

ofMany genes involveda systematicand used in model plants

are available,

by isolation

tags ("gene tagging")

elements used in these systems are class II transposable

retrotransposons

easy

followed

genes becomes more important.

as well as classical

En/Spm, and Mutator

Resources,

have been cloned using this method.

of sequencing

mutants are being produced

project

elements

genes using these sequences as molecularmethods

of AgrobiologicalJapan.

of mutants induced

most important

geneticof plant genes

Hirochika

Department

recentanalysismutagenesis,by tissue

culture(3)
andas some of them normallycopy number can be low, so that it is

responsible

for thecan be isolated

by using

7 is highly

under tissue

activeit has been used to generate a large collection

specificmutation,

the simple
culture(6)

RT-PCR
conditionsof random insertionsforsaturation

mutagenesis.

200,000

insertions

genetic

studies.

A collection

was generated,of 30,000

and the lines

Current status of these studies

regenerated

rice lines

carrying

about

are being used for forward and reverse
will be presented.

Genomics as a tool for an efficient utilisation
using potato and wheat as examples

of genetic

resources

G. Wenzel, A. Lossl, U. Frei, V. Mohler, S.LK. Hsam,X.Q.Huang,F. Thummler, FJ. Zeller
Department for Plant Sciences
Technical University of Munich
D-853 50 Freising-Weihenstephan
Introduction
Classical and biotechnologicalbreeding techniques rely on the production and use of variability followed by the selection of the better plant type. The big challenge of the past scientificcentury was the discovery and the programmed use of the phenomenon of genetic recombination. Thus, it became possible to start the process of combination breeding in a scientific
way
and to predict the results of a logical crossing program. The former simple selection was
backed by recombination and in parallel breeding changed from traditionalroutes to a sophisticated industrialised
process. Traits or genes up till that time part of nature owned by nobody,
became a precious good sliding out of the public domain into the private sector. But a successful new variety is never better in only one trait; it is the result of the art to combine numerous
useful genes in one genotype. This should underline - right at the beginning - the only relativevalue of a single gene, and in consequence the only limited importance of both: primitive cultivars or wild varieties with just several interesting
genes on the one hand and gene transfer
handling only a few genes of interest on the other hand.
A higher plant contains 20 000 to 50 000 genes; recombination is today and in the future thecentral process and thus good luck, the green thumb, is an important prerequisite
for success,like it has been since man settled about 10 000 years ago. It should be stressed that eventhough there is a spectrum of new biotechnologies,
the present breeding progress is the resultof classical breeding, and this will continue. Genomics is an addition to classicaltechniques
only, and will never replace them. Thus, a prerequisite for a successful plant breeding industry
is the establishmentof classical breeding programs. But merchandisingof biodiversity,
genotypes or ultimately genes will speed up and alter agricultural research, breeding and agribusiness.
Newapproaches
It is good luck that the new biotechnological
techniques arose just when the number of breeding aims and the need to produce more food from less land increased. Genomics is one piecein the mosaic for increased food production. Before using genomics, it should be checked,
however, carefully whether it is cheaper, and/or faster, securer, more durable or not less sustainable than classical approaches. It would probably be a waste of time and money if it isapplied just because it is more fashionable.
If the use is done in the right manner, it is expected that the incorporationof DNA technologies will be the most efficient way to combine
economic and ecological aims.
The production of the first transgenic cultivars being now in the market was driven by available technicalpossibilities.
The structural analysis and subsequently
the functional
gene
analysis was much faster in bacteria, and in consequence it was easier to transfer genes identified in viruses and bacteria than such from higher plants. Now heavy research is going on in
the functional analysis of higher plants to identifying
enzymes, the field of proteomics. As
soon as its regulation and the metabolisms is under control, the area of metanomics, an effi-cient improvement of existing cultivars will be the result, particularly
in characters also ofinterest to the consumer with the consequence of an easier acceptance of new products. The
possibilityof converting our descriptive
research field to a constructive one, using genomics,
proteomics and metanomics increases the value of the genetic resources of higher plants as theprinciple source for desired genetic informations.
Gene transfer is not the only branch of genomics. Presently tools helping to improving selection are of greater immediate importance. And thus DNA probes, can be used as markers forthe identificationof genes responsible
for traits of interest. Most of these probes are still only
detecting monogenic traits. An increasing interest exists, elaboratingprobes form oligo- or
polygenic characters. A clear reproducible
correlation between probes and polygenic, quantitative traits (QTLs) would result in a tremendous improvement of breeding programs. Presently, the most powerful applicationof such specific probes and molecular markers is opened
up by marker aided selection (MAS).
Besides MAS gene probes are also a useful tool in plant genetic resources collections.
The
easiest approach is to use DNA fingerprints to estimate genetic diversity. From comparison ofsuch patterns genetic similarities
can be estimated which in turn can serve as the basis for further multivariate
statisticalanalysis. A dendrogram can make such relationships
visible. Sucha molecular tool will be helpful for 1. Detecting duplicates;
2. Ensuring a representative
core
collection;
3. Facilitating
the evaluation process. In the long run the present seed gene banks
might be backed by molecular gene banks, and eventually replace them.
This is not only true for the genetic information of Th the nucleus by also for the cytoplasmic
information. We used e.g. 180 dihaploid
potato clones and 144 German cultivars to analyse
their mitochondrial
genome with 1 1 homologous mtDNA-probes (Lossl et al. 1999). They
could be classified
on the basis of RFLP patterns into fife classes. A dendrogram on the basisof their similarity
showed the genetic distances of the clones used. Some pattern types could
be traced back to Solanum phureja and S. andigena, while others had a typical S. tuberosum
pattern. By somatic fusion it was possible to combine different mitochondrial
types and it waseven possible to find recombinationsof the mitochondrial
genome. Such new mt types contained recombination-specificrepeats homologous to Petunia and Nicotiana.
The somatic fusion allowed in the presence of nearly isogenic nuclear genomes the estimationof the contributionof mt genomes to starch production. Evaluation of cytoplasmic types leadto the conclusion that in starch content the types from non tuberosum Solanum species have asignificant
advantage to the tuberosum type cytoplasm.. In somatic hybrids an interaction between starch content and different mt-plastid combinations could be found. During the in vitro
phase a selection for optimised organellar segregation took place (Lossl et al. 2000).
These results underline the chances opened up by diploid potato species in a gene bank notonly for improved clones using the nuclear genome but also the importance of the cytoplasm.
For such approaches molecular markers are an essential tool.
Using genomics on the nuclear level the problem of QTLs is most prominent at this moment.
We concentrated on the estimation of genes contributing
to Phytophthora resistance.
By theuse of differential
techniques genes can be identified
which are expressed in only very minuteamounts by their enrichment of up to 1000 fold (Diatchenko et al. 1996). Using this techniquewe were able to detect genes in potato whose expression is specificallyinduced by the infec-

tion with Phytophthora(Thiimmler
and Wenzel 2000). A plasmid library was constructedwhich represented about 100 000 primary transformants, and which was expected to be greatly
enriched for clones carrying sequences of Phytophthora-induced
genes. Of 96 clones randomly
picked and analysed, 58% turned out to code for seven known pathogen related proteins. One
novel gene turned out to be clearly differentiallyregulated and thus represents a new PR-gene.
The other 42% of the clones coded for weakly expressed genes, half of them represent genes
not present in the public databases, ad the other half exhibit homology to known enzymes
partly with relation to pathogen resistance. Two genes carry LRR domains. This experiments
demonstrate that gene libraries generated by SSH method represent excellent sources for theidentificationof genes involved in defence against pathogen attacks. Particularly
in finding
new sources for resistances in the collectionof wild potato species such an approach is highly
recommended.
Mapping monogenic traits is easier, however, crops exist which have a more complex genome
like the hexaploid
wheat. Chromosomal location of markers is a essential step for genotyping(e.g. Zeller et al. 1998). For Triticum aestivum "Chinese Spring" we used 256 primer combinations with the AFLP technique using nulli-tetrasomic
stocks (Huang et al. 2000a). The
chromosome and arm assignment of AFLP markers provided a valuable tool for anchoring
unknown linkage groups in chromosomes from experimental wheat crosses involving genebank samples. Similar approaches were applied for mapping the Pm 24 locus by bulked segregant analysis in an F2 progeny from the cross Chinese Spring (susceptible
to powdery mildew) x Chiyacao (resistant).
An allele of a microsatellite
locus located 2.4cM from Pm24 wasshown to be diagnostic and therefore potentially
useful for pyramiding two or more genes for
powdery mildew resistance in a single genotype (Huang et al 2000b). Further, we mapped
powdery mildew and leaf rust genes on the IRS arm of the wheat-rye translocation
chromosome T1BL.1RS in relation to the Sec-1 locus and AFLP and FLP markers , employing segregating F3 populations.
Integrationof molecular markers indicated that Pml7 lies between the
Lr 26 and Sec-1 locus, with both resistance genes allocated distallyto the Sec 1 locus in thesatellite of the IRS arm (Hsam et al. 2000).
Using this information it was possible to combine three Pm genes in one line which is now
under variety test. In wheat we succeeded in combing different powdery mildew resistancegenes - showing the same phenotype - by MAS, hoping that such a pyramided resistance will
be more durable.

New biotechnologicalprocesses are expected to speed up the breeding process. Thus, replacement of old cultivars may become faster. To check whether the fear of increased loss of
biodiversity
by this higher breeding speed is correct, in our institute Benedikte Hatz comparedby the use of molecular markers the gene basis of more than 200 accessions of barley (Hatz
1997). Using 50 DNA-probes in a wide range of different barley varieties, land races and lines
reaching from wild varieties till cultivars,
she could demonstrate that although phenotypicvariability
is lost, the genotypic variability
is not narrowed. Comparing the similarity of the
DNA composition by measuring genetic distances, it became evident that newer and older
types do not form any clear-cut genetic clusters. The danger that genetic variability
is lost due
to the selection process is not as large as anticipated.
Conclusion

10
Only by new ideas the natural evolution can be turned into an anthropocentric
direction thathelps man to survive. This was and is the motivation for today's and forthcoming plant breeding. With increasing understandingof the genetic basis of breeding - and the most advanced
one is the combination of genomics, proteomics and metanomics - genetic resources harbouring such genes are gaining more importance, at least for the immediate future. On the long run
their importance will decrease since more and more gene functions will be uncovered andphysical gene constructs are kept in molecular gene banks. Probably each new cultivar will bepolished with several transgenes. This means, however, that the vast majority of the genes will
still be brought together by recombination and consequently progress in plant breeding demands a powerful classical program. Such basic genotypes are modified with several additional genes via transformation.
In addition to the molecular gene banks, increasing knowledge on the physiology
of genefunctions is generated, as e.g. for disease resistances. This will allow eventually the modification of such genes and the programmed construction of new resistance genes, which reduces
the importance of genetic resources. As long as this is only fiction and as long as man cannot
construct new genes but just new genotypes and since nobody knows the needs of tomorrow,genetic resources are a precious good. Due to the high costs which have to be invested for
isolationand due to the rule that precious things have their price, patenting is a fair way forprotection.
Anyone keeping patented gene collections
should, however, be obliged to give
licences. The price for such a licence might be paid under special circumstances also by the
public. Particularly
since the amount of food and feed stored world-wide would last in thecase of a bad harvest only for about 60 days! A clever usage of genes in gene banks is one of
the best paying strategies to win the battle against stress, pests and diseases.
Literature
Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lykyanov K, Gurskaya
N, Sverdlov ED, Siebert PD ( 1996) Suppression
subtractive
hybridisation:a method for generating
differentiallyregulated or tissue-specific
CDNA probes and libraries.
Proc. Nat. Acad. Sci. USA. 93: 6025-30.
Hatz B (1997)...
Hsam SLK, Mohler V, Hartl L, Wenzel G, Zeller FJ (2000) Mapping of powdery mildew and leaf rust resistancegenes on the wheat-rye translocatedchromosome T1BL.1RS
using molecular and biochemicalmarkers. Plant
Breeding 1 19: 87-89.
Huang XQ, Hsam SLK, Zeller FJ, Wenzel G, Mohler V (2000a) Molecular mapping of the wheat powdery mildew resistance gene Pm24 and marker validation for molecular breeding. Theor.Appl.Genet.
101 : 407-414.
Huang XQ, Zeller FJ, Hsam SLK, Wenzel G, Mohler V (2000b)
Chromosomal location of AFLP markers incommonwheat utilising nulli-tetrasomic
stocks. Genome 43 : 298-305.
Lossl A, Adler N, Horn R, Frei U, Wenzel G (1999) Chondriome-typecharacterisationof potato: mt and novei
plastid-mitochondrial
configurations
in somatic hybrids. Theor.Appl.Genet.
99: 1 -1 0.
Lossl A, Gotz M, Braun A, Wenzel G (2000) Molecular markers for cytoplasm in potato: male sterility
and contribution of different plastid-mitochondrial
configurations
to starch production. Euphytica (in press).
Thummler F, Wenzel G (2000) Function of genetic material: From gene structure to gene function. Prog. Botany
61:54-75
Wenzel G, Schwarz G, Thummler F (1999) Approaches to understand the action of genes responsiblefor disease
resistancein plants. In: Different
pathways though life. Denke A. et al. eds.pp299-317,
Lincom Europa,
Miinchen.
Zeller FJ, Hunag XQ, Paderina EV, Collaku A, Kowalczyk K, Aslam M, Preusha H, Hsam SLK (1998)
Indentificationof powdery mildew resistance genes in common wheat {Triticum aestivum L.em.Thell.).
XII. Cultivars
and landraces grown in Mediterranean countries. Plant Gent. Res. News Lett. 1 16:5-8.

llTracking alienapproaches

introgression

into

the

rice

genome throughF.

D. S. Brar1,

International

Rice Research Institute, Los Banos, Laguna
2West African Rice Development Association,
Bouake, West Africa

molecular

M.Abbasi1, J. Talag1, E. Enriquez1, Z. Li1, M. Jones2 and G. S. Khush1

The genus Oryza to which cultivated rice (p. sativa 2n=24) belongs has 24 wild
species.
These wild species representing AA, BB, CC, BBCC, CCDD, EE, FF, GG and
HHJJ genomes (2n=24 or 48) are an important reservoir of useful genes for resistance tomajor diseases, insects and tolerance to abiotic stresses.
A series of interspecifichybrids,
monosomic alien addition lines and advanced backcross progenies (introgression
lines) havebeen produced through embryo rescue from crosses of elite breeding lines with wild species
representing different
genomes. Intergenerichybrid between O. sativa and Porteresia
coarctata, a tetraploid salt tolerant species has also been produced. Backcrossing of this F1
with the recurrent rice parent has been unsuccessful, probably due to strong incompatibility
barriers.
Introgression lines through direct crosses have been produced between indica rices
and closely related AA genome wild species such as O. rufipogon, O. longistaminata and O.
glaberrima.
Genes for resistance to brown planthopper, bacterial blight, blast and tungro
have been transferred from different wild species into elite breeding lines of rice. Some
promising breeding lines derived from the crosses of O. sativa x O. officinalis and O. sativa x
O. rufipogon have been released as varieties for commercial cultivation.
Some of theintrogressedalien genes (BphlO, Xa2\, Pi9) have been tagged with molecular markers.
Molecular analysis revealed introgressionof small chromosome segments from distantly
related species (CC, EE, FF genomes) into rice. In majority of the cases, one or two markers
were introgressed with flanking markers negative for introgression.
Such small alienchromosome segments introgression
in the face of limited homoeologous chromosome
pairing is being investigated.
Through flourescence in situ hybridization(FISH), parental
genomes have been characterized in several interspecific and backcross progenies involving
different genomic constitutions.
Chromosome pairing between A and E genomes wasanalyzed through FISH. The results showed both autosyndetic (AA, EE) and allosyndetic(AE) pairing.
Alien chromosome segments introgressedfrom the wild species into rice
could also be located through FISH. We are using AA genome wild species to identify andintrogress yield enhancing loci/QTL into rice. Preliminary results indicate that introgression
fromAA genomewild species {p. rufipogon, O. longistaminata) can enhance grain yield ofrice. A large number of introgression
lines from crosses of O. sativa x O. glaberrima havebeen produced and are being evaluated for transfer of tolerance to abiotic stresses from O.
glaberrima into the recurrent parent, O. sativa.
Dihaploid lines (DH) have been produced
through anther culture from the FIs of O. sativa x O. glaberrima.
Microsatellite
markers

12
show extensive polymorphism between O. sativa and O. glaberrima.
These markers areused to determine introgression
from O. glaberrima into O. sativa using advanced backcrossprogenies and DH populations.

13

Genetic

architecture

and complexity as revealed at the molecularin wild and cultivated
rice

level

Y.SanoPlant Breeding Lab. , Faculty of Agriculture,
Hokkaido Univ., Sapporo, 060-85 89 Japan
Keywors : Rice, Waxygene, genome architecture, repetitive
sequences, recombination
Genetic diversity
at the molecular level generally decreases due to genetic
bottlenecks
during domesticationin crop plants, however, phenotypic diversity increases
during domestication
as a general trend in crop plants.
The discrepancy between phenotypicand molecular levels is needed to be resolved in order to understand genepools of crop plants.
Here we examined allelic differentiation
at wx and its flanking region for understandingsofthe genomic complexity observed in wild and cultivated rice.
Allelic

diversty at the wx locus
The Wxgene plays a major role for eating quality of rice since it encodes
ADPglucose starch glycosyl transferase and controls amylose content in the endosperm. So
far, 5 different Wxalleles (Wxa, Wx1",Wx\ Wxop
and wx) were defined on the basis of theamount of the gene product.
The different alleles regulate the level of the Wxproductresulting in a range of variation in amylose content.
Wxhpredominates in the Japonica typeof O. sativa while Wxapredominates in the Indica type and its relatives.
The difference inthe cw-acting function is due to a point mutation of GT to TT at the 5' junction of the first
intron in the Wxgene, which results in an inefficient
splicing for the mature transscript in theendosperm with Wxb(Hirano et al. 1998).
It was predicted that various Wxalleles were
derived from Wxaduring domestication since the wild relatives carried only Wxa. We
determined sequences of Wxgenes of 2 1 wild and cultivated strains and constructed aneibour-joining
tree (Fig. 1). On the basis of Wxsequences, Indica strains were separated
from a cluster containing Javanica and Japonica types.
An exception was a waxyIndica
strain (SIN-1 1) which was included in the latter cluster.
On the other hand, strains of O.
rufipogon which is the progenitor of O. sativa tended to be dispersed in different clades.
African cultivated rice (O. glaberrima, GL) and the progenitor (WBA) formed a clade,although they were close to Japonica type rather than Indica type.
When allelic states of Wx
and the sequence at the 5 ' junction of the first intron were taken into account, the phylogeny
for the Wxgene fitted to the expectation that allelic diversity increased during domestication.
Wxop
and Wx1"both carried GT at the juction, suggesting they were derived from Wxa. The
former seemed to have occurred in Indica type although the latter in Japonica or Javanica type.
Wxbalso seemed to have been derived from Wxa,possibly during the domestication
process ofJaponica type. TT at thejuction
was also deteced in 3 waxy strains all of which were
included in a clade containing Japonica type. The 3 waxy strains were considered to have
independent origins because they had different mutaions within the Wxgene. These results
might give a possibility
that the wx gene in SIN-1 1 is a result of introgression
from a waxy
Japonica strain to an Indica type.

Fig. 1.
Neighbour-joining
tree for wxbased on the transcribedregion (exon 2 - exon 13)
in wild and cultivated rice. O. sativa (SIN, Indica type; SJV, Javanica type; SJP, Japonicatype), O. glaberrima (GL), O. rufipogon (WR), O. barthii (WBA) and O. punctata (WPU)
were used for the study.
Type of Wxalleles and the sequence at thejunctionof the 1st
intron are also indicated.
Bootstrap values (1000 times) are shown below the blanch(Mikami and Sano, unpublished).
Genomic structure surrounding the Wxgene
Recent interests in plant evolution have been focused on a rapid changes in
repetitive
sequences between genes during speciation.
To investigate the genomic
organization of the region surrounding the Wxgene, contiguous clones covering a total of
260-kb were constructed using a BAC library (Nagano et al. 1999).
We devided the 260-kb

15
region into 60 segments to use as probes to determine their repetitivenessby means ofsouthern hybridization.
The hybridization
patterns obtained by probing with these fragments
were classified
into 4 types depending on their repetitivenesswithin rice genome (Fig. 2).
About 70 % of the region was regarded as highly repetitive
sequences which were dispersed
within the genome. In addition, the repetitivenessof each fragments was compared among
AA-genomespecies in Oryza. Highly repetitive
sequences of class 4 (Fig. 2) showed no
obvious difference in the hybridization
pattern among the AA-genome species, suggestingthat those highly repetitive
sequences are well conserved in the closely related taxa.
However, one sorted into class 3 showed copy numbers varying among the AA-genome
species.
The lines belonging to O. sativa, O. rufipogon, O. meridionalis,and O.
longistaminata
possessed high-copy numbers of this fragment, whereas only a few bands
were detected in the lines from O. glaberrima, O. barthii, and O. glumaepatula (Nagano et al.
2000).

Distributionof genetic recombination
around the Wxgene
Meiotic recombination does not occour uniformly along the chromosomes
although our knowledge on recombination is limitted in crop plants.
The Wxgene gives aunique opportunity to examine the frequency of intragenic recombination within locus, so wecompared the frequencies of recombination within the Wxgene and in the flanking region(Khin et al. 1999).
From pollen analysis in hetero-allelicwxplants, the average frequency ofrecombination within the Wxgene was estimated as 27. 1 kb/cM which was about 10 times
higher than that based on the whole rice genome (250-kb/cM).
Six single-copy clones fromthe 260-kb contig were used to estimate frequencies of recombinationin the 195-kb around
the Wxgene. Examinations in a cross ofT65wjc and IR36 revealed that the recombination
frequency within 195-kb region was estimated as 148 kb/cM.
This value was two times
higher than the genome avarage, but five times lower than that estimated within the Wxgene.
The distributionof recombination sites seemed to be evenly distributedin the 195-kb region.
Thus, the present results suggest that the region within the Wxgene is markedly active formeiotic recombination compared with the flanking region which contains repetitive
sequenses.
Recombination is supposed to be important for evolution as well as breeding since adaptive
genes are accumulated into the same chromosome mainly by meiotic recombination.
Whether or not the recombination frequency varies between species is also an urgent problem
to be solved in order to use alien genes efficiently.
References
Hirano, H.-Y.,M.Eiguchi and Y. Sano. 1998.
of the Waxygene at the posttranscriptational
Mol. Biol. Evol. 15:978-987

A single base change altered the regulationlevel during the domesticationof rice.

Khln-Tidar,

Y. Tanaka, T. Inukai

and Y. Sano.

in the 195-kb region surrounding
Nagano, H., S. Kawasaki, Y. Kishima
vicinity

1999.

the wx gene in rice.
and Y. Sano. 2000.

of the waxy locus among the Oryza species

of variable

regions.

Theor. Appl.

Nagano, EL, L. Wu, S. Kawasaki,of the 260-kb

surrounding

Distribution

ofrecombination

sites

Rice Genet. Newslett. 16:59-61
Structuraldifferencesin thewith the AA-genome: identification

Genet. 100:376-383.

Y. Kishima

and Y. Sano.

1999.

Genomic organization

the waxy locus in a Japonica

rice.

Genome 42: 1 121-1

126

Fig. 2. Classificationof the DNA segments using Southern blotting patterns for the 26Gkb contig around the wx locus. (A) The panels exhibit the typical hybridization
patterns
for the 4 classes.
(B) Distribution
pattern of repetitive
sequences in the 260-kb reigon.

17

The Introgression
SugionoMoeljopawiro1,

1 RIFC Biotechnology,
2 Plant Breeding Department,of QTL from Oryza rufipogon to IR64
Endang M. Septiningsih2

and Susan R. McCouch2

Jalan Tentara Pelajar 3A, Bogor 161 1 1, Indonesia
Cornell University,
252 Emerson Hall, Ithaca, New York
14850, USA.

Introduction
Rice is the world's most important food crop and the staple food in Indonesia.
Thecountry is currently
self-sufficientin rice, producing
48.8 million
tons on 10.6 million
hectares of land.
Rapid population
growth is putting
increasingpressure on the foodproducing resources of Indonesia and new crop varieties and cropping systems are needed tomeet consumer demand. Rice production
alone will have to increase to 53 million tons per
year by 2000 in order to maintain self-sufficiency.
New paradigms
of international
cooperation
are needed and new strategies
for plant improvement capable of producing
highlyproductive,
nutritious,
and resource-efficient
crop varieties
must be developed.
Familiarity
with a variety of approaches
to genetic enhancement of crop plants wereincreasingly
essential to those involved in agriculturalresearch.
Genetic diversity
is a prerequisite
for increasing
yields and for stabilizing
productionin the face of disease epidemics and fluctuating
environmental conditions.
Vast reservoirs ofgenetic diversity
have been collected and are maintained in germplasm collections
around theworld. However, there has been relatively
little utilizationof these genetic resources toenhance the performance of elite rice cultivars,
other than as a source of single genes fordisease and insect resistance.
Broadening the genetic base of the rice breeding program inIndonesia would have positive effects on the sustainabilityof the breeding effort as well as on
the stabilityof performance in both the short and the long term. New strategies
that
simultaneously
target variety development and diversificationof the gene pool are urgently
needed.
Two well-saturated
RFLP maps of rice are available(Causse et al, 1994; Kurata etal, 1994),
and a microsatellite
map providing
genome-wide coverage of rice had beendeveloped at Cornell University.
These maps contain closely linked, codominant loci thatcan be monitored for linkage to genes controlling
virtually
any character important to crop
plants (Tanksley,1993).
These maps, if used in conjunction
with traditional
breeding
techniques,
can allow researchers to locate and selectivelytransfer genes that will improveyield (Xiao et aL, 1995), quality (Ahn etal, 1993; Harrington et al, 1996; Yano et al, 1988)
and adaptability
to different
production
conditions(Wang et al, 1994; Champoux et al,
1995; Li et al, 1995; McCouch and Doerge, 1995; Ray et al, 1996; Redono and Mackill,1996).
By targeting
previously
untapped sources of genetic variation,
molecular marker
analysis
offers an effective way of bringing
valuable new genes into the gene pool andsimultaneously
improving crop performance (Xiao et al, 1996).
The objectivesof this research are 1) to facilitate
the utilizationof exotic rice
germplasm, 2) uncover positive alleles which otherwise would be overlooked based on thephenotype of the parent, 3) to identify
molecular markers for the alleles of interest to aid inmarker-assisted
variety, and 4) to initiate
the development of near isogenic
background as the foundation for multi-location
evaluation and variety release.

lines

in IR64

Materials

and methods
A marker-assisted backcross breeding strategy was being used to rapidly discover andtransfer yield-enhancing
QTL alleles from wild or unadapted species of Oryza into locally
adapted indica cultivars widely grown in Indonesia. One elite indica variety, IR64, was usedas female parents in crosses with Oryza rufipogon (Accession #IRGC105491).
BC2F2
families were analyzed for yield and yield components in replicatedfield trials in Bogor andSukamandi, and the same families were analyzed with RFLP and microsatellite
markers as
the basis of QTL identification.
Characterizationof populationsin 2 locations in Indonesia
were used to determine if the effects of the QTL are similar across locations (G X E).
BC2F2 families are being developed according to the scheme outlined in Figure 1.

Figure 1. Flow chart of population

development.

Parental survey filters containing
IR64, O. rufipogon and the corresponding
Fl's wereprepared. RFLP markers from the rice molecular linkage map (Causse et aL, 1994) had been
selected at 20 cM intervals throughout
the genome and were scored for polymorphism
between pairs of parents. Microsatellite
markers had also been selected to provide saturated
genome coverage. The use of microsatellites
was expected to increase the probabilityofdetecting polymorphism
between parental lines and to increase the efficiencyof the mapping
effort.
Populations
consistingof BC2F2 families were evaluated for their agronomic traits
based on field experiments conducted at the Muara Experiment Station in Bogor and at the
Research Institutefor Rice in Sukamandi.
Two replications
were evaluatedin eachenvironment and plots consistedof 3 rows of 25 plants/row.
A standard field evaluation
system was used to permit a comparison of genotype x environment interaction(G X E)
among these populations.

19
The phenotypic and genotypic data were used as the basis for QTL analysis to identify
putativemarker-designatedregions of chromosomes associatedwith yield and yieldcomponents in the Bogor and Sukamandi growing environments. Standard analyticalprocedures for identifying
QTL (interval
analysis;
ANOVA) using a variety of software
packages (Q-Gene; C. Nelson, Plant Breeding
Dept, Cornell Univ., pers. comm.) and
MAPMAKER/QTL (Lincoln et aly 1992) were employed in the analysis.
Results

and Discussion
Asian and American rice breeders have made virtually no use of Oryza rufipogon incommercial inbred or hybrid development.
This can be attributed
both to the manyagronomically
undesirable
traits apparent in progenies of these materials, and to breeders'inability
to identify useful genetic variation based on field evaluation of such progenies.
Preliminary
QTL analysis
revealed potentiallybeneficial
QTL alleles from O.
rufipogon. A region on chromosome 5 provided an increase in yield from the O. rufipogon
introgression,and an O. rufipogon introgression
on chromosome 7 provided an increase in
grains per plant. In addition,
despite the late heading date of O. rufipogon, three putative
heading date QTL introgressedfrom O. rufipogon provide earliness, located on chromosomes
1, 8, and ll. A QTL allele from O. rufipogon on chromosome 1 provides an increase in grain
weight and panicle length.
As expected, some negative traits were also contributed by O.
rufipogon, including a large increase in plant height on chromosome 1, in a similar region as
the sd-1 locus. Careful selection
against these negative regions will be needed to avoid
weedy characteristics
in the improvement process. Several grain quality QTL were detected,
including increased amylose content on chromosome 6 from O. rufipogon, possibly explainedby the waxy locus.
The linkage map for the IR64/0. rufipogon QTL mapping populationof 285 BC2F2
families
genotyped at Cornell contains 126 markers. Most of these (92 markers) aremicrosatellite
markers that had been previously developed at Cornell.
The microsatellite
markers provided a higher rate of polymorphism than RFLPs, and also were able to be runusing a high-throughput
protocol. In addition, 34 RFLPs were added to fill gaps in the map,and to aid in comparisons in previous rice QTL studies, as well as comparative maps in thegrasses.
A QTL introgressedfrom O. rufipogon on chromosome 5 was significant
forincreased yield (LOD = 3.02) from the field data from Sukamandi, Indonesia.
This same
region also had slightly
smaller QTL for increased grains per plant and seed set from O.
rufipogon (LOD = 2.62 and 2.60, respectively).
The percent variance explained (R2) for thisyield QTL is 4.9%.
A QTL introgressedfrom O. rufipogon on chromosome 7 was significant
forincreased yield components: spikelets
per plant (LOD = 5.53; R2 = 8.0%) and grains perplant (LOD = 3.61; R2 = 4.8%) from the field data from Bogor, Indonesia. This same region
also had slightly
smaller QTL for yield from O. rufipogon (LOD = 2.55, R2 = 3.1%). At thesame time, this same region had a QTL for smaller grain weight from O. rufipogon (LOD =4.49;
R2 = 6.1%). Therefore, the O. rufipogon alleles in this region contribute a larger
number of smaller grains. More work would be needed to determine if pleiotropy
or multiple
genes in this region are causing these effects.

11.1%).

20
Multiple QTL were detected near the end of the long arm of chromosome 1 in bothfield environments (Sukamandi and Bogor, Indonesia).
Very large plant height QTL weredetected with increased height from O. rufipogon, with 46.6% variance explainedin theSukamandi field trail, and 31.9% explainedin the Bogor trial (LOD = 45.46 and 29.13,
respectively).
Previous reports have detected a gene for semi-dwarf habit (sd-1) in thisregion. Panicle length QTL from O. rufipogon were also detected in this region (Sukamandi:
LOD = 3.17, R2 = 3.9%; Bogor: LOD = 4.48, R2 = 6.7%). In addition, QTL for increased
grain weight from O. rufipogon were detected in this region (Sukamandi:
LOD = 2.81, R2 =4.8%; Bogor: LOD = 7.86, R2 =At this time, it is not known whether pleiotropyormultiple genes are causing these QTL effects.
Three QTL were detected where the O. rufipogon allele provides
earlinesswhen
compared to the IR64 background,
all of which were detected in the Sukamandi fieldenvironment. These were located on chromosomes 1, 8, and ll (LOD = 4.35, R2 = 7.3%;
LOD = 3.93, R2 = 6.0%; and LOD = 7.49, R2 = 9.8%, respectively).
A QTL for increased amylose content from O. rufipogon was detected at the top ofchromosome 6 (LOD = 14.7, R2 = 18.2%). Previous reports have located the waxy gene inthis region. In a different region of chromosome 6, a QTL was detected for gel consistency,with IR64 providing
an increase in gel-mm (LOD 4.23, R2 = 5.5%).
A QTL of large effect was detected on chromosome 7, with O. rufipogon providing
red grains (LOD = 27.0; R2 = 36.8%). As this is a negative trait from O. rufipogon, thisinformation can be used to select against O: rufipogon introgressions
at this location in future
breeding efforts.
A QTL for milled grain quality was detected on chromosome 2, where IR64 increases
the percent of whole, unbroken grains (LOD 3.64, R2 = 5.5%).
Conclusion•E Wild species, such as O. rufipogon, do harbor genes capable of enhancing yield in
cultivatedrice.
•E This molecular breeding strategy can simultaneously
identify and transfer these desirable"wild QTL" into elite breeding lines.
•E With the use of saturated linkage maps, it is possible
not only to dissect quantitative
traits, but also to effectively
reconstruct a genotype of interest via marker-assistedtransferof target QTL.
References
Ahn, S., J.A. Anderson, M.E. Sorrells, and S.D. Tanksley (1993)
Homoeologus relationships
ofrice, wheat and maize chromosomes. Molecular and General Genetics 24 1 :83-490.
Causse M, TM Fulton, YG Cho, SN Ahn, K Wu, J Xiao, J Chunwongse, Z Yu, PC Ronald, S B
Harrington, GA Second, S R McCouch, and SD Tanksley(1994)
Saturated molecular mapof the rice genome based on an interspecificbackcross population.
Genetics 138: 1251-1274.
Champoux, M.C., S. Sarkarung, Dj. MacKill, J.C. O'Toole, N. Huang, and S.R. McCouch(1995).
Locating genes associated with root morphology and drought avoidance in rice via
linkage to molecular markers. Theor.Appl.Genet.
90:969-981.
Harrington S., H.F.J. Bligh, W. Park, Clones and S.R. McCouch (1996)
Linkage mapping ofstarch brancing enzyme HI in rice. Theor Appl Genet (submitted).

21
Kurata N, Nagamura Y, Yamamoto K, Harashima Y, Sue N, Wu J, Antonio BA, Shomura A,
Shimizu T, Lin SY, Inoue T, Fukuda A, Shimano T, Kuboki Y, Toyama T, Miyamoto Y,
Kirihara T, Hayasaka K, Miyao A, Monna L, Zhong HS, Tamura Y, Wang ZX, MommaY,
Umehara Y, Yano M, Sasaki T, Minobe Y (1994)
A 300 kilobase interval genetic map ofrice including
883 expressed sequences. Nature Genetics 8:365-372.
Li, Z., S.R.M. Pinson, J.W. Stansel, and W.D. Park (1995).
Identificationof quantitative
trait loci
(QTL) for heading date and plant height in cultivatedrice (Oryza saliva. L.). Theor. Appl.
Genet. 91:374-381.
McCouch SR and RW Doerge (1995) QTL mapping in rice. Trends in Genet 1 1:482-487.
Ray J.D., L. Yu, S.R. McCouch, M.C. Champoux, G. Wang, and H.T. Nguyen (1996)
Mapping
quantitative
trait loci associatedwith root penetration
ability in rice {Oryza sativa L.) Theor
Appl. Genet 92:627-636.
Redoiio, E.D. and DJ. Mackill(1996)
Molecular
mapping of quantitative
trait loci in japonica
rice. Genome 39:395-403.
Tanksley, S.D. (1993)
Mapping Polygenes.
Annu. Rev. Genet. 27:205-233.
Wang, G., DJ. MacKill,
J.M. Bonman, S.R. McCouch, M.C. Champoux, and RJ. Nelson(1994).
RFLP mapping of genes conferring
complete and partial resistanceto blast in adurably resistant rice cultivar. Genetics 136: 1421-1434.
Yano M., K. Okuno, H. Satoh and T. Omura (1988)
Chromosomal location of genes conditioning
low amylose content of endosperm starches in rice, Oryza sativa L. Theor. Appl. Genet
76:183-189.
Xiao J, J Li, S Grandillo,
SN Ahn, SR McCouch, SD Tanksley, L Yuan (1996)
A wild species
contains genes that may significantly
increase the yield of rice. Nature.

22

Evolutionary
relationships
among Oryza species inferredelements associated with the catalase gene CatA

from transposable

Masao Iwamoto
Department of Genetic Resources II, National Institute ofAgrobiological
Resources, Tsukuba,
Ibaraki 305-8602,
Japan
Key words
Catalase •EEvolution •EOryza •ERice •ETransposable element

Abstract
Intron-2 of the rice (Oryza sativd)
CatA catalase gene is similar in nucleotide
sequence to p-SINEl, a retroposon.
To examine when the p-SINEl-like
intron was inserted
into CatA during the evolutionary
divergence of Oryza species, and to elucidate
theevolutionary
relationships
among Oryza species using the intron as a marker, polymerase
chain reaction (PCR) analyses were performed.
The results indicatedthat all the Oryza
species with the AA genome have the CatA ortholog with the intron, whereas other Oryza
species have the CatA ortholog without the intron.
These findings indicate that intron-2 of
CatA is a good marker for distinguishingrice with the AA genome among Oryza species.
Tourist-OsaCatA, a miniature inverted-repeat
transposable
element (MITE), was found in the
5 -flanking
region of the rice CatA. PCR and sequencing analyses revealed that the AA
genome Oryza species, except O. longistaminata,
contain the full-length
Tourist element andthat O. longistaminata
and other Oryza species contain the partial element.
This Tourist
element is distributedin all four Oryza species complexes in the Oryzeae tribe.
Aphylogenetic
tree of the CatA orthologs in Oryza species based on the nucleotide sequences of
the Tourist elements was constructed.
The CatA orthologs formed four clusters, each ofwhich corresponded to one of the Oryza species complexes.
Although the CatA orthologs in
O. longistaminata
accessions were in a cluster with those in the other AA genome species,
they were placed near those of the species in the O. officinalis complex. The Tourist
elements associatedwith CatA and its orthologs
are thus useful for examining theevolutionary relationships
among Oryza species.
Intr oduction
Oryza sativa, rice, is one of the species classified
into the genus Oryza. Oryza
species can be taxonomically divided into four species complexes: the O. sativa, O. officinalis,O. meyeriana and O. ridleyi complexes (Vaughan 1989).
The O. sativa complex consists of
the Oryza species with the AA genome, and the O. officinalis complex with the BB, CC, EE,
FF, BBCC and CCDD genomes.
Aggarwal et al. (1997)
proposed
new genomic
designations
based on the results of total genomic DNA hybridization:
GG for the O.
meyeriana complex and HHJJ for the O. ridleyi complex.
Catalase (H2O2:H2O2 oxidoreductase,
EC 1. 1 1. 1.6; CAT) catalyzes the dismutation ofhydrogen peroxide, one of the activated oxygen species, into oxygen and water, and is foundin a wide range of organisms from aerobic bacteria to higher plants and higher animals.
O.
sativa has three catalase genes, as is the case for other higher plants examined to date. Oneof the rice catalase genes, CatA (Higo and Higo 1996), has a unique intron (intron-2 as shownin Fig. 1), which appears to have been derived from the insertion of a retroposon,
p-SINEl(Iwamoto et al. 1998).
In addition, a MITE named Tourist-OsaCatA was also found in the
5 -flanking region of rice CatA (Fig. 1).
In this study, to elucidate
the evolutionary
relationships
among Oryza species,
phylogenetic
analyses were performed using nucleotide sequences of the /?-iS77VE7 -like introns

23
and the Tourist elements associatedwith CatA and its orthologs(Tourist-CatA),
evolutionary pathway of Oryza species was inferred from these results.

and theFig. 1
Exon-intron structures of O. sativa (Os) CatA. Exons and introns areindicatedby boxes and thick lines, respectively.
Tourist-OsaCatA and p-SlNEl-hke intron
are shown by the shaded boxes. The designationof the location of Tourist-OsaCatA refers
to its distance in base pairs from the CDNAstart site of CatA.

p-SINEl-tike intron
To examine whether CatA orthologs in Oryza species have the p-SINEl-like intron,
PCR analyses of 39 accessions from 17 Oryza species were performed (Iwamoto et al. 1998;
Iwamoto et al. 1999a).
Agarose gel electrophoresisof the PCR products revealed that all AA
genome Oryza species had a major band of about 400 bp, indicating the presence of the intron.
In O. rufipogon W0120, there are two major bands of about 400 and 450 bp. When the PCR
products of six accessions of O. longistaminata collected from various regions of Africa were
electrophoresed,
there were several major bands in two accessions (C101229
and C104977).
In the Oryza species with genomes other than the AA genome, one major band of about 300
bp, the size of which was equivalent to that of the PCR product of the CatA ortholog without
the intron, was detected irrespectiveof genome type.
A phylogenetic
tree of the CatA orthologs in the AA genome Oryza species wasconstructed based on the nucleotide sequences of the PCR products of the CatA orthologs.
In the tree, the CatA orthologs were roughly divided into two groups: those in the
Asian/Oceanian
Oryza species, and those in the African species.
Furthermore, theAsian/Oceanian
Oryza group could be divided into two subgroups: the CatA orthologs ofthree varieties of O. sativa formed one subgroup, and those of O. nivara and O. rufipogon, allof which are wild Oryza species distributedin Asia and Australia, formed the other.
Tourist- CatAPCR analyses of the 37 accessions of 17 species were performed (Iwamoto et al.
1999b).
Agarose gel electrophoresisof the PCR products revealed that the AA genome
Oryza species, except for the O. rufipogon W0157 and O. longistaminata
accessions, had amajor band about 400 bp in length, suggesting the presence of the Tourist element. On theother hand, the accessions of the O. officinalis, O. ridleyi and O. meyeriana complexes and O.
longistaminata
had a major band of about 250 bp. Sequencing revealed that the Oryza
accessions showing the 400-bp PCR products had Tourist element sequences almost identicalto Tourist-OsaCatA.
On the other hand, the accessions showing the 250-bp PCR products
had partial sequences of the element.
A phylogenetic
tree of the CatA orthologs in Oryza species was constructed based on the
nucleotide sequences of the conserved region of Tourist-CatA (Fig. 2). The CatA orthologs
formed four clusters, each of which corresponded to one of the Oryza species complexes.
The agreement between complexes and clusters
confirms
that the structuringofFig. 2 Phylogenetic
tree, constructedby the neighbor-joining
method, based on the
nucleotide
sequences of the conserved region of the Tourist-CatA elements.
Horizontal
distances are proportional
to evolutionary
divergence among the nucleotide sequences.
The
numbers at the nodes are the bootstrap values from 1000 replicate samples.
Genome types
are indicated in capital letters.
Oryza species into clade complexes is biologicallyjustified.
Although the CatA orthologs in
O. longistaminata
accessions were in a cluster with those in the other AA genome species,
they were relatively
distant from the orthologs of the other species in the O. sativa complex
and were located closer to those of the species in the O. officinalis
complex.
The25
phylogenetic
tree showed that the CatA orthologs in the species of the O. sativa complex are
evolutionarilyclose to those in the species of the O. officinalis complex, and are most distantfrom those of the two accessions of the O. meyeriana complex.
It seems likely that Tourist-CatA was already present in the 5 -flanking region of the
CatA ortholog in the common ancestor of Oryza species, and that partial deletionof theTourist-CatA occurred in the ancestor(s)of 0. longistaminata
and the species in the O.
officinalis,O. ridleyi and O. meyeriana complexes during the course of evolution of Oryza
species.

Acknowledgments
I would like to thank my collaborators.
Ministry of Agriculture,
Forestry and Fisheries,

This work was supported
Japan.

by grants from theReferences
Aggarwal, R. K., D. S. Brar and G. S. Khush. 1997. Two new genomes in the Oryza complex
identified
on the basis of molecular divergence
analysisusing total genomic DNA
hybridization.
Mol. Gen. Genet. 254:1-12
Higo, K. and H. Higo. 1996. Cloning and characterizationof the rice CatA catalase gene, ahomologue of the maize Cat3 gene. Plant Mol. Biol. 30:505-521
Iwamoto, M, M.Maekawa, A. Saito, H. Higo and K. Higo. 1998. Evolutionary
relationshipof plant catalase genes inferred from exon-intron structures:
isozyme divergence after
the separation of monocots and dicots.
Theor. Appl. Genet. 97:9-19
Iwamoto, M., H. Nagashima, T. Nagamine, H. Higo and K. Higo. 1999a. p-SINEl-like
intronof the CatA catalase
homologs and phylogenetic
relationships
among AA-genome
Oryza and related species.
Theor. Appl. Genet. 98:853-861
Iwamoto, M., H. Nagashima, T. Nagamine, H. Higo and K. Higo. 1999b. A Tourist element in
the 5 -flanking
region of catalase gene CatA reveals evolutionary
relationships
among
Oryza species with various genome types.
Mol. Gen. Genet. 262:493-500
Vaughan, D. A. 1989. The genus Oryza L.: Current status of taxonomy. IRRI Res. Paper Ser.
138

26

The construction(Instituteof Genetics,

and identification

ChineseBin Wang
Academy of Sciences,of plant

BAC library

Beijing

100101,P. R. China)

Abstract:
The constructionof artificial
chromosomal library is a key step for plantgene isolation.
Bacterial
Artificial
Chromosome (BAC) library is the most widelyused library in plant molecular biology
research. In this presentation,
rice BAC
library was taken as an example to illustrate
the procedure and the methodology for
plant
BAC library
construction,
identification,screening
and BAC contig
development. A new method for the preparationofHMW (Mb size) DNA and large
inert DNA fragments for BAC library construction
was introduced.
The HMWDNA
prepared with this method is pure enough, without LMWnuclear DNA and organelle
DNA contamination.
The constructed rice BAC library consistedof 18,432 BAC
clones that were stored in 192 pieces of 96-well plate and 48 pieces of384-wellplaterespectively.
The whole BAC library was transferred onto 24 pieces of 12x8 cm highdensity membrane filters, in which each BAC clone was duplicated.
The averageinsert size of the BAC clones is about 120 kb. The total capacity of the BAC libraryis 5.14 times equivalentof rice haploid
genome. The possibility
in success of
screening with single-copy
probe is about 99%. In stability
test, five BAC clones
were transferred
generation after generation till to 100 generations.
The digestion
patterns at generation
time 0 and 100 of the BAC clones were compared, no
significant
difference was found. This result indicatesthat the host cells ofE. coli canstable maintain the BAC clones till 100 generations.
382 BAC clones were screened
with ctDNA and mtDNA probes, no positive clone was found, this result indicatedthat the BAC library was without significant
contaminationof organelle DNAs. The
BAC library was screened with three molecular markers linked to rice Ef(t) gene, 7
different
positive BAC clones were identified,
and two primary BAC contigs weredeveloped with these 7 BAC clones. Then, the two primary contigs were jointed
together through chromosomal walking. A BAC contig encompassing the Ef(t) gene
locus was developed which consisted of 3 BAC clones covering about 250 Kb. The
Ef(t) gene was harbored in the BAC clone 2706. Later, the BAC library was screened
with molecular markers linked to rice tmsl gene, a BAC contig encompassing thetmsl gene locus was developed which consisted of 5 BAC clones covering about 400
Kb. The tmsl gene was harbored in the BAC clone 14K4.
The problems and possible
resolutions
met in the process of BAC library
construction were discussed.
Key words: Rice;gene

Artificial

chromosomal library;

BAC library;This work was supported by China State High Technology
Foundation International
Rice Biotechnology
ProgramEf(t) gene; tmsl

Program and Rockefeller

27

1. Introduction
BAC is the abbreviationof Bacterial
Artificial(Chromosome, it is a kind ofcloning vector for library construction.
Because this vector originatedfrom F' factor
of bacteria (E.coli F' strain), so it got the name of BAC. BAC library is a kind of
Artificial
Chromosomal Library. Libraries
constructedusing BAC as vector are
called BAC libraries.
There are several kinds of artificial
chromosomal libraries
now,such as YAC library, BAC library,
PAC library, TAC library, MAC library. The
historicaldevelopment of artificial chromosomal libraries
is shown as follows:
Librari es :Developed in:

YAC->B AC->PAC->BmAC-»TAC
1988
1992
1994
1996
1996

Among them, BAC library is the most widely used one, because it with someadvantages over other libraries,
such as: easy to manipulate,
low chemerism, clear
genetic background, stable (over 100 generation),high transformation
efficiency andwith reasonable insert size. BAC library is becoming a useful and necessary tool forplant molecular genetics research, such as: genome research, gene map based cloning(MBC), gene fine mapping, BAC contig construction,
BAC transformation
and BAC
FISH. Two new vectors, BIBAC and TAC, are derivativesof BAC and speciallydesigned for plant gene transformation.
2. Basic procedure
The basic

for BAC libraryprocedure

construction

for BAC library

construction

diagram.

Fig. 1. Procedure

diagramfor BAC library

construction

is illustrated

in the follow

28
From this diagram, it is known that the preparationofBAC vector DNA andinert DNA are the key steps for constructing
a good BAC library. In our laboratory,
the BAC vector DNA is usually prepared by ultra-highspeed centrifiigationin CsCl2
gradient,
and digested
with restriction
enzyme Hind III, then dephosphorylatedtoprevent from self-recycling
and stored as small aliquot at -70 °C. The insert DNA(100-200Kb) was prepared from HMW(Mb size) DNA isolated from contact nuclei
after in situ digestion
by Hind III followed by two-size fractionation
via PFGE. The
size-fractionatedDNA fragments in gel slices were recovered by electroelution.
Finally,
the recovered insert DNAs were ligated to the dephosphorylated
BAC vector
DNA with T4 DNA ligase. The ligation mixture was used to transform the competentcells of E. coli strain DH10B by electroporation.
The recombinants
were selectedbased on CmRand white colony. After enough qualified
BAC clones were obtained,
they were distributed
into both 384-well and 96-well plates respectively,
and stored at-70°C.
3. Identificationof the constructed

BAC library

A qualified BAC library must be good in representativeof the plant genome
regarding to its capacity and reality. Based on this principle,
in practice, BAC library
was usually
identified
base on the following
parameters:(1). The insert size
distribution;(2). Percentage of empty clones; (3). Total capacity;(4). Reality;(5).
Clone stability;(6). Level of ctDNA and mtDNA content. The identification
resultsof the constructed rice BAC library are as follows:(1). The insert size distribution:
DNAs were extracted
from 110 randomly
selected
BAC clones (fromdifferent
ligations)of the library. The isolated DNAs were completely
digested
with
Not I and analyzed by PFGE. The insert size of BAC clones ranged in 50-250 Kb,
and over 70% of the BAC clones have inserts larger than 100 Kb. The average insert
size of the BAC library is about 120 Kb.
(2). Percentage of empty clones:
In the 206 BAC clones tested, only one was found without
Therefore, the percentage of empty clones is about 0.5%.

sufficient

insert.

(3). Total capacity:
The total clone number of the BAC library is 18,432, the average insert size of
the BAC clones is about 120 Kb. So that: 120 (Kb) X 18432/430000(Kb)=5.14
Therefore, the total capacity of the BAC library is 5.14 times equivalentofrice haploid genome. The possibility
in success of screening with single-copy
probe
is about 99%.
(4).

Reality:
In order to confirm the reality of the insert DNA ofBAC clones, 36 randomly
selected BAC clones were digested with Not I and subjected
to PFGE analysis. The
digested
BAC DNAs were Southern blotted and hybridized
with total rice genomic

29
DNA. All of the inserts from the 36 BAC clones were hybridized.
indicate that the BAC clones contain really rice genomic DNA

These results(5). Clone stability:
In stability
test, five BAC clones were transferred generation after generation
til to 100 generations.
The digestion
patterns at generation time 0 and 100 of each
BAC clone were compared, no significant difference was found. This result indicatesthat the host cells ofE. coli can stable maintain the BAC clones till 100 generations.
(6). Level ofctDNA and mtDNAcontent:
382 BAC clones were screened with mixed probes from sorghum chloroplast
psbA gene and maize mitochondrial
atp6 and 18S+5S genes. No positive clone wasfound, this result
indicatedthat the BAC library
was without
significant
contamination of organelle DNAs.
4. Make high-density

filters

The constructed rice BAC library consisted of 18,432 clones that were stored
in 192 pieces of 96-well plate and 48 pieces of 384-well plate respectively.
The
whole BAC library was transferred onto 24 pieces of 12x8 cm high-density
HybondN^ membrane filter, in which each BAC was duplicated.
Eachpiece offilter contains
1536 BAC clones.
5. BAC library

screening

The BAC library was screened with three molecular markers linked to rice
Ef(t) gene, 7 different positive BAC clones were identified,
and two primary BAC
contigs were developed with these 7 BAC clones. Then, the two primary contigs were
jointedtogether through chromosomal walking. A BAC contig encompassing the Ef(t)gene locus was developed which consisted of 3 BAC clones covering about 250 Kb.
The Ef(t) gene was harbored in the BAC clone 2706. Later, the BAC library was
screened with molecularmarkers linked
to rice tmsl gene, a BAC contig
encompassing the tmsl gene locus was developed which consisted of 5 BAC clones
covering about 400 Kb. The tmsl gene was harbored in the BAC clone 14K4.
6. Storage
48 piecesof the BAC clones

All of the 18,432 BAC clones were stored in 192 pieces of 96-wellof384-wellplate respectively.
They were all stored at -70 °C.

plate andThe constructionof BAC library from rice line 5460F will facilitatethephysical
mapping and the isolationof Efft) gene as well as the tmsl gene by using
map based cloning approach.

30

Rice centromere structure with repetitive
artificial
chromosomes (RACs)
Nori Kurata, Tadzunu Suzuki, Ken-ichi Nonomura
National Instituteof Genetics, Yata 1, 1.1.1, Mishima,e-mail: nkurata @lab.nig.ac.jp
Key words : rice, centromere,

repetitive

sequences

Shizuokasequence, artificial

and construction

41 1-8540,

of rice

Japan.

chromosome

Centromere is characterized
by several structural and functional features which shouldbe necessary for its own roles, such as kinetochore
formation,
chromatid cohesion andchromosome separation.
Centromeres consistingof multiple repetitive
sequences form quite
specializedorganization
to express their function.
Centromeres of most organisms analysedso far span from hundreds kilobases
to more than several megabases as the regions being
highly heterochromatic,late DNA replicating
and suppressed in genetic recombination.
Theregion showing centromere characteristics
is composed of many kinds of repetitive sequences,a part of them is specific to the organisms and others are commonto many organisms.
In this paper, we analyse multiple repetitive sequences composing rice centromeres andtheir organizationin the centromere structure.
Several commonrepetitive sequences to othercereal centromeres may indicate structural
origin of cereal centromeres.
This may alsosuggest a probabilityof functional
replacement of a cereal centromere sequence with the ricecentromere composed of multiple commonrepetitive sequences.
In this context, constructionof a rice artificial
chromosome is one of challenging
trials for introducing
huge size genetic
material as a mini-chromosome to different
species across genus. The events which would
occurr both in the nucleus and in the transgenic plants possessing
artificial
chromosomes ofdifferent species or genus should also present useful information about interactions
betweenchromosomes of different species and so indicate something about differentiationof species.
Meanwhile, rice or cereal artificial
chromosomes might become a well organized tool for
introducing
hopeful chromosome segments to other cereal species to generate novel type
Gramineae species.
Identificationof Centromere Repetitive Sequences
Wefirst searched for centromere specificrepetitive
sequence in rice by screening
CENP-B (centromere protein B) binding consensus sequence with PCR amplificationof thegenomic sequences.
This consensus sequence of CENP-B box had already identified
to beincluded in the CCS 1 (cereal centromere specific)
family sequences isolated from several cereal
plants (Aragon-alcaide
et cd. 1996).
We isolated another new short sequence and revealed thatthe short sequence was a part of the longer 1.9kb repeat unit of centromere specific location inrice (Nonomura and Kurata, 1999)
The 1.9kb repeat unit, RCE1, was estimated to be present
in several hundreds to one thousand copies in the rice genome. The 14kb centromere
genomic clone having a couple of copies of RCE1 was isolated and sequenced.
Structure ofthis clone is summarized in Fig. 1 and showed several characteristicfeatures, such as high AT
content sequences, some of them were indicativefor MAR/ARS-Or functions, and othercentromere repetitive
sequences. Three RCE1 copies located on the 14kb fragment were
present several kbs apart from each others.
At the same time, Dong et aL (1998) reported other several centromere specific repetitive
short sequences in rice.
Most of them were sequences commonly located on the multiple
cereal centromeres and one 170bp short tandem repeat was specific to the rice centromere but
not detected in other cereals.
In addition,
we have recently identified
other repetitive
unit ofTy3-gypsy
type RT (riverse
transcriptase)of RIRE3 which was also clusteredin thecentromeric region in rice. We further planned to investigate
wide-range structure of ricecentromeres and screened genomic YAC clones using RCE1 and RCS2 repeat units.
Severalrice YACs which carry both RCE1 and RCS2 repeat sequences were isolated.
One of them
revealed to have been already landed on a candidate centromere region of the chromosome 5.
So we targeted on the centromere of chromosome 5 to analyse its structure in detailFig. 1. The 14kb centromere sequence with multiple repetitive
units specific for centromeres.
The upper row shows a restriction
map showing the positionsof several characteristic
motifs.
The large diamonds indicate
Topo-II
boxes (GINa/tAt/cATTNATNNg/a);
the smalldiamonds
indicate core sequences of the Topo-II box consensus (AtA^/CATT);
the squres
mark A boxes (AATAAAT/CAAA)5 circles T boxes (TTa/tTT/aTTt/aTT)?
and stars T boxes(TTA/ITT/ATTT/ATT),and starsBUR sequences (AATATATT or AATATT). The AT content
was calculated as the average for each 20-bp window.
The arrows in the plot indicate regionswith more than 90% A/T content.
Centromere Structure of the Rice Chromosome 5
On the centoromere region of chromosome 55 we reconstructed
YAC and BAC contigs andanalysed in detail for the distributionof repetitive
sequences and in the centromere characters
geneticallyand physically.
Fig.2 shows a summary of the centromere structureandcharacterizationof the rice chromosome 5. The single locus at 53.7 cM possessed a large

Fig.2.

Genetic

and physicalstructureof the centromere region of chromosome 5.

32
number of DNA markers and occupied more than 2.3 Mb. This locus is includedin thecandidate CEN region restricted
cytologicallyand the locus scattered with over twelve DNA
markers could not divided into smaller genetic segments by recombinationeven using near 500
F2 segregants.
This means high suppressionof genetic recombination
along the region of
more than 2.3 Mb. Three YAC contigs (BAG clones were also arranged but are not shownhere) spanning this region revealed to carry multiple
repetitive
sequences characteristic
tocentromeres.
Physicalmaps using YAC and BAG contigs were searched for the distributionof repetitive
sequences of RCE13 RCS2 and other repetitive
unit ofRIRE 3. Fig. 3 shows apart of distributionof the repetitive
sequences in this region.
RCS2 tandem repeats located as
two or three blocks of several to scores Kb length and RCE1 repeats of more than ten copies
were distributednear RCS2 with decreasing
number depending on the distance from RCS2
blocks. Many copies of RIRE3 repeats were also detected in this region.

Fig. 3. Physicalmap of a part of CEN5 distributedwith centromere specificrepetitive
sequences.
All results
obtained here suggested
the region around RCS2 repeat blocks was mostprobable candidate of structural and functional core of the rice centromere 53 CEN5. Then we
chose the YAC or BAG clone located on this region and possessed both RCE1 and RCS2
sequences to construct rice arfificial chromosome (RAC).
Construction of RACs: Rice Artificial Chromosomes
Artificial
chromosomes need to be composed of several elements; centromere, telomere,
replication
origins and other unknown factors for stable maintenance In the cell nucleus.
We
have already cloned centromere and telomere sequences but not replication
origin (On).
However a large size genomic DNA fragment of centromere region should contain Its own Ori,so no additional
Ori would be needed.
As shown in Fig. 4, a left arm and a right arm
carrying selection markers necessary for the selection both In yeast and rice cells and telomeresare now trying to replace arms of the centromere YAC clone (360 Kb) by homologous
recombinationin yeast cells (retrofitting
method).
Another trial to use a shorter centromere
sequence of BAC clones also being carried out. Once the constructionof RACs would bedone, introductionof huge size Intact RACs Into rice cells and regenerationof those cells will
be challenged by employing as many as possible methods.
What Is a minimum functional
unit/factor
for artificial
chromosome?
Which region isessential
for mitotic or meiotlc functionof the centromere ? How does the chromosome
organize Its own behaviour in the nucleus especiallyIn the different
species,genomes orgenus?
We expect to have a fundamental knowledge about the mechanisms of chromosome
behaviour
togetherwith to generate
novel methodology
using artificial
chromosome
intruduction
into far-related
species.

Fig. 4. Constructionof a rice artificial
chromosome by retrofitting
the centromere
with RAC left and right arms. Armvectors were orglnated from Adam et al. 1997.

YAC clone

Acknowledgements:
This work was supported by the Grant 0946006 for Scientific
Research,from the Ministryof Education,
Science and Culture of Japan, and by the Program for
Promotion of Basic Research Activities for Innovative Biosciences.
We thank Drs. Gehard
Adam and Hisako Ohtsubo for providing
us retrofitting
vectors and RIRE DNAs and also
thank Rice Genome Research Program, NIAR and Clemson University
Genome Projects forproviding us YAC and BAC clones and informations,
respectively.

References :Adam G? Mullen J A, Kindle K L. 1997.
Retrofitting
YACs for direct DNA transfer intoplant cells. Plant Journal ll:1349-1358.
Aragon-Alcaide
L. , Miller T, Schwarzacher T, Reader S, Moore G. 1996. Acereal centromericsequence.
Chromosoma 105:261-268.
Dong F5 Miller J. T, Jackson S. A, Wang G. L, Ronald P. C, Jiang J. 1998.
Rice (Oryza
sativa) centromeric regions consist of complex DNA Proc. Natl. Acad. Sci. USA 95:81358140.
Nonomura K and Kurata N. 1999. Organizationof the 1.9-kb repeat unit RCE1 In thecentromeric regin of rice chromosomes.
Mol Gen Genet. 261:1-10.

34

DNA MARKER-BASED ESTIMATES OF COANCESTRY
PARENTAL CONTRIBUTIONSIN RICE
Dindo

A. Tabanao1,

1 ,4Philippine
2 University
3 University

Azucena L. Carpena2,

Rex N. Bernardo3,

LeocadioAND
S. Sebastian4

Rice Research Institute,
Munoz, Nueva Ecija, Philippinesof the Philippines
Los Banos, College, Laguna, Philippinesof Minnesota, St. Paul, MN

Keywords: coancestry,

microsatellites,

genetic

relationships,

molecular

markers

The coefficientof coancestry was calculatedusing DNA marker data in order to circumventthe limitations
associatedwith the pedigree-based(fy) approach.
Microsatellite
markeranalysis of eight modern rice varieties and their 25 progenitors
generated a total of 559 alleles
from 119 loci. Marker estimates(jfy) were computed for all 528 pairwise
genotype
combinations
using a tabular method. The mean values offy and jfy were 0.0649 and 0.0452,
respectively,
jfy correlated highly significantly(r = 0.74) with/y, and it deviated > 0.10 fromfy in only 44 (8.3%)
genotype pairs. Also, the jfy dendrogram agreed closely to the fy
dendrogram (r* = 0.65). jfy was, in fact, more suitable (r* = 0.89) than fy (r* = 0.78) tocluster analysis. Comparison of the estimates of parental contribution(k) revealed slight todrastic differences
between Mendelian expectations
and marker-based values. The differences
between pedigree and marker estimates of coancestry and parental contributions
are due torandom drift and human selection during inbred line development.
The close agreement andhighlysignificant
correlationof jfy to fy indicate
its validity
as a measure of genetic
relatedness,while its immunity to assumptions of the pedigree method and appropriatenesstomathematicalclassification
imply its superiority.

INTRODUCTION
Measures of genetic relatedness
are very important in plant breeding-related
activities
and are necessary in applications
involving
varietal
identification,
intellectual
property
protection,and utility
patenting.
The development of molecular markers has enabled plant
breeders to determine the genetic relationships
among plant germplasm at the level of the
DNA itself. DNA-based markers have the advantage of being virtuallyunlimitedin numberand independent of environmental
effects. The question then would be which similarity
index
is more accurate and meaningful to use.
The coefficientof coancestry (/) is the most widely-used
and accepted measure ofgenetic
reiatedness
in plant breeding.
However, its total dependence
on the parentage
informationof the germplasms under comparison limits its use and accuracy in terms of nonsatisfactionof assumptions,such as equal parental genomic contributions
and homozygosityof parental lines.
1 Plant Breeding and Biotechnology
Division,
Philippine
Rice Research Institute,
Maligaya,Munoz 3 1 19, Nueva
Ecija, Philippines
2 Department of Agronomy, Universityof the Philippines
Los Bafios, College 403 1, Laguna, Philippines
3 Department of Agronomy and Plant Genetics, Universityof Minnesota, 41 1 Borlaug Hall, 1991 Buford Circle,
St. Paul, MN 55108-6026
4 Executive Director, Philippine
Rice Research Institute,
Maligaya,Munoz 3 1 19, Nueva Ecija, Philippines

35
The use of simple sequence repeats (SSRs), also known as microsatellites,
are gaining
wide acceptance in the analysis of genetic relationships
because of their ability to detect large
numbers of discrete
alleles
repeatedly,
accurately,and efficiently.
The genetic profiles
produced by SSRs can be used in conjunction
with pedigree and performance data to
document ownership and protect intellectual
property rights.
In DNA profiles, the proportion of alleles that are common between two genotypes isknown as marker similarity(S). Lynch (1988)
elucidated
the expected value of S as a functionof/and the conditional
probability
that marker alleles are alike in state (ais) given that they
are are not ibd (9). Given this theory, Bernardo (1993) proposed estimating
the coefficientofcoancestry by using DNA marker data based on the fact that DNA marker data are a direct
sampling of the genome itself, and that/is
the probability
that two alleles at the same locus
are identicalby descent (ibd). This marker-based approach would circumvent the limitations
associated with the classic pedigree-based
approach. This study was conducted (1) to computethe coefficientof coancestry of selected rice germplasm using microsatellite
DNA marker
data and (2) to assess the validityof the marker-based estimate relative to the pedigree-based
method.

MATERIALS AND METHODS
The study made use of 8 Philippine-released
rice varieties
and their 25 initial,
intermediate
and immediate parents. These were grown for DNA sampling using a CTAB
procedure. A total of 118 SSR primer pairs were used to survey the microsatellite
polymorphismof the sample materials, following
the concentrationsof PCR components andtemperature profile recommended by Research Genetics (Huntsville,
AL). PCR products werepartitioned
in 4% polyacrylamidegels. Silver staining followed the protocol provided by the
manufacturer (Promega Corp., Madison, WI). Allelicbands were denoted, from the fastest to
the slowest migrating fragments, by letters a, b, c, and so on.
The coefficientsof coancestry
was computed for all 528 pairwise
genotype
combinations,according to a procedure based on a tabular analysis developed by Emik andTerrill (1949).
The inbreds were first sorted from oldest to newest, so that an inbred was listed
before any of its progeny. Startingfrom the oldest inbred, the coefficientof coancestry
between / andy was calculated as
fij = hfaj+

Xffbp 0 <fij,Xa, Xb < 1

where faj was the coefficientof coancestry between a and y; fy was the coefficientofcoancestry between b andy; and, Xa and Xb are the marker-based parental contributionsof aand b, respectively,
to /. A Fortran-based computer algorithm (Bernardo
et al.9 2000) was
employed for this procedure.
For the pedigree-based
estimates, the parental contributions(Xa and Xb) were taken fromexpected Mendelian values (e.g., 0.50 for an F2-derived inbred and either parent). For themarker-based estimates, the parental contributions
were calculated as Xa = (Sat - &&&)/[(1Sab)2] and Xt = (Sbt - SaiSab)/[(l- Sab)2], where S is the proportionof marker variants shared
between the genotypes in subscripts
to the total number of marker variants detected.

36
A dendrogram was constructed from/y and x/*y using average linkage method. Then, thetwo types of estimates were compared at the individual
values level, at the matrix level, and at
the dendrogramlevel. To compare two matrices, the matrix correlation
coefficientwascomputed using SPSS 10.0 for Windows. To compare two classifications,cophenetic
valueswas generated from each of the two dendrograms and were then analyzed for correlation.
The goodness of fit of a tree matrix to the original
similarity
matrix was tested bygeneratinga cophenetic matrix from the dendrogram and taking the cophenetic
correlation
coefficient(/**) by comparing the similarity
and the cophenetic
matrices. Since the two
matrices are not independent,
the critical values ofr* given by Lapointe and Legendre (1992)
were used for tests of significance.

RESULTS AND DISCUSSION
One of the markers generated two amplifications,
resultingto 1 19 SSR loci in all. The
number ofalleles
detected per locus ranged from 2-1 1, with an average of4.7 and a total of
559 alleles.
The number of SSR loci per chromosome ranged from 5-21, the average being 10loci per chromosome. The average polymorphism
information content of the SSRs was 0.59.
The mean values offy and x/*y, computed in all 528 pairwise
genotype combinations,were 0.0649 and 0.0452, respectively,
xftj deviated by > 0.10 from/y in only 44 pairs (8.3%).
Taking agreements at the matrix level, the correlation
ofjfy with/y was highly significant(r =0.74,/?

< 0.01).

The deviationsof marker estimates from pedigree estimates
are due to directional
selectionand genetic drift which occur during inbred line development.
These processes
disrupt the actual proportion of loci at which two homozygous cultivars carry alleles that are
ibd (Cox et #/., 1985). Selection
is especiallyintense in plant breeding programs, favoring
those alleles,and consequently
other adjacent chromosomal segments, that are preferred by
the breeders, jfy has an advantage over/y in that it takes into account the effects of selection
and genetic drift by including
the genomic contributionsof the parents.
Results of mathematicalclassification
revealed that the jfij dendrogram had a close
agreement to the/y(r* = 0.65) dendrogram. Results also revealed that jfy (r* = 0.89) was
more suitable to cluster analysis than/y (r* = 0.78). This is an important implication
becausebreeders usually classify
lines or varieties,
especiallywhen in large numbers, into groups
through some cluster analysis techniques
in order to depict a graphicalrepresentationof their
genetic interrelationships.
In terms of parental contributions(X), the marker estimates revealed the effect of
directional
selection on the contributionsof parents to their progeny. Results showed that thediscrepancies
between pedigree and marker estimates ranged from 0.0045 to as much as
0.5878.
The pedigree-based
coefficientof coancestry, on the other hand, uses Mendelian
expectationsof 0.50 for either parent of a biparental
cross and its derivative
and 0.75 for a
direct selection
from its landrace-an assumption that would somehow compromise its
accuracy. This is especiallythe case in modern crops that have undergone intensive genetic
improvement over the years.

37

The highly significant
correlationof jfy tofy indicate its validity
as measures of genetic
relatedness,while its immunity to assumptions of the pedigree method and appropriatenesstomathematical classification
imply its superiority
over the pedigree-based
method.

Acknowledgment
This project was funded by The Rockefeller
Network, and Philippine
Rice Research Institute.
Literature

Asian Rice Biotechnology

Cited

Bernardo, R. 1 993. Estimationof coefficient
Theor. Appl. Genet. 85:1055-1062.
Bernardo,

Foundation,of coancestry

using molecularmarkers in maize.

R., J. Romero-Severson, J. Ziegle, J. Hauser, L. Joe, G. Hookstra and R.W. Doerge.
2000. Parental
contribution
and coefficientof coancestry
among maize inbreds:
pedigree,
RFLP, and SSR data. Theor. Appl. Genet. 100:552-556.

Cox, T.S., Y.T. Kiang, M.B. Gorman and D.M. Rodgers. 1985. Relationshipbetween coefficient of parentage and genetic similarity
indices in the soybean. Crop Sci. 25:529-532.
Emik,

L.O. and C.E. Terrill.
1949.
coefficients.
J. Hered. 40:5 1-55.

Lapointe,

FJ. and P. Legendre.
1992. Statisticalsignificanceof the matrix correlation
coefficientfor comparing independent
phylogenetic
trees. Syst. Biol. 4 1 :378-384.

Lynch,

M. 1988.
599.

Estimationof relatedness

Systematic

procedures

by DNA fingerprinting.

forcalculating

Mol. Biol.

inbreeding

Evol.

5:584-

38

Genetic Analysis and Breeding Improvement ofa Single Tiller Mutant (sitl) in RiceQIAN Qian1,

LI Xueyong2,

ZENG Dali1,

OUYANG Jian2, Teng Sheng1,

Li Jiayang2

1 China National Rice Research Institute, Hangzhou 3 10006, P. R. China
2 Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, P. R. China

Introduction

Several tilleringmutants including
three few tiller mutants (rcn(reduced
culm number)7,
rcn2 and rcn5) induced by gamma-ray irradiation
or EMS had been found, rcn mutants had
only 1-2 tillers, but under high temperature, they almost restored normal phenotype. Each rcn
mutant is controlled by a single recessive gene. Classical genetic mapping results showed thatrcnl and rcn5 located on chromosome 6 and rcn2 located on chromosome 4. In 1990, we
found a new natural single tiller mutant from variety H89025 in Hainan Province, China. Thismutant was stable in different conditions.
Molecular markers such as RFLP, RAPD, STS, SSR etc. have been widely used in gene
mapping of rice recently. Genes controlled many important traits had been located on ricelinkage map. In present paper, the single tiller mutant was used as material to demonstrate thegenetic and physiologicalmechanism of single tiller. Genetic analysis of single tiller mutant
was undertaken, single tiller gene (sitl) was mapped by using SSR, STS, CAPS, and RFLP
markers, the content of hormones was analyzed. Breeding improvement was on the way.
Materials

and Methods

Characteristicsof the sitl
Single tiller mutant, sitl and H89025 were planted in paddy in CNRRI. Each was planted 5lines, 6 plants per line, with the density of 18x20cm (except in "plant density" test). Twelve
plants in the middle were selected. The tiller development process of sitl and H89025 wasinvestigatedevery 15d after they were transplanted.
The agronomic traits were also
investigated.
Influence of environment factors on the tiller number of sit 1
1. Influence of sowing time
sitl and H89025 were sowed 5 times with an interval of one month, from 1, April to 1,
August.
2. Influence of nitrogen fertilizer
Three levels of nitrogen fertilizer(150, 300, and 450 kg/ha respectively)
were designed inthree concrete grooves.
3. Influence of plant density
Four planting densities (16x18,
18x18, 18x20, and 20x20cm, respectively)
were used.
Endogenesis hormone content investigation
Four grams of leaf from plants in flourishing tillering period was used to investigate content
of IAA, CTK, GAs and ABA by ELISA method. Every investigation
repeated four times.
Genetic analysis
sit 1 was crossed with H89025, four japonica varieties Chunjiang
2, Chunjiang
129,
Taichung 65, and A-5, two wide compatible varieties, 02428 and Neiken 1, and three indica

39
varieties, Zhong 156, Minghui 63, and Guangluai 4.
Gene mapping
F2 population
derived from sit 1/Minghui
63, including
74 recessive lines was used as
mapping population. Ninty SSR markers, which covered the whole rice genome were used for
initialmapping. STS, CAPS, and RFLP markers were also used according to reported
protocol for further mapping.
Breeding improvement of sitl
sitl was used to select lines with single tiller, big panicle, big grain, and high seed-setting.
Because the seed setting of sitl was low, Minghui 63 and IR 64 were used for continue backcrossing.

Results
Characteristics
of sitl
The agronomic traits of sitl and its original parent H89025were showed in Table 1. There
was no significant difference between the two lines on plant height, panicle length and thelength of internodes. But there was difference in tiller number, for H89025, it was 13.3/plant,
whereas that was only 1.6/plant for sitl, and sitl had no flourishing tillering period, its tiller
number remained approximately 1.6. Although the pollen fertility of sitl was normal, the seedsetting was only 5.7%.
Table 1. The agronomic traits of sit 1 and its original

parent H89025.

Influence of environment factors on tiller number of sitl
In the range of safe temperature, the influence of sowing date to tiller number of sitl and
H89025 was not significant.
It was well know that nitrogen fertilizer
promoted tillering. Three levels of nitrogen (High,
Middle, and Low) were set according to local fertilizer
level. Tiller number of H89025 wasincreased obviously with the increase of nitrogen, whereas the tiller number of sit 1 wasalmost unchanged.
The tiller number of sitl was stable in different planting density, while for H89025, it wasslightly
increased in low plant density.
Hormonecontent analysis
There were no significantly
differencesin the contents of IAA, GAs, and ABA between sit
1 and H89025, but ZR content of sitl was 6.2 times higher than that of original
parent
H89025.
Genetic analysis
sitl was crossed with 10 varieties.
All Fi showed normal type. The segregation of single
tiller in all F2 populations
was shown in Table 2. The segregation ratios of these crosses
indicated a significant
deviation from 3 normal types to 1 single tiller type. It was indicatedthat single tiller was controlled by single recessive gene.

Table 2. The segregationof normal and single tiller

types in different

cross combinations.

Mapping of sitl geneF2 population
derived from sitl/Minghui
63 was used for gene mapping. Forty-fivepolymorphic
SSR markers and bulk segregation analysis were used for initial mapping. RM3
found on chromosome 6 was found to be linkage with sitl. The recombination frequency of
RM3 in F2 recessive population was 23.6%.
RM253 (SSR marker), G122 and G12 (STS marker) were selected for further study. The
recombination frequency of RM253 was 47.6%, indicatedthat RM253 was not linked withsitl. G12 was transferred
to CAPS marker, and the recombination
frequency was 12.2%.
G122 was a dominant marker that produced a band of 1.1 Kb in S and no amplified
production in M. There was only 1 line showed M type in 74 lines, and the recombination
frequency was ll.6%. According these results, it could be concluded that sitl was locatedbetween RM3 and G12 on chromosome 6 (Fig.l).
RFLP markers between RM3 and G12, including S12715,
S1437, R2266B, R1559, R2549
and T6 were selected for further mapping, sitl was finally mapped between S1437 and R1559,
the distances of 1.5 cM and 2.8 cM, respectively.
S1437 and R1559 were in the same YAC
clone (Fig.2).

Fig 1. Initial
6.

mapping of sitl on chromosome

Fig 2. The molecular map of sitl on
chromosome 6.

41

Breeding improvement
Seed setting of single tiller lines in BC4F1 segregation
population of sitl/Minghui
63 wasimproved. The highest seed setting was about 40%. In the combination of sitl/IR 64, the seedsetting in BC4F1 segregation
population
was about 70%. Seed setting reached approximate
90% in some lines with 2-3 tillers.

Discussion
The ecologicalcharacters of rice plant type including
plant height, the No. of tiller, and thetiller angle influenced the ability of growth competition. Plant type improvement was one of
the most important factors to elevate the grain yield. In late 1950s, Chinese rice breeders
developed a number of wide adapted semi-dwarf varieties. In 1960s, IR8 was developed in
IRRI, and spread rapidly in Southeast Asian. These semi-dwarf rice varieties with sdl, whichwere called as Green Revolution, elevated the grain yield greatly. Huang YX (1983) proposed
to select cluster, rapid growth type, which was clustering in vegetative phase and growing fast
in reproductive
phase. Many super high-yield
varieties, which was representativeof Teqing 2,
had been developed according to this proposal. Yang SR et al. (1992)
developed
a newreduced tiller variety, Shennong 366, as a main parent of ideal plant type. IRRI began super
rice program in 1991. A new ideal plant type of reduced tiller number and large panicle wasdeveloped. The grain yield would be increased 20-25%. China Super Rice Research Program
had also been started in China. It can be predicted that the new plant type varieties will beapplied in rice farming in several years.
The grain yield of maize, which also belongs to the Gramineae, is great larger than that ofrice. The evolution course of plant type of maize was multi-panicle,
weight panicle, andsingle panicle. This suggested that it would be possible that rice plant with single tiller had
higher grain yield. Searching and studying single panicle germplasm may be one of the key
works to make a new breakthrough in rice yield in 21 century.
Our mutant sitl showed several differenceswith the few tiller mutants reported to date.
First, sitl was not few tillers, but was lack of tillering
ability. Second, most of the few tiller
mutants were temperature sensitive, the tiller number would increase almost to normal typewhen it was in high temperature, whereas sitl remained stable in different
temperature
conditions.
Third, few tiller mutants were produced by artificial
mutation (gamma ray andEMS), sitl was from natural mutation, the economical traits of sitl was better than those of
few tiller mutants.
It was indicatedthat sit 1 was a cytokinite-overproduction
mutant. Cytokinite
was very
important to tillering.
Generally, high cytokinite
content wass beneficial to tillering.
But our
results suggested that cytokinite
restrained tillering
when the content was too high.
Fine mapping was the base of marker assistant selection (MAS) and map-based cloning.
Some major genes for blast resistance had been fine mapped, and these results could be usedin MAS. Xa21 and Xal for bacteria blight resistance, Pib for blast resistance and dl for dwarf
had been cloned through map-based cloning method. In present study, sitl has been mapped
between S1437 and R1559 on chromosome 6. S1437 and R1559 were in the same YAC clone.
This result provided a powerful tool to clone this gene. The seed setting of single tiller wasincreased greatly after breeding improvement. Now, weplan to make further crosses between
single tiller and big panicle lines to improve the panicle trait. This work is continuing.

42

Evaluation and Utilizationof Genetic Resources
A case study for salt tolerance in barley
Kazuyoshi Takeda
Research Institutefor Bioresources,
Okayama University,
Kurashiki 7 10-0046, Japan
Keywords : Salt tolerance, QTL, Genetic resources, Barley
Introduction
Efficiencyof plant breeding or selecting
super genotype is depending upon the efficiencyof selection and the magnitude of genetic variation existing in the breeding population.
Thus the diversityof genetic resources is vitally important for the future plant breeding.
Above all improving stress tolerance is impoetant for low input sustainableagriculture.
Wehave collected baley germplasm from allover the world and evaluated their genetic
traits from view point of phylogeny
and plant breeding.
In this paper I will report the genetic variation and inheritanceof salt tolerance in barley.
Materials and methods
About 6,700 cultivars and 350 wild accessions of 15 Hordeum spesies were examined
for the salt tolerance at the germination and the seedling stage.
Doubledhaploid(DH)
poputation
crossedbetween Steptoe/Morex
(S/M)
and
Harrington/TR
306 (H/T), offered by North American Barley Genome Mapping Project were
examined for mapping QTL of salt tolerance.
Diallelanalysisand parent-offspring
analysis
were conducted
for clarifingtheinheritanceof the trait.
For evaluating
the salt tolerance at germination,
seeds were germinated at 25°C in 1.0,
1.5 and 2.0% NaCl solution for 10 days in Petri dishes. Then seedling growth or germination
speed was scored 1 (not germinated in 1.0% NaCl solution)
to 6 (well germinated in 2.0%
NaCl solution).
For evaluating salt tolerance at seedling stage, seeds were planted in seedling box, rised
to three-leaf
stage, then isrigetedwith 500m mol or 1,000m mol NaCl solution for three
weeks. Materials were scored 1 (susceptible)to 5(tolerant).
Results
Salt tolerance at germination
Vavietal variation of 6,7 12 cultivars
for salt tolerance at germination
showed a normal
distribution(Mano et al. 1996).
In general, six-rowed varieties
were more tolerant than two-rowed ones, naked types
were more tolerant than covered ones. Rondomly selected 268 varieties
were germinated insalt and polyethyleneglycol
solutions.
Collelation
coefficient
between them was as high as430.789,

showing

the mechanism of salt stress at the germination

Most tolerant
selected.

varieties

were repeatedly

wereand from Japan,

Korea, China and Nepal.

Six ofvariety

could germinate

from sea water.

were generally

more suscsptible

than cultivars

varieties

them were covered. The most tolerant
Wild
stage

baley

accessions

to osmotic reaction.
varieties

They were all six-rowed

evaluated

is relating

and nine super tolerant

at germination(Mano and Takeda 1998).
Diallelanalysis

revealed

that salt

toleranceat germinationis controlled

by multiple

correlation

coefficient

genetic factors (Mano and Takeda 1997-c). Parent-offspring(F2/F3)
heritabilityof the trait was around 0.5 (Mano and Takeda 1997-a).
In S/M DH populationa major QTL, explaining

centromere of 5H. And it was allelicexplaining
allelic

41% of variation,

47% of variation,

was detectedto ABA response. In H/T DH population

was found on the terminalornear thea major QTL,of the long arm of 5H. And it was also

to ABA response (Mano and Takeda 1997-b).

Salt tolerance

at seedlingstage

A total of 6,68 1 cultivars
Repeated selection detected

showed a normal distribution
eleven super tolerant varieties.

for salt tolerance at seedling stage.
All of them were six-rowed andcovered (Mano and Takeda 1995).
Correlation

coefficient

betweenthe salt

toleranceat germinationand seedlingwas-0.061 (n=6,646),
showing the mechanism of tolerance is defferent at the stages.
Salt tolerance of wild accessions was generally higher than cultivars (Mano and Takeda
1998).
Dialelfactors

analysis

revealed

that salt

toleranceat seedlingstage is controlled

by multiple(Mano and Takeda 1997-c).

By triplicated
QTL analysis,a stable QTL on chromosome 1H was detected in S/M
populationand one on chromosome 5H in H/T population(Mano and Takeda 1997-b). They
were independent
Summary
Varietial
analysisof QTL for salt tolerance

variation

revealed

that it is underditected on 5H.
Varietal variation
under the control
respectivel
Saltof salt tolerance

at germination

the controlof salt toleranceof quantitative

at germination.

at seedlinggenes. A stableshows a normal distribution.

of quantitative

genes.

Significant

DiallelQTL wasstage shows also a normal distribution.
QTL was found on 1H (S/M)

It is

and 7H (H/T),y.
tolerance

former is relating

at germination

is independent

to osmotic reaction.

The latterof salt tolerance

at seedling

may relate to Sodium toxicity.

stage.

The

44
References
Mano, Y. and K. Takeda 1995. Varietal variation
tolerance in barley seedlings.
Bull. Res. Inst. Biores.,Mano, Y., H. Nakazumi

and effect of some major genes on salt
Okayama Univ. 3(1) : 71-81.

and K. Takeda 1996.

major genes on salt tolerance

Varietalvariationat the germinationstage

in barley.

1991-a.

of salt tolerance

in and effect
Breeding

Scienceof some46(3):227-233.
Mano, Y. and K. Takeda
parent-offspring
Science47(4)correlation

response

and the seedling

1997-b.

Mappingstage in barley

Mano, Y. and K. Takeda 1997-c.

based(Hordeum

vulgareL.). Breeding

quantitative

trait

for salt

tolerance

DiallelEuphytica

103 : 137-141.

loci(Hordeum

vulgaleanalysisof salt tolerance

seedling stage in barley (Hordeum vulgare L. ). Breeding Science
Mano, Y. and K. Takeda 1998. Genetic resources of salt
species.

at germination

in barlyon: 353-358.

Mano, Y and K. Takeda
germination

Heritability

and salectionL.). Euphytica

at

94 : 263-272.

at germination

47(3) : 203-209.
tolerance
in wild

and theHordeum

45

Sourcing,

evaluating

and utilizing

biodiversity

in crop genomes

Pat Heslop-Harrison
and Trade Schwarzacher
Universityof Leicester, Leicester LEI 7RH, UK
E-mail:Website:PHH4@le.ac.uk and TS32@le.ac.uk
FAX: +44/0 116 252 2791

www.le.ac.uk/biology; this will include

some of the presentation

Global problems and genomic answers
The subject of this meeting itself recognizes that we are at a turning point where theinformation and technologies
derived from molecular biology and genomics can bedirected towards solving real problems in the development and improvement of crops.
Major international
concerns about the sustainabilityof industrialized
agriculture havecometo the fore, ranging from its impact on rural economies and ecosystems in themost developed countries, through to effects on social structures in the poorest
countries. Genome technologies
are able to provide quantitative
measurements of theways that biodiversity
is being eroded by effects both of changes in agricultural
practices and of humankind's impact on the global climate. Genome technologies
asapplied to the development of new crop plants are versatile and can be directed toalleviate problems in appropriate timescales. The power of genome technologies meanthat even global problems can be analysed on a local scale and research fromindividuals
can be used to build towards global answers. In this talk, we will aim torecognize the major opportunities
for genome technologies
to solve global problems,
while showing examples of our detailed analyses of a few specialized
situations
that
contribute to answering these problems.

The structureof the plant genome

The contributionof genes to biodiversity
has been widely considered, but genes andassociated low-copy DNA sequences make up a small fraction of most plant genomes.
Repetitive sequences, ranging from microsatellites
or simple sequence repeats of motifs
one to four base pair long, to repeat units longer than 10,000 bp, represent the majorityof most genomes, and each motif may be repeated hundreds or thousands of times in
the genome (Schmidt and Heslop-Harrison,
1998; Heslop-Harrison,
2000). Repetitive
sequences include retrotransposons
and retroelements, genes such as the 5S or 45S
rDNA complexes and intergenic spacers, and tandemly repeated elements that foldaround nucleosomes. Repetitive DNA, the focus of our research, in the genome isimportant for evolutionary,genetic, taxonomic and applied studies and it provides
polymorphic
markers. Some repetitive DNA sequences are highly conserved among
eukaryotes and prokaryotes, and are even recognizable as remnants of the RNA-based
world that preceded the origin of DNA. Other sequences, such as some tandem repeats,differ between even closely related species, where the genes show little greater
variation than is found among alleles within a species. Our work focuses on the nature,
diversityand evolution of the major repetitive
sequences in the genome, and their
physical and functional relationships.

46

Biodiversity

and plant phylogenetics

Biodiversity
can describe both the range of species within an ecosystem and the range
of genes present within a species. Both forms of biodiversity
are available to breedersof crops, but we need to define the extent and nature of the diversity as well as thestrategies
to utilize
it in crop plant improvement. Contrasting
requirements
andpossibilities
are present in different species, depending on their relationships
with otherwild and cultivated species, the amount of selection already carried out by humans, andthe requirements from the crop. Understanding
the biodiversity
at the species level is
assisted by knowledge of the basis of evolution of species at the genome level. A strong
basis in phylogeny - natural taxonomy - is valuable to determine how diversity can beassessed and used. Methods of molecular systematics are giving new insight into therelationships
and evolution of the whole plant kingdom, aspects of which are
unexpected from morphologicalexamination alone.
Retrotransposons
are nuclear DNA sequences that encode enzymes for theirown amplification,
through an RNA intermediate,and for their reinsertionin thegenome. Different classes, includingLINEs, copia and gypsy element classes, andpararetroviral-likesequences are present in all plant groups examined. Our evidence
indicates that all retrotransposonclasses were present at the base of the phylogeny ofplants, although parts of their sequences were already distinct from those present inother kingdoms (Friesen et al 2000). Gymnosperms represent an important and ancientgroup of species, still dominant in many ecosystems around the world and of higheconomic value for timber and pulpwood. The species present in Northern andSouthern hemispheres are different, and the relationships
between different groups are
not entirely clear. Multi-colour fluorescent microarray hybridization
potentiallyhasmajor applications
in studying the evolution of DNA sequences in genomes, whether inthe short times associated with disease syndromes or plant breeding, over the many
generations associated with generation of biodiversity,
or in the time-scales associatedwith species evolution. I will present results showing the diversity of retroelements indifferent
gymnosperm species,
analysedby both membrane and microarrayhybridization,
which suggest the relationships
between the major gymnosperm groups
and emphasize the diversity present early in plant evolution. The analysis of diversity
and evolution of ubiquitous retroelement sequences and families has been particularly
valuable within the gymnosperm species where relationships
are unclear, partly
because of the huge genome size (eight times larger than human). The screening
capacity of microarrays means that the structure of families of different
repeatedelements, including
their abundance and detailed
sequence, can be assayed in acomparative and massively parallelmanner. Thus even small differences
betweenorganisms can be detected in sequences that are parts of the most rapidly evolving
genomic structures,
where gene sequences will remain closely similar.
Major
advantages of fluorescent microarray hybridizations
include the scale and number ofclones that can be analysed, the ability to hybridize three to five probes simultaneously,
the control available over hybridization
stringency,
and finally the quantitative
data
about both relative and absolute hybridization
strengths.

Biodiversity

at the species level

Knowledge of phylogeny and relationships
enables the definition of species groups.
Within such groups of crop plants, it is valuable to have a picture of the diversity
present in the group of species that constitute a gene-pool than can be accessed bysexual crossing. We will show examples using total genomic DNA as a probe to47

differentiate
genomes in species which are readily hybridised, and where breeders are
keen to transfer chromosome segments into new plant breeding lines.
Individual
repetitive
DNA sequences motifs can be used to examine thegenomic diversity within a group of related species. In wild Hordeum taxa representing
the four basic genomes of the genus, Southern and fluorescent in situ hybridization
wasused to examine genomic relationships
and diversity of rDNA and two repetitive DNA
sequences (Taketa et al 2000). One sequence showed between 30 and 60 hybridizationsites on chromosomes of all diploid species studied, but hybridization
patterns differedamong the species. A second, less abundant sequence, enabled conclusions about
relationships
between and within the cultivated barley group and the wild Hordeum
species. The presence of a sequence in a tetraploid
cytotype of H. murinum but its
absence in the diploid
cytotypesuggested that the tetraploid
was not a simple
autotetraploidof the diploid,
but had additional
biodiversity
perhaps influencing
its
success as an ephemeral, colonizing species. Data about the inter- and intra-specificvariationof repetitive
DNA sequences gives information
about both theinterrelationshipsof the species and the evolution of the repetitive sequences.
Diversity
within species is widely used in conventional
plant breeding
programmes, with the aim of bringing together optimum combinations of genes fromdifferent varieties or occasionally different species. We have used repetitive sequences
as markers for evolution in breeding lines to look at the organization of major repetitive
DNA sequence classes in seven related wheat lines involving the introductionof genesfor resistance to eyespot, from the wild, tetraploid
wheat Aegilops ventricosa (Bardsley
et al 1999) into bread wheat. Within the pedigree, very high levels of polymorphism
were found in the distribution
patterns of two different repetitive
sequences, withindividualarms showing up to five different variants. Given the relatively low level ofvariation between breeding lines found for most RFLP probes, this was unexpected, but
the results allowed the parent-of-origin of individual
chromosome arm segments to be
determined in many of the varieties. Further comparison of pairs of varieties revealed
that the number of arms sharing distribution
patterns for one repetitive
sequence
showed a close correlation with the relationshipof the varieties. No relationshipbetween shared repetitive
sequence distribution
and coefficientof parentage wasevident using the other sequence as probe. These results firstly show that the repeat
sequences are useful to track chromosomes through a breeding programme, andsecondly that different
repetitive
sequences show contrasting
rates of evolution.
Although both sequences were useful markers to help identify the parent of origin of achromosome arm in a particular hybrid-derived line, one showed more rapid evolution
and hence not the level of heritabilityof the other.
A remarkable source of biodiversity
is in landraces. A Portuguese landrace of
wheat, Barbela, shows impressively
wide adaptation
to different
environments,
producing worthwhile crops in low fertility conditions, often associated with acid soils.
In the 1930s, Barbela was the most cultivatedwheat in Portugal showing winterhardiness in the northern inner region and drought tolerance in the semi-arid and hot
regions of the south (Ribiero-Carvalho
et al. 1996). We will discuss work showing thelevels of diversity
present within this landrace using polymorphic
microsatellite
markers. Market requirements for uniformity mean that diverse landraces are under
threat in many regions, and genomic technologies
are proving valuable to assess thevariation present in these lines, to find markers for following
specific traits andcharacters and to identify what must be targeted for conservation.
We see exciting developments in using microarrays to screen biodiversity
present in germplasm banks. The reciprocal nature of microarrays is a major advantage:

48

one marker (the probe) can be scored on DNA from thousands of accessions (the target
on the array), or, in the alternative experiment, thousands of markers (now the target on
the array) scored with DNA from a single accession (potentiallya new accession in a
genebank).

Utilizingbiodiversity

There are multiple ways that biodiversity
can be used in plant breeding today. Longdistance transfer of characterized
genes is a reality through transformation technology,
but the genes and their functions must still be identified,often in germplasm distantfrom a crop, and considerable
technology is often required to identify
key loci fordisease resistance,stress tolerances or quality factors. Other technologies,
including
induced mutations, have potential to generate new diversity,
particularly
to correct
specificvarietal weaknesses. It can be suggested that there is a need to increase thediversity of species that are used as crops - in most of the world the trend has been toincreasing
reliance on smaller numbers of species, and these are more and more
selected from a narrow and uniform germplasm base. However, crops with limited
markets are often high-value and cash-generating
to farmers. Knowledge of the crops
and their relatives,
combined with assistancegiven by breeders using advanced
technologies
can have rapid results in increasing the productivityof such crops andcorrecting
minor agronomic weaknesses. A final challenge
for breeders is theunderstanding,manipulation
and utilizationof the genomic responses of plants to achanging and apparently more variable climate in many of the most productive cropareas.

References
These describe the general area of research, hut are not specific citations
for new work to bepresented.
Bardsley D, Cuadrado A, Jack P, Harrison G, Castilho A, Heslop-Harrison JS. 1999. Chromosome
markers in the tetraploid
wheat Aegilops ventricosa analysed by in situ hybridization.
TheorAppl Genet. 99: 300-304.
Friesen N, Brandes A, Heslop-Harrison JS. 2000. Diversity,
origin and distributionof retrotransposons(gypsy and copia) in Conifers. Molec. Biol. EvoL, subject to revision.
Heslop-Harrison, J. S. (2000) Comparative genome organizationin plants: from sequence and markers tochromatin and chromosomes. Plant Cell 12: 617-635.
Heslop-Harrison JS. 2000. RNA, genes, genomes and chromosomes: repetitive
DNA sequences inplants. Chromosomes Today 13: 45-56+ plate VI.
Ribeiro-Carvalho
C, Guedes-Pinto H, Harrison G, Heslop-Harrison
JS. 1996. Wheat-rye
chromosome translocations
involving small terminal and intercalaryrye chromosome
segments in the Portuguese
wheat landrace Barbela. Heredity 78: 539-546.
Schmidt, T. and J. S. Heslop-Harrison (1998)
Genomes, genes and junk: the large scale organizationofplant chromosomes. Trends Plant Sci. 3: 195-199.
Schmidt A, Doudrick RL, Heslop-Harrison JS, Schmidt T. 2000. The contribution of short repeats of low
sequence complexity to large conifer genomes. TheorAppl Genet 101 : 7-14.
Schwarzacher T, Heslop-Harrison JS. 2000. Practicalin situ hybridization.
203+xii pp. Bios: Oxford.
Taketa S, Ando H, Takeda K, Harrison GE, Heslop-Harrison JS. 2000. The distribution,organization
andevolution of two abundant and widespread repetitive
DNA sequences in the genus Hordeum.
Theor. Appl Genet. 100: 169-176.

49

Genomeanalysis

in barley

and its impact on the utilization

A. Graner, D. Smilde, A. Drescher, K.-P. Pleissner,of genetic resources

F. Ordon* and W.Michalek

Institute of Plant Genetics and Crop Plant Research (BPK), Corrensstr. 3, 06466
Gatersleben,
Germany* Institute of Crop Science and Plant Breeding I, Heinrich-Buff
Ring 26-32, 35392
Giessen, Germany

Introd uction
Driven by modern agricultural
practices and continuous genetic improvements grain yield ofbarley has almost tripled within the past century and continues to reach new heights. Targeted
breeding has led to the development of cultivars tailored for diverse environments on allcontinents and for different purposes such as feed an brewing. With the increasing yield, thegenetic variability
present on barley fields gradually
decreased, since landraces and local
varieties were replaced by cultivars, which usually consist of a single genotype. Extrapolated
from data available for the production of certified
seeds, 83% of the total German winterbarley acreage of 1.4 million ha are planted to only 15 and 92% of the spring barley acreage(0.8 mio ha) are planted to ll cultivars
only. Even worse, the analysis of both pedigrees andmolecular data shows that many, if not most, of these cultivars share related pedigrees, thus
further reducing the effective population
size. Evidently,
the genetic uniformityof todays
barley fields is in contrast to the wealth of genetic resources of barley maintained in ex situ
collections
amounting to more than 370,000 accessions worldwide. Based on case studies
regarding resistance to various fungal and viral pathogens, the present paper will highlightsome of the activities
in various fields of genome analysis which have been initiatedtopromote the deployment of genetic resources of barley in practical breeding programs.
Resistance Breeding
Breeding for disease resistance consists of two steps - the identificationof a resistance donor
and the transfer of the corresponding
gene(s) into a breeding line. For a long period of time,the development of resistant germplasm was dependent on singularities
such as the repeatedoccurrence of a disease in consecutive years and the availabilityof resistant germplasm. The
latter might have been present in landrace populations or happened to be present in an exotic
germplasm accession obtained by expeditions
into the centers of diversity,
as they were
performed since the early 1920ies (Vavilov1997). Later, systematic activities
have been
initiated
to evaluate germplasm collections
for the presence of resistance genes to various
fungal and viral pathogens (e.g., Proeseler et al. 1999).
The availabilityof a comprehensive set of mapped markers of barley and other related grass
species (http://wheat.pw.usda.
gov/ggpages/maps.html)
has greatly promoted the investigationof the genetic basis of resistance to a range of fungal and viral pathogens. Thus, the genetic
localizationof a gene of interest, which forms the prerequisite
for its systematic manipulationin the breeding process, can now be achieved with unprecedented precision.
In addition,
selectable
markers have been developed that allow a reliable selection for genes that would be
difficultto score by conventional means.
Molecular Mapping and Marker Development
Historically,
the first phase of molecular mapping of disease resistance was mainly aiming toprove the principle,
i.e. to show that any gene can be accurately localized
and that tightly
linked molecular markers can be identified(for review see Graner et al. 2000a).
Recent50

mapping activities
have a strong focus on agronomicallyimportant pathogens and theidentificationand mapping of those resistance genes that protect the plant against a broad
range of pathotypes.
This is obviously
a time consuming, multistep process ranging from theidentificationof appropriate
resistance donors to the development of a selectable marker. It
requires a close collaboration
between barley breeders, phytopathologists,and geneticists.
In this context major emphasis has been put on the identificationof major genes conferring
resistance to the leaf rust pathogen Puccinia hordei. In addition
to the race specific genes
Rph2, Rphl2, and Rph9 (Borovkova et al. 1997, 1998), selectable
markers were recently
developed for the genes Rph7 and rphl6 (Ivandic et al. 1998, Graner et al. 2000b). To date,
no P. hordei pathotypes have been observed in Europe that have overcome the latter two
genes. Therefore, their combination
in.one genotype might represent an efficient
way toincrease the shelf live of its resistance. To this end, codominant PCR-markers were developedfor both Rph7 and rph!6, now permitting a marker assisted combination without any progeny
testing.
Similar to Erysiphe graminis (powdery mildew) P. hordei represents an example,
where the genepool of cultivatedbarley is largely depleted of useful resistance genes andresearch activities
have to focus on the identificationof novel alleles from wild barley
Hordeum spontaneum as is the case the with rphl6. In this context, the availabilityofselectable
markers forms a prerequisite
for the rapid introgressionof the corresponding
genes
into adapted barley germplasm.
Barley yellow mosaic (BaYMV) and barley mild mosaic virus (BaMMV) are soil-borne
pathogens. Both are transmitted
to the roots of winter barley seedlings by the fungal vector
Polymyxa graminis. However, in contrast to the fungal pathogens mentioned above, a seriesof resistantaccessions could be identified
in the gene pool of cultivatedbarley (Ordon &Friedt 1993). Genetic analysis and molecular mapping has led to the localizationof a series ofresistance genes on chromosomes 1H (Graner et al. 1999a),
3H (Graner & Bauer 1993,
Graner et al. 1999b, Iida & Konishi 1994 ), 4H (Bauer et al. 1997) and 7H (Saeki et al. 1999).
Resistance of European cultivars is mainly based on the rym4 locus in the telomeric portion ofchromosome 3HL conferring immunity to BaMMV and BaYMV. The practical value of therym4 locus is restricted by the fact that this locus does not encode resistance to a second, morevirulent strain of BaYMV, termed BaYMV-2. On the other hand, the ryrn5 locus, which maps
in the same marker interval as rym4 (Graner et al. 1999b),
confers resistance to all strains of
the BaMMV/BaYMV complex, including BaYMV-2. Hence, this locus is increasingly
introgressed to replace rym4. A second locus conferring resistance to all European strains of thevirus complex was recently detected on chromosome 4H where it turned out to be allelic to
the already described rymll locus. Its introgression
into elite germplasm will represent analternative to rym5.
Tests for allelism revealed, that rym4 and rym5 carry identical genes with respect to resistanceto BaMMV. Moreover, no recombinants with altered resistance specificities
were detected in
more than 1000 F2 derived progeny lines from crosses between each of the two resistancedonors with a susceptible
parent. This leaves open the question of the molecular structure of
the rym4/rym5 resistance locus, whose different specificities
could be due to either a set ofclosely linked genes or multiple allelism (Graner et al. 1999a).
In addition to immunity, partial resistance to BaMMV has been observed. Here, the presenceof the recessive resistance gene leads to a prolonged latent period. Genes conferring partial
resistancehave been mapped to the centromeric region of chromosome 1H and tochromosome 4HL (Bauer et al. 1997, Graner et al. 1999a).
It is presently
unclear, whether
partial resistance may represent just a "leaky" version of an immunity type of allele, or if it isbased on a different mechanism such as an impaired translocation of virus particles
from theroot into the upper parts of the plant.

51
Map Based Gene Isolation
As reported above, the genetic analysis of the rym4/rym5 locus provided evidence of its
complex nature. However, due to the finite resolutionof genetic experiments, nothing is
known about the physicalstructure of the locus and the structure of the corresponding
gene(s).
To facilitatea map based isolationof the rymAlrymS locus, high resolution
maps
were constructed and additional
markers are being developed for the target interval. First
estimates
give a genetic vs. physicaldistance ratio of about 1-2 Mb/cM, which roughly
corresponds to the estimate of Kuenzel et al. (2000) for this chromosomal region.
Evidently,
the map based isolationof a gene in barley requires considerable
efforts andattempts have been undertaken to use the small rice genome as a platform to identify the rice
homologue of a gene before getting back to barley. While such a homology based cloning
approach has worked well for the isolationof the Rht-1 dwarfing gene in wheat (Peng et al.
1999), the approach has failed so far regarding the isolationof a disease resistance gene inbarley. In spite of a highly conserved synteny between barley and rice in the chromosomal
region harbouring
the barley Rpg-l resistancegene, sequence analysis of a rice BAC
containing markers flanking Rpg-l did not lead to the detection of the rice homologue (Han etal. 1999). This failure might be the result of a rapid evolution and genomic reorganizationof
disease resistance genes (Leister et al. 1998). In order to test this hypothesis,
more data areneeded regarding
comparative
mappingof resistancegenes in barley
and rice.
Notwithstanding
these limitations,the vast amount of resources available for the rice genome(including
the complete sequence that is expected to be available soon) will be exploited in
the future to gain more rapid access to the genes in the complex Triticeae genomes.
Systematic Approaches to Gene Isolation
Technical progress regarding DNA sequence analysis has prompted the establishmentof acomprehensive collectionof partiallysequenced cDNAs commonly termed ESTs (expressedsequence tags). Despite the presence of regions that are enriched in genes, the large size of
cereal genomes, presently prohibits the establishmentof a genomic sequencing effort. Hence
ESTs provide an economic alternative to identify a significant
portion of the genes present inbarley. Recently, an EST sequencing effort has been initiatedanalyzing
three different
libraries (caryopses, etiolated
leaves, roots). At present some 13,000 sequences have been
generated from 7,500 CDNA clones, which represent about 4,200 tentative genes. Sequence
data and BLAST-X results are available from a web server via the homepage of the IPK(http://www.ipk-gatersleben.de/).
The availabilityof comprehensive EST collections
will
facilitate
the transfer of genetic information to and from related species such as rice and corn.
These approaches are based on marker synteny in selected chromosomal areas. Using aninterspecificapproach for the development of a high density map of the Rh/Pt resistance locus
on barley chromosome 3H (Graner et al. 1996; Graner & Tekauz 1996) an efficient marker
saturation of the target interval in barley was achieved by the implementationof an in silico
approach that exploits sequence homology between barley and rice ESTs. Thus, the target
interval in rice and barley could be downsized to about one centiMorgan and a 400 kb BACcontig in rice was established.
Conclusions
Since most major resistance genes act in a race specific way and will be overcome by time,
there is a permanent demand for the identificationof novel genes/alleles.
For several
pathogens including leaf rust and mildew the gene pool of cultivated barley is largely depletedof useful resistance genes and increasing efforts are required for the identification
andcharacterizationof any additional
gene. Fortunately,
wild barley H. spontaneum, and more
recently H. bulbosum, have proven to represent rich sources of resistances to almost any

52pathogen thus attracting much attention in evaluation programmes. The systematic use of thelatter will criticallydepend on further improvements regarding the generation of fertile
hybrids and the availabilityof data regarding
the recombinationof its chromosomes withthose of H. vulgare.
Prior to mapping and breeding,resistancegenes have to be identified
in germplasm
collections
like needles in a haystack. Hence, the evaluation of genetic resources presently
forms the rate limiting
step in terms of the identificationof novel resistance genes. In the
future, the isolation of an increasing number of resistance genes may lead to the development
of diagnostic
markers that can be used for a rapid differentiationof alleles in germplasm
collections
thus increasing the throughput by avoiding lengthy tests for allelism. In the longrun, the development of disease resistant barley germplasm is expected to benefit from thegain in knowledge that will result from the structural and functional analysis of resistancegenes. However, map based gene isolation,in particular,when it requires a chromosome
walking step, is a slow process that presently requires extensive resources. The ability tointegrate the resources and the wealth of genomic information available from related species
like rice and corn will facilitate
the development of less expensive approaches for geneisolation.
In this context barley ESTs, apart from being a resource in itself, will act as thevehiclesrequired to import and make use of the genomic information from other plant
species.
References
Bauer, E., Weyen, J., Schiemann, A., Graner, A and F. Ordon (1997).
Theor. Appl. Genet. 95, 12631269.
Borovkova, I.G., Jin, Y., Steffenson,
B.J., Kilian,A., Blake, T.K. and A. Kleinhofs(1997).
Genome40,236-241.
Borovkova, I.G., Jin, Y. and BJ. Steffenson(1998).
Phytopathology
88, 76-80.
Graner A. and E. Bauer (1993).
Theor. Appl. Genet. 86, 689-693
Graner, A., Foroughi-Wehr,
B. and A. Tekauz (1996).
Euphytica
91, 229-234.
Graner, A. and A. Tekauz (1996).
Theor. Appl. Genet. 93, 421-425.
Graner, A., Streng, S., Kellermann,
A., Proeseler,
G., Schiemann, A., Peterka, H. and F. Ordon(1999a).
J. Plant Diseases& Plant Protection
106, 405-410.
Graner, A., Streng, S., Kellermann,A., Schiemann, A., Bauer, E., Waugh, R., Pellio, B. and F. Ordon(1999b).
Theor. Appl. Genet. 98, 285-290.
Graner, A., Michalek,W., and S. Streng (2000a)
Barley Genetics
VIII (S. Logue, ed.), Dept. of Plant
Science, Waite Campus, Adelaide,
Australia
pp. 45- 52.
Graner, A., Streng, S., Drescher, A., Jin, Y., Borovkova, I. and B.J. Steffenson(2000b).
Plant Breed.
119,389-392.
Han, F., Kilian, A., Chen, J.P., Kudrna, D., Steffenson,
B., Yamamoto, K., Matsumoto, T., Sasaki, T.
and A. Kleinhofs(1999).
Genome 42, 1071-1076.
Iida, Y. and T. Konishi (1994).
Breeding
Science 44, 191-194.
Ivandic,
V., Walther, U. and A. Graner (1998).
Theor. Appl. Genet. 97, 1235-1239.
Kuenzel, G., Korzun, L. and A. Meister (2000).
Genetics
154, 397-412.
Leister,D., Kurth, J., Laurie, D.A., Yano, M., Sasaki, T., Devos, K., Graner, A. and P. Schulze-Lefert(1998).
Proc. Nat. Acad. Sci. 95, 370-375.
Ordon, F. and W. Friedt (1993).
Theor. Appl. Genet. 86, 229-233.
Peng, J., Richards,
D.E., Hartley,
N.M., Murphy, G.P., Devos, K.M., Flintham,J.E., Beales, J., Fish,
L.J., Worland, A.J., Pelica,F., Duraialagaraja,
S., Christou,P., Snape, J.W., Gale, M.D. and N.P.
Harberd (1999).
Nature 400, 256-261.
Proeseler,G., Habekuss, A., Kastirr, U., Graner, A. and K. Hammer (1999).
J. Plant Diseases & Plant
Protection
106,425-430.
Saeki, K., Miyazaki,
C, Hirota, N., Saito, A. and K. Ito (1999).
Theor. Appl. Genet. 99, 727-732.
Vavilov, N.I. (1997).
Five Continents.
Intl. Plant Genetic Resources Institute,
Rome.

53

Molecular

diversityand evolution

in the genus Hordeum

T. Komatsuda
National Institute of Agrobiological
Resources, Department of Genetic Resources n, Tsukuba
305 8602, Japan, (e-mail: takao@abr.affrc.go.jp)
Key words: Triticeae,

phylogeny,

barley,spike morphology,

gene mapping.

Introduction
HordeumL. belongs to the tribe Triticeae, which includes a number of important cereal crops
such as wheat {Triticum spp.), rye (Secale cereale) and barley (Hordeum vulgare). The genus
Hordeum comprises 32 species, 45 taxa, including diploid, tetraploidand hexaploid with a basic
chromosome number of x=7 (Bothmer et al. 1995). Based on the entire study of meiotic pairing,
Bothmer and his co-workers recognized four genomes in Hordeum and they were designated I(if. vulgare and H. bulbosum), Xu (H. murinum), Xa (H. marinum) and H (all the other
Hordeumspecies). The differentiationof the four genomes has been supported by cytological,
biochemical
and molecular marker analyses (reviewed by Bothmer et al. 1995).
H. vulgare includes ssp. vulgare, the cultivars and landraces, and ssp. spontaneum, thewild progenitor of cultivated barley. H bulbosum has a good crossablity
with the cultivatedbarley and, even though its chromosomes are mostly eliminated in the young hybrid embryos, its
chromosomes can be substituted and chromosomal segments can be integrated into barleygenome (Pickering
2000), thus H bulbosum forms the secondary gene pool for barley. Allother species belong to the tertiary genepool. Hordeum is a good case study for evolutionary
patterns within genus including diploids and polyploids.
Study of diversity between and within
species would be significant,particularly,
for the origin of cultivated barley. Recent studies on
nuclear gene sequences revealed unexpected views of diversity and evolution of the genus
Hordeum.The objective of this paper will be to discuss results we have obtained from a seriesof our studies (Komatsuda et al. 1999b, Tanno et al. 2000, Komatsuda et al. in preparation).
Choosinga gene for phylogenetic
study
The diagnostic morphologicalcharacter of Hordeum is the possession of three one-flowered
spikelets(triplet)
at each rachis node. In a triplet, the central spikelet is fertile and able to develop
into grain, while the two lateral spikelets are usually sterile or rudimentary. Fertile lateral
spikelets
occur only in some forms of if. vulgare and in some forms of the wild species as H.
bogdanii. Fertility of lateral spikelets is one of the important genetic traits for domestication ofbarley and probably for evolution of wild species of Hordeum. The lateral fertilityof H.
vulgare is controlled predominantly
by the vrsl locus. We constructed a high resolution map of
the vrsl locus and found a nuclear DNA, cMWG699, closely linked to the vrsl locus(Komatsuda et al. 1999a). This DNA is single copy in nuclear genome of H. vulgare as
analyzed by Southern hybridization,and this DNA revealed a high nucleotide sequencehomology (80%) to the chloroplasttranslation
elongation factor EF-G gene (Hernandez et al.
1993). Allele specific primer pairs designed for this sequence generated a single copy DNA
fragment not only from Hordeum, but also from Psathyrostachys
Nevski, Triticum L. and
Secale L. These results suggested that this DNA sequence is useful for comprehensive
phylogenetic
studies in the genus Hordeum and probably in Triticeae.

54
Diploid
species of Hordeum
Phylogenetic
analysis of the nuclear DNA sequence was performed for studying the evolutionary
history of the four basic genomes of Hordeum. PCR-amplification
applying allele specificprimer pair was successful to generate a single DNA from all the 20 diploid Hordeum species.
The amplified DNA was around 930 bp long. Among the DNA an intron part of around 350 bp
long was sequenced and compared. Alignment was able to be done manually, the number of
insertion/deletion
gaps was only eight. Psathyrostachys
was put as an outgroup for phylogenetic
inference.
Parsimony tree, based on substitutionsof nucleotide, indicated that the genome of genus
Hordeumprobably diverged into two groups first; one is a clade containing I and Xu, and theother including H and Xa (Fig. 1). The clade of each group was highly supported by bootstrap
analysis. Then each group was differentiated(or probably is differentiating)mutually to loose
meiotic pairing in their hybrids. I and Xu were resolved, and within the I genome H. vulgare
and H. bulbosum formed sister group. In compare to the clear resolution of I and Xu,
separation of H and Xa was not so clear, since the clade of H group was less supported.
Another parsimony tree including both base substitutions
and insertion/deletion
events showedthat the Xa genome to be included in the large, highly homogeneous H genome group. Eachparsimony tree suggested the close phylogenetic
relationshipof the H and Xa genomes.
The results of this analysis supported the four basic genomes. The more important result
obtained from this study, however, is that Hordeum diverged into two gropes. H murinum ismost closely related to the cultivated barley. Based on the morphology, ecology and distribution
H.murinumhas been classified in the same section Hordeum as H. bulbosum and H vulgare(Bothmer and Jacobsen 1985). However, non of the biochemical and molecular marker studies
supported this classification,and this study would be the first to support the classification,section Hordeum as including H murinum,H bulbosum and H vulgare. This gene treeneeds to be compared with a second tree, preferably from a different chromosome. The
phylogenetic
tree represented here was different from that inferred using rpoA gene sequence on
chloroplast
genome (Petersen and Seberg 1 997), indicating the different phylogeny between
nuclear and chloroplast
genome in Hordeum.
H. marinum
This species is of great interest as a valuable source for salt tolerance (Bothmer and Jacobsen
1985, Mano and Takeda 1998). This species consists of diploid and tetraploids,
and the origin of
the tetraploid is a topic of much debate, alloploid or autoploid.
Genetic variabilityof H marinum ssp. marinum was studied using the same DNA andthe same methodology. A total of 65 accessions of this diploid taxon were analyzed with the
PCR-amplification
followed by restriction
analysis. The result showed a diagnostic Mspl sitewhich distinguishes
ssp. marinum into two clades. The two clades showed disjunctdistributions,the Iberian Peninsula (type 2) and other regions i.e. Turkmenistan, Jordan,
Turkey, Greece, Italy, Germany, France and Majorca (type 1). The two forms were separated by
the Pyrenees and the sea between Valencia and Majorca, Balearic Islands. To elucidate thesequence variation, 1 1 accessions of type 1 and 4 accessions of type 2 were chosen to represent
maximumgeographicaldistribution
within the native area. The entire DNA sequences, being 932
bp long, were compared. The type 1 and type 2 showed clear differences.
All the 1 1 accessionsof type 1 shared identical sequence in spite of their wide distribution
from France to
Turkmenistan. On the other hand, 4 accessions of type 2 revealed considerable variation of
nucleotide. The two forms may represent two ecotypes previously not recognized. This result

Implies that the ssp. marinum, especiallyin the Iberian Peninsula, might include more nucleotide
variation which have not been detected in this study.
Compared to the ssp. marinum, the ssp. gussoneanum was not variable. Diploid taxon(23 accessions)did not show sequence variation, and tetraploidtaxon (20 accessions)generated
two classes of DNAs but showed no variation within each class. The important point was that
one of the classes (class a) revealed the same nucleotide
sequence as diploid taxon of ssp.
gussoneanum, indicatingthat diploid of ssp. gussoneanum is the immediate donor of one
genome of tetraploid(Fig. 1). The other class of the tetraploid(class b) remained in the sameclade as H. marinum, Indicatingthat H. marinum Is monophyletic.

Fig* 1. Phylogenetic
tree of the diploid
Hordeum species (except a tetraploid
H. marinum wasincluded)based on base-substitutionof the intron region of the cMWG699 clone. The mostparsimonious
tree is shown (length
71, CI = 0.90? RI = 0.96). Numbers above branches indicate
bootstrap
values obtained from 100 bootstrap
replicates.

theH. vulgare
Phylogenetic
analysis of the nuclear DNA sequence was performed to study the origin of 6rowed barley. This study included 280 landraces (ssp. vulgare) from Eurasia and Africa,
consistingof 204 six-rowed and 76 two-rowed accessions, and 183 wild or weed barley (ssp.
spontaneum) from mid east and Morocco. These materials were analyzed with the PCRamplification
followed by restriction
analysis with TaqI, and representative
accessions were
provided to the sequencing analysis. The sequence analysis showed that wild or weed barley has

56
a larger variation
than cultivatedbarley. Two-rowed landraces
had a larger variation
than sixrowed landraces.
The important
point is that there are only two lineagesof six-rowed barley. The
major type had the same DNA sequence with that of one wild barley originatedfrom
Turkmenistan.
This material
was of var. proskowetzii
form showing intermediately
developed
lateralspikeletswith elongatedawns. The other type was minor and originatedfrom some
European countries
and Morocco. This type had the same DNA sequence with that of Moroccan
weed barley discoveredfrom barley field by Molina-Canoet al. (1982).
The divergencetime ofthe two types was calculated
as more than 100 thousand
years, being much longer than thehistoryof barley cultivation,
indicatingthat the two types probably
pre-existed
in wild barley.
This study thereby
showed a diphyletic
origin regarding
six-rowed barley.
H. bulbosum, H. murinum and many other speciesof genus Hordeum have somewhat
developed
lateral spikelets
and the awn of lateral spikelets
are elongatedor at least the lemma are
tip-pointed.
The common progenitor
of barley had been postulated
to have intermediately
developed
lateralspikelets,
because the lateral
spikeletsof H. vulgare ssp. spontaneum are too
small to evolve into forms of six-rowed barley. From these reasons, the most primitive
form ofssp. spontaneum has been postulated
to be similar
to the var. proskowetzii
form (Takahashi
andTomihisa
1970, Bothmer and Jacobsen
1985).
The results obtained
from this study supported
thehypothesis,
because the Turkmenistanmaterial
had lateral spikelets
intermediately
developedwithelongatedawns.
Acknowledgements
The research grant from Science and Technology
gratefully appreciated.

Agency (Japan-Sweden

Bilateral

International

Joint Research)isReferences
Bothmer, R. von, N. Jacobsen. 1985. Origin, taxonomy, and related species. In Rasmusson DC (ed) Barley ASA Agronomy Monograph 26: 19-56.
Bothmer, R. von, N. Jacobsen, C. Baden, R.B. J0rgensen, I. Linde-Laursen. 1995. An ecogeographicalstudy ofthe genus Hordeum. 2nd edition. Systematic and Ecogeographic
Studies on Crop Genepools. 7.
International
Plant Genetic Resources Institute, Rome.
Hernandez, T.J., C.A. Breitenberger, A. Spielmann, E. Stutz. 1993. Cloning and sequencing of a soybean nuclear
gene coding for a chloroplast translation elongation factor EF-G. Biochim. Biophys. Acta 1 174, 191-194.
Komatsuda, T., W.Li, F. Takaiwa, S. Oka. 1999a. High resolution map around the vrsl locus controlling
twoand six-rowed spike in barley, Hordeum vulgare. Genome42:248-253.
Komatsuda, T., K. Tanno, B. Salomon, T. Bryngelsson,
R. von Bothmer. 1999b. Phylogeny in the genus
Hordeumbased on nucleotide sequences closely linked to the vrsl locus (row number of spikelets).
Genome42:973-981.
Mano, Y., K. Takeda. 1998. Genetic resources of salt tolerance in wild Hordeum species. Euphytica 103:137-141.
Molina-Cano, J.L., C. Gomez-Campo,J. Conde. 1982. Hordeum spontaneum C. Koch as a weed of barley firlds
in Morocco. Z. Pflanzenziichtg
88:161-167.
Petersen, G., O. Seberg. 1997. Phylogenetic
analysis of the Triticeae (Poaceae) based on rpoA sequence data.
Molecular Phylogenetics
and Evolution 7: 217'-230.
Pickering, R. 2000. Do the wild relatives of cultivated barley have a place in barley improvement? Barley Genetics
VIII. Proc. 8th Internationalbarley Genetics Symposium, Adelade. 1 :223-230.
Takahashi, R. and Y. Tomihisa. 1970. Genetic approach to the origin of two wild forms of barley, laguncliforme
Bacht. and proskowetzii Nabelek (Hordeum spontaneum C. Koch emend. Bacht.). Barley Genetics ll:5 162.
Tanno, K., S. Taketa, K. Takeda, T. Komatsuda. 2000. Multiple origins of six-rowed cultivated barley as indicatedby a study on a DNA marker closely linked to the vrsl locus (row type gene). Barley Genetics VIII. Proc.
8th Internationalbarley Genetics Symposium, Adelade. 2:62-64.

57

Preservation,
Evaluation and Utilizationof Economically Valuable
Plant Germplasm in Bulgaria - Present and Future Prospect.
InstituteIvan Atanassov, Atanas Atanassov,of Genetic Engineering,
2232 Kostinbrod-2,

BULGARIA

The geographic
situationof Bulgaria has made it one of the "germplasm rich"countries. Although it has relatively
small territory (111,000
km2 of which not morethan 40% arable land) the country is well known for its most diverse climatic and soil
conditions in the Balkan region. This climatic diversity
allows growing of more than
75 cultivatedcrops. The transition
period Bulgaria has passed during the last decade
has influenced negatively the development of its agriculture
and industry. Particularly,
this is true for preservation
and utilizationof Plant Genetic Resources (PGR). Strong
unfavorable trends have been observed in the applicationof the plant breeding andpractical utilizationof PGR: limitationof the number of breeding lines developed andtheir use; insufficientexpertise
and applicationof modern methods for effective
evaluation of the breeding material; diminishing
the practical utilizationof industrial,
medical and rare plants. As a consequence, resistance to environmental stress factors,
such as drought, frost, diseases and pests, salt and acid soils was decreased andproductivity
fell down by an average of 15-20 % in almost all the crops. That is why,building
up and implementationof national program for preservationand efficientutilizationof existing PGR is one of the strategic
directionsof the development ofagriculture science in Bulgaria presently.
I. Current status and problems of preservation,
management and utilizationof
PGR in Bulgaria.
There is one "base" PGR collection in Bulgaria, maintained at the Institute forIntroduction
and Plant Genetic Resources, Sadovo. In addition several active
collectionsof breeding lines and wild relatives of respective crop plants exist in thebreeding and research institutes
in the frame of National Center of Agricultural
Sciences and Bulgarian Academy of Sciences. The main disadvantagesof all thesecollectionsare:(1) lack of appropriate
molecular, and modern cytogenetic and biochemical
characterizationof the accessions;(2) lack of comprehensive information system supporting effective utilizationof thecollection and promoting the international
cooperation;(3) existing collections
are preserved and maintained only by traditional
methods andpreservationof plant and DNA samples at ultra low temperatures is generally not
employed;(4) important medical and rare endemic plants are generally not evaluated andpreserved.
II. PGR-program of the Instituteof Genetic Engineering:targets and directionsof development.
The Institute of Genetic Engineering was establishedat the Agricultural
Academy in1985 as a National Centre for plant cell and molecular genetics research and coordinates
plant biotechnology
activities
in the country. The IGE cooperates andperforms joint research with more than fifty foreign research centers, universities
andcompanies. Taking advantage of its scientificand technologicalcapacity, the IGE
actively
participatesin the work on PGR utilization,and has made particular
contributions
to several strategic areas:

58-molecular, cytogeneticl
and biochemicalevaluationof existing
germplasm
collections
and new accessions: As a result of long lasting efforts for development
and adoption of up-to-date experimental
methods, presently
IGE employs highly
experienced
research staff. Most of the modern experimental
approachesfordevelopment and applicationof molecular markers, and cytogenetic
and biochemical
analyses of plants are routinely used in the laboratories
of IGE. Based on this, IGE
currently increases its involvement in evaluationof existing PGR collections
withmodern molecular methods and provides the respective
expertise to other national
breeding and research institutes.
-establishmentof modern germplasm databank
Possessing
its own, Internet
connected, computer network and a wide experience in experimental data processing,
IGE initiates
the establishmentof modern germplasm databank, accumulating the datafrom molecular, cytogenetic
and biochemicalevaluationsof existing
germplasm
accessions. Further connections to international
PGR-networks and Internet accession
to the information system will be built up.
-improving and expanding PGR collection ofIGE. Supporting the practical utilizationof existing PGR, the IGE established
its own active collectionof breeding lines of
economicallyimportant species. After determinationof phyto-sanitary
status of theaccessions, they are being used for further in vitro multiplicationof planting material.
The main present task related to the active collection
is development of appropriate
molecular markers for proving of accession authenticity.
Except the collection of cropand industrial
plants, IGE has also establisheda unique collectionof medicine andendemic plants, and has developed efficienttissue culture procedures for in vitro
multiplication,
regeneration and culture for most of them. Presently, IGE expands andimproves the collection
through involving new species and samples from various
regions
and performing
initialphysiological,
biochemical
and molecularcharacterizationof accession. The establishmentof respective databank based on theobtained data is in progress.
preservation ofplant and DNAsamples at ultra low temperatures: IGE maintains its
ownultra low temperature collection of DNA samples used in the research work. The
institute will promote expanding of the collection with DNA samples related to PGRutilization.
promoting networks for Plant Genetic Resources for food and agriculture
Serving as a National Center for plant biotech research, IGE has establishedclose
contacts and collaborations
with national breeding and research institutes
and foreign
plant biotech institutes
from Europe, Japan and USA. Based on establishedlinks,
networking capacity and experience, IGE initiates realizationof national and regional
networks for preservation and utilizationof Plant Genetic Resources.
III. Strategic
objectivesof IGE.
IGE has defined now strategic objectives
based on its own strengths and limitation:
1. Enhancing the capacity of national plant genetic resources in order to respondeffectively
to clients' needs and to emerging challenges (genomics and proteomics
programs, etc.)
2. Expanding the global knowledge of plant genetic resources on the development ofproper agriculturalresearch policy in the country.
3. Improving the access to this knowledge and its dissemination.

59

Germplasm Diversity

in its Evolutionary

Joanne A. Labatej,

Steve Kresovicht,

Context:

U.S. Maize as a Case Studyand Kendall

R. LamkeyJ

Nineteenth century U.S. farmers and breeders made a significant
contribution to world
agriculture
by developing
a unique race of maize {Zea mays L.), known as Corn Belt Dent.
Breeding material descended from this race represents virtually
all of the U.S. production, as
well as the bulk of the feed grown in other temperate regions, accounting for more than 50% of
the annual global supply. The Corn Belt Dent racial complex is a collectionof open-pollinatedpopulations
that were developed during the 1 800s by mass selection of material derived fromcrossing two distinct races of maize, the Northern Flints and the Southern Dents (Anderson and
Brown 1952). The two progenitor races have been described to be so different that, relative to thevariation found within the wild grasses, they would be considered different species and possibly
members of different genera (Anderson and Brown 1952). This suggestion was based on
taxonomicallyimportant traits such as inflorescencestructure, internode pattern and proportion,leaf shape, and cytology. Flint maize exhibits traits for adaptation to the cool, moist New
England climate. The typical hard, smooth, and shallow kernels are spaced widely on the ears,
allowing them to ripen earlier, be resistant to moulds, dry out quickly, and withstand early fall
frosts without injury so they can germinate well the following
spring. This is in contrast to dent
types with the seed arranged compactly on the ear, and deep, rough (dented) grain containing
softer starch. On average, flints were discovered to mature relatively
earlier than dents, withdents yielding
more because of the longer growing season (Jones et al. 1924).
The open-pollinatedvarieties that were highly successful in spreading through the U.S.
Corn Belt during its settlement trace back to crosses between flint and dent types. The most
famous example of this is illustrated
by the story of Robert Reid, who serendipitously
crossed a"semi-gourdseed" dent with a "Little Yellow" flint in Illinois
in 1847 to give rise to Reid Yellow
Dent (Troyer 1999).
Open-pollinatedvarieties were rapidly replaced by hybrid cultivars obtained by crossing
highly inbred lines in the 1920s and 1930s. It was difficult to find a farmer's field ofopenpollinatedmaize in the U.S. by the 1940s (Anderson 1944; Jones and Everett 1949). The
potential
narrowing of the germplasm base was immediately
recognized, as it was pointed outthat 6 popular four-way hybrids traced their ancestry back to only two open-pollinatedpopulations(Anderson 1944). The narrowness of the genetic composition in U.S. maize is still aconcern, with the relative proportionof Reid Yellow Dent derived germplasm in modern hybrids
estimated to be 50% (Troyer 1999). It has been asserted that this genetic base must be broadenedin order to decrease the crop's susceptibility
to a variety of risks such as disease epidemics(Committee on Genetic Vulnerabilityof Major Crops 1 972).
Given the breadth of what is currently available in germplasm banks it is impossiblefor
any one breeding program to evaluate all of it for possible incorporation,
to ensure a broad
germplasm base and continued crop improvement. We present a strategy we used for gathering
pertinent information from various sources to describe a set of Corn Belt Dent accessions, withthe long term goal of exploiting
them for novel genetic variation. Our immediate goal is to go| Institutefor Genomic Diversity,
Biotechnology
Bldg., Cornell University,
Ithaca, NY, 148532703. % USDA-ARS, Dept. of Agronomy, Iowa State Univ., Ames, IA, 5001 1-1010.
keywords: maize, population genetics, germplasm conservation

60
beyond a simple molecular genetic characterizationof the material and understand historical,
ecological,
and human influences regarding the origins of the variation discovered within andamong accessions. While highlighting
the findings of our survey of Corn Belt Dent germplasm,
wewill provide an overview of how such data can be processed and point out the electronic
resources that we found to be valuable.
MATERIALS AND METHODS
Wegenotyped 461 plants sampled from a diverse array of Corn Belt Dent-related
germplasm (44 open-pollinatedCorn Belt Dents, 8 Northern Flints, 4 Southern Dents, 1
synthetic,and 12 inbred lines) at 20 microsatellite
loci widely distributed
throughout the
genome. Allele frequency data were used for i) estimating
descriptive
statistics
and F-statistics
using GDA v. 1.0 software (Lewis and Zaykin 1999). i ) constructing
a consensus neighborjoining
tree (Saitou and Nei 1987) using PHYLIP v 3.57c software (Felsenstein1995) and, iii)
Mantel tests (Mantel 1967) of the null hypothesisof independence
between genetic distance andgeographic
distance using GENEPOP v 3. Id software (Raymond and Rousset 1995).
RESULTS AND DISCUSSION
Most molecular population genetics data sets are initiallyanalyzed to describe genetic
variation in terms of diversity
measures and genetic distances, population
structure andclustering
patterns, and Hardy-Weinberg and multilocus (linkage)equilibrium.
Many software
programs to carry out such analyses have been developed for personal computers. Their
implementationof powerful statisticaltechniques
and user-friendlinessmake them an attractive
alternative
to performing calculations
on spreadsheets or by writing simpler programs foroneself. One considerationin selectinga software program is the type of molecular genetic
marker that was assayed. Markers can be described as dominant (e.g., RAPDs) or codominant(e.g., microsatellites),and data may be collected in the form ofhaplotypes
or diploid genotypes.
The programs TFPGA (Miller
1997), Arlequin (Schneider
et al. 1997), GDA (Lewis and Zaykin1999), GENEPOP (Raymond and Rousset 1995), GeneStrut (Constantine
et al. 1994), andPOPGENE (Yeh and Boyle 1997) can all accommodate a variety of marker types, are freely
available
for downloading from the Web, and were recently reviewed and compared to eachother (Labate 2000). An excellent program for clustering
analysis is PHYLIP (Felsenstein1995).
Examining population structure in the Corn Belt Dent germplasm by F-statistics,
we
discovered that the majority of variation (85%) was shared among open-pollinatedaccessions.
Cluster analysis showed strong support (1 00%) for the separation of flint from dent germplasm
in a neighbor-joining
tree (Saitou and Nei 1987). The divergence between flint and dent did not
result from uniquely defining alleles in each cluster, but rather, the flints contained a subset(90/130)of the total alleles found in the dents. Many of the accessions were apriori known to beclosely related to each other. Some were different strains of the same variety, or in some cases avariety was purported to be have been selected from another variety. Clustering
analysisfrequently
supported known relationships,
but overall the accessions were so closely related thatclusters of varieties became increasingly
less distinct
as more strains were added to the analyses.
The Germplasm Resources Information Network (GRIN) web server (http://www.arsgrin.gov/)
provides information for approximately
10,000 genera and 435,000 accessions in the
National Plant Germplasm System. Many quantitativedescriptive
measures are available foraccessions. For maize they fall into a range of categories,
such as, chemical composition,diseaseand insect resistance,
phenology, morphology, growth habit, quality, and productivity.
The61
accessions that we genotyped originatedfrom a.broad geographicalarea across the U.S., from
northern NewJersey to western Nebraska, and from northern Michigan to southeastern Kansas.
Wehypothesizedthat geographicalorigin of an accession should be correlated with its relative
time to maturity, and downloaded pollen shed data (number of days from planting to when 50%
of observed plants have shed pollen) from GRIN for all accessions in our study for which it wasavailable(27 accessions).
All of these data were collected for the LAMP (Latin American Maize
Project)
study 10 01 in Ames, Iowa in 1987 (USDA, ARS 2000) so could be directly comparedto each other. A significant
correlation(r = 0.7624, d.f. = 25, P < 0.01) between days to pollen
shed and maturity zone of the strain's geographicalorigin was found, reflecting
a north/southcline in relative maturities.
This finding implied that there are still genetic differencesamong theaccessions that reflect their original
adaptation to local growing conditions.
We postulated thatthree forces have primarily
shaped allele frequencies in the accessions i) pedigree,ii) migration
into the Midwest Corn Belt from the east coast, and iii) selection
by farmers for local adaptation.
Because clustering analysis had revealed that genetic compositionsof the accessions had been
substantiallyinfluenced
by pedigree, we selected 1 1 accessions
that were believed to be fairly
distinctfrom each other in their pedigrees to look for evidence of migration and selection.
Historicalaccounts provided by 1 9th and early 20th century state agriculturalexperiment station
bulletinswere a valuable resource in providing
approximate year and geographicalorigin ofvarieties. Many of these accounts gave direct testimony to gene flow, often the crossing of
pioneer introduced seed with a local strain of maize to obtain a superior and subsequently
improved variety. Influence of migration on allele frequencies should be evidenced by a test for
isolation
by geographicaldistance. Precise estimates of geographic
distance between all pairs ofaccessions were obtained using two Web-based tools. The first, the Geographic Names
Information System, can provide information on 2 million physical and cultural geographic
features in the U.S. The Federally recognized name of each feature and the feature's location by
State, county, and geographic
coordinates can be obtained (information
on 3.5 million
geographic names outside of the U.S. and Antarctica can be obtained from the GEOnet Names
Server, http://l64.2 14.2.59/gns/html/index.html).
The second essential tool to obtain data for theisolation-by-distancetests was the "Surface Distance Between Two Points of Latitude andLongitude" (http://www.vsv.slu.se/johnb/java/lat-long.htm).
This was used to compute all
pairwise distances between places of origin of accessions in units of nautical miles. A Mantel test
(Mantel 1967) was used to test the null hypothesisof independence between two semimatrices,
one containing the 55 pairwise genetic distances (FStI(\-Fst))
and the other containing the 55
pairwise nautical miles distances. Statisticallysignificant
correlations
were found between
genetic distance and geographicaldistance (r = 0.5219, P = 0.03). The data displayed
evidenceof north to south rather than east to west genetic differentiation
because the flint accessions (allbeing from the north) were geneticallysimilar but geographicallydistant from each other fromeast to west. When flint accessions were removed from the analysis the significant
correlation
between genetic and geographicaldistance disappeared(r = 0.2 167, P = 0.28).
Therefore, the microsatellite
data did not reveal adaptive genetic differentiationof thevarieties from north to south, although it was inferred to be present from pollen shed data.
Historicalorigins of the accessions, in terms of the flint/dent dichotomy and more recent
pedigree relationships,
dominated the profile of the molecular genetic composition of the Corn
Belt Dent open-pollinatedpopulations.
This was not unexpected because selection for local
adaptation will have acted in specific ways on relatively
small genomic regions while history anddemography influenced the entire genome in similar ways. Genotyping of accessions using

62
densely spaced, highly polymorphic
genetic markers will be necessary
involved
in local adaptation.
These results provide a framework withinand study of the Corn Belt Dent populations
can be carried out.

to reveal genomic regions
which further sampling

REFERENCES
Anderson, E. 1944. Sources of effective germplasm in hybrid maize. Ann. Mo. Bot. Gard.
31:355-361.
Anderson, E., and W.L. Brown. 1952. Origin of Corn Belt maize and its genetic significance,p.
124-148.
In J.W. Gowen (ed.) Heterosis.
Iowa State College,Ames, IA.
Committee on Genetic Vulnerabilityof Major Crops. 1972. Genetic Vulnerabilityof Major
Crops. NAS, Washington,
DC.
Constantine,
C.C., R.P. Hobbs, and AJ. Lymbery. 1994. FORTRAN programs for analyzing
population
structure from multilocus
genotypic
data. J. Hered. 85:336-337.
Distributedby theauthor at http ://wwwvet. murdoch. edu. au/vetschl/imgad/GenStrut.htm.
Felsenstein,J. 1995. PHYLIP Version 3.5c. Genetics Dept, Universityof Washington,
Seattle,WA.Distributedby the author at http ://ibbrain.
ibb.waw.pl/docs/PHYLIPdoc/index.html.
Jones, D.F., and H.L. Everett. 1949. Hybrid field corn. Connecticut Agric. Exp. Stn. Bull. 532.
Jones, D.F., W.L. Slate, and B.A. Brown. 1924. Corn in Connecticut.
Connecticut
Agric. Exp.
Stn. Bull. 259.
Labate, J.A. 2000. Software for population
genetic analyses of molecular marker data. Crop Sci.
inpress.
Lewis, P.O., and D. Zaykin. 1999. Genetic data analysis.
Computer program for the analysis ofallelic
data. Version 1.0 (dl2).
Dept. of Ecology and Evolutionary
Biology,
The Universityof Connecticut,
Storrs, CT. Distributedby the authors at http://alleyn.eeb.uconn.edu/gda.
Mantel, N. 1967. The detection of disease clusteringand a generalized
regression
approach.
Cancer Res. 27:209-220.
Miller, M.P. 1997. Tools for population
genetic analysis (TFPGA)
1.3: A Windows program forthe analysis of allozyme and molecular population
genetic data. Biology Department,
Arizona State University,
Tempe, AZ. Distributedby the author at
http ://www.public.
asu. edu/~mmille8.
Raymond, M., and F. Rousset. 1995. GENEPOP (version 1.2): Population
genetics software forexact tests and ecumenicism. The J. ofHered. 86:248-250.
Distributedby the authors at
http ://www. cefe. cnrs-mop. fr.
Saitou, N., and M. Nei. 1987. The neighbor-joining
method: A new method for reconstructing
phylogenetic
trees. Mol. Biol. Evol. 4:406-425.
Schneider,S., J.-M. Kueffer, D. Roessli, and L. Excoffier. 1997. Arlequin ver. 1.1: A software
for population
genetic data analysis.
Genetics and Biometry Laboratory, UniversityofGeneva, Switzerland.
Distributedby the authors at http ://anthropologie.unige.ch/arlequin.
Troyer, A.F. 1999. Background ofU.S. Hybrid Corn. Crop Sci. 39:601-626.
USDA, ARS. 2000. National Genetic Resources Program. Germplasm Resources Information
Network - (GRIN). Lamp evaluation in Ames, Iowa in 1987. [Online
Database]
National
Germplasm Resources Laboratory,
Beltsville,
Maryland. Available:
http://www.arsgrin.gov/cgi-bin/npgs/html/eval.pl?49
1 3 02 (09 October 2000)
Yeh, F.C. and Boyle, T.J.B. 1997. Population
genetic analysis of co-dominant and dominantmarkers and quantitative
traits. Belgian Journal of Botany 129: 1 57. Distributedby theauthors at http ://www.ualberta.
ca/~fyeh/index.htm.

63

DiversityLimof Garcinia

spp. and Interspecies
Analyses

Sompong Te-chato , Mongkol

Relationships

by DNAand Mii Masahiro2

department
of Plant Science, Faculty of Natural Resources, Prince of Songkla
University,
Hat Yai, Thailand, 90112
2Laboratory of Plant Cell Technology, Faculty of Horticulture,
Chiba University,
648
Matsudo-city, Matsudo, Japan, 271

Corresponding

author: tesompon@ratree.

psu.ac.th

Abstract
Floraland fruit morphology
of Garcinia spp. have some differencesandsimilarities
in number, shape and color. Among those species, mangosteen contains a
concentrated yellow latex affecting the quality of its fruits. In case of pollen viability,
itis interestingthat a high number, 93-100%, of viable pollen were found in cha-muangand pawa. Ploidy level study of the species by flow cytometry revealed that pawa havethe smallest DNA content while the other three species, (mangosteen, somkhag and madun) have a DNA content two times higher. Ma-phut has the highest DNA content,
which was three times higher than that of pawa. From sequencing of genomic DNA
using specific primer, it was found that mangosteen had a close relation with pawa,followed by somkhag and ma-phut.

Introduction
Garcinia belongs to the family Guttiferaeor Clusiaceae.
The genus arose insoutheast Asia, ranging from the southern part of Thailand and peninsular Malaysia to
Indonesia and to some parts of the Philippines.
However, the exact origin of the genus isstill debated. According to Whitmore (1973), the genus is listed as having 39 species withanother ten unidentified
or unnamed in this region. Among these species, only a few are
known and used in daily life in Thailand (Lim et ai, 1986), namely G. mangostana L., now
being grown as an orchard fruit tree, G. atroviridis
Griff, G. speciosa Wall., G. cowaRoxb.
and G. dulcis Kurz. The latter four species possess both medicinaland horticulturalproperties.
G. atroviridis
Griff, G. dulcis Kurz and G. cowa Roxb contain a highconcentration of hydroxy citric acid (HCA), especiallyin the fruit and leaf. This chemical
regulates some enzymes in the hydrolysis
pathway in order to efficiently
convert fat or fatty
acid into energy. G. speciosa Wall, can grow well under drought conditions,
so it is used asa rootstock for mass propagation of mangosteen.
Garcinia spp. have very small chromosomes. Most cytology
researchers haveencountered difficultyin identifying
distinctdifferencesamong the species. Metaphase
chromosomes spread well but staining
is poor leading to difficultyin counting andidentifying.
Richards (1990)
reported using morphologicalcharacters for identification
among the three species of Garcinia. He showed that four characters; latex and petal color,sessile stigma and fruit color of G. mangostana resemble G. malacensis. Another three64
characters, stigma, stamen/staminodelobe, and fruit show that G. mangostana resembles G.
hombroniana. Based on these similarities
he stated that G. mangostana is a hybrid between
G. hombroniana and G. malaccensis.
Classifying
the relationshipamong these species by biotechnologicalmethod has
not been conducted. All plant parts contain a high content of yellow latex which hampers
the extractionof good quality
DNA. Before further deteriorationof the forest causes
extiction of these species, it is prudent to classify and collect them for further improvementof the species, especiallymangosteen.

Materials and Methods
Comparison study on floral and fruit morphology of Garcinia

spp.

In this investigation,
five speciesof Garcinia,
namely G mangostana L.
(mangosteen),
G. speciosa Wall (pawa), G. atroviridis
Griff (somkhag),G. dulcis Kurz
(ma-phut),
G. cowa Roxb (cha-muang) and G. schomburgkiana(madun) were collectedfrom the Plant Science Research Station, Prince of Songkla University
campus, and privateorchards in the Narathiwat Province. Both vegetative parts (leaves)and reproductiveparts(flowers,
fruits and seeds) were collected and used. Seed germination tests were conductedin both nursery bed and test tube.

Nucleus preparation

for flow-cytometry:

Leaf samples collected from field-grown mature mangosteen, pawa, somkhag, maphut and madun trees were cleaned by rinsing under running tap water. A small sample ofleaves (approximate
1 mm2) was chopped into DAPI or Partec buffer solutions under cool
condition (usually
carried out on ice). The filtrate was then fed through a flow cytometerand the content of DNA in the nucleus determined.

DNA extraction

and phylogenetic

study

The surface of young leaves of all the Garcinia spp. samples were rinsed withtap water many times to remove surface contamination. Approximately
20 mg of clean
leaves were put in a 2 ml Eppendorf tube and crushed in 150 |HL tris-EDTA (TE) buffer
and DNA extraction was carried out by minipreparation
method.
The DNA isolated from leaf sample of each species of Garcinia according to
the methods describedearlier was amplified by the polymerase chain reaction (PCR)
technique with specific primer localizedin chloroplst.
Two set of primers; rbcL andpsbA, each consistingof forward (FWD) and reverse (RVS) direction,
were employed:
rZ?cL-FWD (5 '-TTGGCAGCATTCCGAGTAA-3 ' ), rbcL-RVS (5 ' -TGTCCTAAAGTTCCTCCAC-3'),
psbA-FWD (5' -CCATGACTGCAATTTTAGAG-3')
and psbA-RVS(5'-ACTTCCATACCAAGGTTAGC-3').
The first set of primers was designed toamplify a 1 1 18 bp fragment, encompassing 78% of the Rubisco large subunit (rbc L)
sequence of tobacco (Shinozaki
et al, 1986). The second set was designed to amplify a
991 bp fragment, including
93% of photosystem II Dl protein gene (psbA).

Results
Comparison study on floral and fruit morphology

of Garcinia

spp.

Reproductive
phase and morphologicalcharacteristicsof the genus Garcinia,
including floral and fruit morphology are summarized in Table 1

65

Table

1. Morphological

Nucleus preparation

charactersof Garcinia

spp.

for flow-cytometry

The results from flow-cytometry revealed that DNA content varied from species tospecies. Among the five species tested, pawa had the lowest content of DNA classified tobe diploid (2n=2x). Three species (mangosteen, somkhag and ma-dun) had a DNA content
two times higher than that of pawa and were classified to be tetraploid(2n=4x). Ma-phut
had the highest DNA content, three times higher than that of pawa, and thus was classified
to be hexaploid (2n=6x) (Fig. 1).

Phylogenetic

study

From the resultclose to pawa, followedof genomic sequencing, it is quite clear that mangosteen was very
by somkhag, ma-phut and ma-dun (Fig 2).

Reference s
Lim, M., Hemapat, T., Wanachit, W. and Suwankiri, P. 1986. Survey and collectionofLansium and Garcinia in Southern Thailand. EBPGR Newsletter
10: 10- 12.
Richards,
A. J. 1990. Studies in Garcinia,
dioecious
tropicalforest trees: the phenology,
pollination
biologyand fertilizationof G hombroniana Pierre. Botanical
Journal of
the Linnean Society 103:25 1-256.
Shinozaki,
K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida,N., Matsubayashi,T.,
Zaita, N., Chunwongse, J., Obokata, J., Yamagushi,
H., Shinosaki,K., Ohto, C,
Torazawa, K., Meng, B.Y., Sugita, M., Deno, H., Kamogashira,T., Yamada, K.,
Kusada, J., Takaiwa, R, Kato, A., Tohdoh, N., Shimada, H., Sugiura, M. 1986. The
complete nucleotide
sequence of tobacco chloroplast
genome: its gene organizationand expression.
EMBO J. 5:2043-2049.
Te-chato, S. 1997. Isolationand culture of protoplastsof somkhag {Garcinia
atroviridis
Griff.) to microcolony.
Songklanakarin
J. Sci. Technol. 19:255-262.

66
Te-chato,
S. 1998. Isolationand culture
protoplastsof mesophyll
protoplastsofmangosteen. Songklanakarin
J. Sci. Technol. 20: 15-20.
Te-chato, S. and Aengyong, W. 1988. Microplant
propagationof mangosteen (Garcinia
mangostana). Songklanakarin J. Sci. Technol. 10:7-1 1.
Te-chato, S. and Lim, M. 1999a. Improvement of mangosteen micropropagation
throughmeristematic
nodular callus formation from in vzYra-derived leaf explant. Scientia
Horticulturae(in press).
Te-chato, S. and Lim, M. 1999b. Plant regenerationof mangosteen via nodular callus
formation. Plant Cell, Tissue and Organ Culture. 59:89-93.
Te-chato, S. Lim, M. and Chumroenruk, M. 1995. Grafting vitro-shootof mangosteen on
rootstock of pawa ex vitro. Songklanakarin
J. Sci. Technol. 17:245-250.
Te-chato, S., Lim, S. and Kulsiroratana,J. 1992a. In vitro micrograftingof mangosteen.
Songklanakarin
J. Sci. Technol. 14:353-359.
Te-chato,
S., Lim, M. and Muangkaewngam, A. 1992b.
Enhanceefficiency
micropropagationof mangosteen through young leaf culture. Songklanakarin
J. Sci.
TechnoL 14:1-7.
Whitmore, T. C. 1973. Garcinia L. In Tree flora of Malaya (ed. T.C. Whitmore) vol. 2, pp
196-225.
Kuala Lumpur: Forest Department,
Ministryof Primary Industries,
Malaysia.

Fig. 1. DNA contents of some species

in Garcinia

genusFig 2 Genetic

relationshipamong Garcinia

spp.

69

Population
strategies

analyses using different molecular techniques to determine
for in-situ conservation - examples from the genera Glycine,
Oryza and Vigna.

Duncan Vaughan, N. Tomooka, A. Kaga, A. Shcherban1, R.Q. Xu2, Y. Jinno3, K. Doi, K.
Kashiwaba NIAR, Tsukuba, Japan;Currently,!Institute of Genetics and Cytology, Novosibirsk,
Russia, tobacco and Health Research Institute, Kentucky, USA, 3Hokkaido PGR Center, JapanThe objectives of this paper are to briefly describe the results of population research thatprovide various types of information on inter and intra-specific variation in wild relatives ofcrops that may assist in developing strategies for in-situ conservation. In this paper we present
results from using 3 types of molecular information to analyse population diversity,
RAPD
diversity of island populations of Glycine tabacina, Vigna reflexo-pilosa and Vigna riukiuensis,
AFLP analysis of the Vigna angularis complex in Japan and sequence data for part of theintegrase coding domain of a gypsy-like retrotransposon found in the Oryza qfficinalis complex.
1. Island populations.
The biology of island populations
has generated much research interest. The results ofstudies of three species that grow on the islands of southern Okinawa, Japan, are presented.
Glycine tabacina is a perennial herbaceous plant with its center of diversity in Australia, but
populations of this species are found over much of the southern Pacific and it grows in Taiwan
and mainland, China and Japan. Miyako island, Japan, is the most northerly location of thisspecies. G. tabacina on Miyako island grows primarily in grass on cliff tops above the sea.
Populations of this species were analysed by RAPD and AFLP methodologies. The results of
RAPD analysis are shown (Fig. 1). Based on 12 primers only one polymorphic band was foundamongMiyako island populations out of a total of 156 polymorphic bands when populations of
G. tabacina from Australia were included in the analysis. Similar results were obtained with
AFLP analysis. These results suggest that populationsof G. tabacina on Miyako island are theresult of founder effect and possibly recent introduction.
Vigna riukiuensis has a rather restricted distribution
in southern Okinawa and Taiwan.
Analysis of populationsof V riukiuensis from 5 islands of the Yaeyama and Miyako islandarchipelagoes revealed clear island to island variation based on both RAPD and AFLP analysis.
The results suggest that there is a floristic barrier between the Miyako and Yaeyama archipelagos.
It is possible that V. riukiuensis is an ancient element of the flora of these islands.
Vigna reflexo-pilosa
is a climbing,herbaceous, tetraploid,
perennial that grows
throughout the islands of southern Okinawa, Japan. The full range of its distribution
is as farsouth as northern Australia and east as far as Myanmar. Southern Okinawa represents its northern
distribution
limit. Analysis of 5 populationsof V. reflexo-pilosa from 5 islands of southern
Okinawa revealed very little variation based on RAPD and AFLP analyses. The intra-specificgenetic diversity based on AFLP data measured by the Shannons diversity index was 0.006 for
V.reflexo-pilosa compared to 0.039 for V. riukiuensis growing over the same islands of southern
Okinawa (Yoon et al. 2000).
The results of analysis of these island populations
suggest that the in-situ conservation
strategy for Glycine tabacina and V. reflexo-pilosa may be similar. That one or two populations
may capture the majority of the genetic variation of these species in southern Okinawa. On theother hand since V. riukiuensis shows genetic differentiation
thus specific populations in both
the Mikako and Yaeyama archipelagos would be need to be conserved to capture the full genetic
diversity of this species.
2. Crop complex populations.
Crop complexes consistingof related wild, weed and cultivatedtaxa are particularly

70interesting to study since they occur in disturbed habitats that can speed up processes of evolution
and enable these processes to be better understood (Harper, 1977). In Japan the azuki bean
complex consists of the cultigen,
Vigna angularis var angularis and its wild relative var.
nipponensis. In addition, to these two varieties, field observations and laboratory analyses havealso recognised weedy and complex populations (Fig. 2) (Xu et al. 2000 a, b). AFLP analyses
of wild and weedy populations and the cultigen from different parts of Japan have shown thatweedy populations have much less intra-population
genetic variation than wild populations andare geneticallyclosely related to the cultigen. Wild populations have greater intra-population
genetic variation than weed populations and the cultigen and wild populations
analysed from
northern and southern Japan are geneticallydifferentiated(Fig. 3).
Several years of monitoring populations
in various parts of Japan have shown that
populations
vary from year to year as a result of various factors such as cutting vegetation by
farmers. Four years of observations of a complex population in Tottori prefecture have shownthat farmers rotate yearly the nearby fields they plant to azuki beans. Plants in different parts ofthe complex population there show evidence of segregation from one year to the next suggesting
gene flow from the cultigen to neighbouring
wild and/or weedy plants.
Aspects to consider when developing strategies to maintain in-situ the dynamic population
structure of crop complex genepools include:
1. Disturbancethat prevents succession; 2. Breeding system(s)and pollinators;
3. Size of
population and population structure.
Ex-situ conservation of seed bearing crops and there wild and weedy relatives is usually
not difficult. However, conservation of germplasm in habitats to maintain micro-evolutionary
processes requires in-situ conservation (Li et al. 2000).
3. Species complex populations.
The Oryza officinalis complex consisting of 10 species and is the largest species complex
in the genus Orzya. This complex with a pantropical distribution
is of particular interest because
4 genomes have been designated for species that occur in this complex (Vaughan, 1994). Our
research objectives were to better understand the evolution and relationshipsof the species in thiscomplex by analysis of all the species and their major geographic groups.
Wesequenced clones fromwithin a new gypsy-like retrotransposon, originallyfound inO. eichingeri,that gave intense hybridization
signals with all species of the O. officinalis
complex (Shcherban et al., 2000). Cloning and sequencing showed that amplified fragments werehighly homologous to each other (75-90%). Analysis of primary sequence data supports previous
reports that vertical evolution has been the major influence on the evolution of gypsy-likeretrotransposons
within Oryza species. Thus we used the primary sequence data to estimate
divergence times of species in the O. officinalis complex. Based on the molecular clock concept
the divergence time between species is inversely proportional
to the nucleotide substitutions
which varies between different genomic regions. We used the average of two reported rates ofsubstitution
to estimate average divergence time between accessions of the O. officinalis complex(Fig. 4). Based on a comparison of sequence data the 3 main species groups separated at about
the same time in the Miocene (25-1 1 mya). This range of dates for the major divergence among
0. officinalis complex species is supported by information from general studies of grass
phylogeny and divergence estimates fromelectrophoresis
studies.
The implicationof the results for in-situ conservation of the species in the Oryza
officinalis complex are:
1. Oryza eichingeri appears to be ancestral to other CC genome species and thus this species
should be a focus of conservation efforts. Further studies of this species are necessary,particularly
in Sri Lanka the only place where it grows close to another CC genome species O.
rhizomatis. The ancestral form of O. eichingeri
was the likely the CC genome donor to theallotetraploid
species of the complex.
2. O. officinalis has distinct genetic variation in Southeast Asia, China and India. Therefore
populations
from these different regions should be conserved in-situ.
Genetic studies of island populations,a crop complex and species complex using various
molecular markers and sequence data can provide useful information for the in-situ conservationof crop relatives. However, while techniques to analyse populations
can give new information
equally critical is knowledge and quality of the genetic resources analysed.
References
Harper, J.L. 1977 The population biology of plants. Academic Press, London. 892 pages.
Li, Y.-C, M.S.Roder, T. Fahima, V.M.Kirzhner, A. Beiles, A. B. Korol, E.Nevo. 2000. Natural
selection
causing microsatellite
divergence in wild emmer wheat at the ecologicalvariable microsite at Ammiad, Israel. Theor. Appl. Genet. 100:985-999
Shcherban, A., D.A.Vaughan and N. Tomooka. 2000. Isolation of a new retrotransposon-likeDNA sequence and its use in analysis of diversity within the Oryza officinalis complex.
Genetica (In press)
Vaughan, D. A. 1994. The wild relativesof rice: A genetic resources handbook. IRRI, Los
Banos, Philippines.
137 pages
Xu, R.Q., N.Tomooka, and D.A.Vaughan. 2000a. AFLP markers for characterizing
the azuki
bean complex. Crop Sci. 40: 808-815
Xu, R.Q., N. Tomooka and D.A.Vaughan and K. Doi. 2000b. The Vigna angularis complex:
genetic variation and relationships
revealed by RAPD analysis, and their implicationforin-situ conservation and domestication.
Genet. Res. Crop Evol. 47: 123- 1 34
Yoon, M.S., K. Doi, A. Kaga, N. Tomooka and D.A.Vaughan. 2000. Analysis of the genetic
diversity in the Vigna minima complex and related species in East Asia. J. Plant Sci. (Inpress)

Fig. 1.(above)
Dendrogram based on Jaccards coefficient
calculated
from RAPD polymorphism detected in Japaneseand Australian populationsof Glycine tabacina (tab) and G.
tomentella (torn).
Fig. 2. (right)Distribution
in Japan of wild O, weedy •Eandcomplex 3 populations based on direct collection.

Fig. 3. Principal Component Analysis plot of 41 populations
belonging to the azuki bean species
complex. The wild, weedy and cultigen populations are marked with '•œ'•›'•£', respectively.
All but one wild populationin quadrates A and B were from northern or southwestern Japan,
respectively.(Modified
from Xu et al. 2000a)Fig. 4. An UPGMA dendrogram of the O. officinalis complex species based on ICD sequencealignment. The nucleotide substitution
numbers are indicated in bold above branches. Numbers
at each internode represent the deduced average time of species divergence in millions of years.

73

Howto control biodiversityof crops by means of biotechnology?
Some approaches to overcoming breeding barriers.
Taiji

ADACffl1},Lanzhuang CHEN2), Sun Hee WOO3), Jotaro All and Mio NAGANOAppliedGenetics and Biotechnology
University,
Gakuen Kibanadai

Key words: breeding

barrier,

Division, Faculty of Agriculture,
MiyazakiNishi 1 -1, Miyazaki 889-2192, Japan

heterostyly,

apomixis,

buckwheat,guineagrass

Introduction
It is true that the conventional
types of plant breeding
have had remarkablesuccess in improving a wide range of crop traits. The crossing of plant forms produces
the recombinationof variation
among genotypes, strains and species. It is still often
desirable,
however, to make sexual crosses between a wild and a crop species that aresexually incompatible,
which means that the cross is not possible
and the germplasm
flow is impeded.
The inability
to undergo normal sexual reproduction
is called a"breedingbarrier".
Therefore, a breeding barrier comes from the fact of reproductive
isolation(Stebbins, 1958).
For a long time great effort and various approaches for overcoming such barriers
have been made by several kinds of techniques,e.g. immature embryo rescue andaseptic culture. Haploid plant production
using pollen and anther culture techniques
opened new avenues in the 1960s.
The success of future plant breeding
programs will focus on the utilizationof"new" biotechnologicaladvances including
genetic engineeringand somatic hybridization. In order to break through the breeding
barriersby means of biotechnology,
there are many factors to be considered.
Success will also depend upon a more detailed
knowledge of reproductive
biologyand breeding systems. From such aspect of views
weare devoting ourselves as the following investigations.
Development and analysis of homostylar
autogamous buckwheat
Programs aimed at buckwheat breeding
and genetic improvement is further
fraught with numerous problems. Prominent among them is the self-incompatibility
phenomenon peculiar to the reproductive
biology of this seed propagated genus. Due toallogamy,
the individuals
in a population
are highly
heterogeneous.
The selfincompatible,
heterostylic
system is a limitation
for breeding
improvement.
Toovercome this problem, remote hybridization
has been attempted by crossing with amonomorphic self-compatible
wild species, F. homotropicum (Campbell,
1995; Wooand Adachi, 1997). The hybrids were successfully
produced through embryo rescue andforwardedto successive
generations
by selfing
as well as back crossing,i.e.
introgressive
hybridization.
The scheme is shown in Fig. 1. Further analyses revealed
the heterostylism
is governedby a single locus S, with three alleles, S, S and s, which
Present addresses1} Laboratoryof Plant Genes and Physiology,
Graduate School of AgricultureandBilogical
Sciences, Osaka Prefecture University.
1-1, Gakuen-Cho, Sakai, 599-853 1
2) Gene Research Center, Miyazaki University.
Gakuen Kibanadai Nishi 1-1, Miyazaki,
889-2192.
3) Kihara Institutefor BiologicalResearch, Yokohama City University,64
1 -1 2,Maiokacho, Totsuka, Yokohama, 244-08 13

respectively
control the F. homotropicum homo-type Sh Sh , F. esculentum (thrum) Ss
and pin ss. Back crossing is being extended for further
generations in order to develop
a stable population
with introgressionof the gene(s) from F. homotropicum
Three
RAPD markers,
OPB1250,

OPP81000

and

GPQ7800,

tightly
linkedto Sh genewere
cloned and characterized.
Nucleotide sequence information,
obtainedfrom each end of thethreelinkedRAPD markers, was used to design
21-mer oligonucleotide
primers for
PCR amplificationof the respective
SCARs. Two of these SCAR primers,
SCB141250 and SCP81000 amplifiedasingle fragment in F. homotropicum
but was absent in F. esculentum. In
one case, SCQ7800, showed
differentsize
PCR fragments
incommonbuckwheat than in F. homotropicum.
This co-dominant marker is useful fordifferentiating
heterozygosity
fromboth types of homozygote (Fig. 2).

Fig. 2.
genomic
amplifiedprimers.

Fig. 1. Scheme
bridization.

of genetic

analysisand introgressivehy-

Agarose gel electrophoresisof SCAR markers converted from RAPD markers. A: PCR products amplified from
DNAs from #284 (ss), #1 (ShSk) and their F2 segrigants(ShSh, Shs, ss) using SCAR primers. B: PCR products
from genomic DNAs from Miyazakizairai(MZ; ss), #1 (ShSh) and their F2 segrigants(SHSh, Shs, fusing
SCAR
The SCAR primers used are indicatedabove the each lane.

Identificationof apomictic
gene in Gulneagrass
Apomlxls Is a mode of reproduction,found In a number of diverse plant species,that bypasses female melosls and syngamy to produce embryos geneticallyIdenticaltothe maternal parent(Asker
and Jeriing, 1992). It causes sometimes, therefore, difficulty
to get recombination
by sexual reproduction.
But apomixis provides
a method forcloning plants through seed and involves the conservation of existing hetferozygosity,itis expected to simplify
the developmentof hybrid cultivars
and the productionofcommercial hybrid seed.
The mechanistic
details and the mode of inheritance of apospory have been studiedin guineagrass(P#«/otfw
maximum). Based on ovary length,
for sampling
different
developmental
stages of embryo sac formation in obligate
and facultative
apomictic
genotypes, a CDNA library was made and a stage-specific
CDNA clone was isolated.
This A2-134 CDNA, designatedASG-1 (Apomixis
SpecificGene),
was found to be
expressed in flower buds of the apomictic but not of the sexual accession. Furthermore,
the gene was also not expressedin buds of the apomicts
until completionofmegasporogenesis
but could only be found during a phase characterized
by theappearance of aposporous initialcells (AICs)of the embryo sac. The appearance of
these cells is strictly limited to apomictic genotypes (Fig. 3).
Sequence analysis revealed that
theA2-134
CDNA (l,177bp)
codes for a protein of 305 amino
acids
with
amolecularmassof34.2kDa.
The amino acidsequence of A2-134
is related
to RD223 a seed-specificand
drought-induced
gene ofArabidopsis thaliana( YamaguchiShinozaki,
1993; Kiyosue et al.,1994),toUSP,
an unknown
seed prote in precursor of Vicia
faba (Baumlein et al., 1991), toa polygalacturonase
1 betachain
precursor(Polyg
1) of
Lycope rsic on
esculentum(Zhengetal.,
1992)
and toADR65 an aexin down-regulated
gene ofGlyctne
max( Datta etal., 1993). Southern blot analysis
showed a different hybridization
pattern atthegenomic
level
when A2-134
CDNA washybridized
withtotal DNAs
isolatedfrom leaves of sexualaswell as apornictic
plants,indicating
thatthe gene(or related
Fig. 3. A) Northernblot of a CDNA done (A2-134)
Isolatedfromfamily members) exists
in bothstage A2 flower buds of apomictlc
guinea grass. At this stage (A2)types. The CDNA library and the
AICs appear in apornictic
but not in sexual genotypes. The hybridization signal corresponds
to this stage and is not visible
during megASG-1 gene should be valuable
aspore (AS1)or sexual embryo sac formation(S2).
C2-112is a
tools
toward understanding
the
CDNA clone found in both genotypes
at stages A2 and S25 respectively, and is used here as a control for clone specificity(B). Ubiquimolecularmechanism of apotously expressed clones hybridize
with AS 1 under the experimental
mixis, particularly
apospory,
conditions
used, demonstrating
sufficient
RNA in lane AS 1 (datanot shown).
and toward thetransfer
of apomixis to important crops.

76
Conclusions
The complementary
applicationof conventional
but standard breeding
techniquesand modern biotechnology
tools in genetic
improvement
program appears to beindispensablefor the achievementof effective
breeding goals in different
plant species.
References
Aii, I, M. Nagano, S.H. Woo, C. Campbell
and T. Adachi : Developmentof the SCAR
markers linked to the ^gene in buckwheat. Fagopyrum 16. 19-22 (1999).
Asker, S. E. and L. Jerling : Apomixis
inPlants
CRC Press, Boca Raton (1992).
Campbell,C. : Inter-specifichybridizationin the genus Fagopyrum. Current Adv. in
Buckwheat Research (Proc. 6th Intl. Buckwhet
Symp., Ina in Japan), 255-263
Datta, N., P. R. LaFayette,
P. A.Kroneer, R. T. Nagao, and J. L. Key : Isolation
andcharacterizationof three families
of auxin down-regulated
CDNA clones. Plant Mol.
Biol. 21, 859-869(1993).
Kiyosue, T., K. Yamaguchi-Shinozaki,
and K. Shinozaki:
Cloning of cDNAs for genesthat are early-responsive
to dehydrationstress (ERDs)
in Arabidopsis
thaliana L. :Identification
ofthreeERDs
asHSP cognategenes.
Plant
Mol. Biol. 25, 791798(1994).
Nair, A., T. Ohmoto, S.H. Woo, and T. Adachi : Amolecular-genetic
approachforhypoallergenic
buckwheat. Fagopyrum 16, 29-36 (1999).
Stebbins,
G.L., Jr. : The inviability,
weakness, and sterility ofinterspecifichybrids.
Adv. in Genet. 9, 147-215(1958).
Woo, S.H. and T. Adachi: Productionof interspecifichybrids between
Fagopyrum
esculentum and F. homotropicum
through embryo rescue.
SABRAO J. 29, 89-95(1997).
Yamaguchi-Shinozaki,
k. and K. Shinozaki:
The plant hormone abscisicacid mediates
the drought-induced
expression
but not the seed-specificexpressionof rd22, a gene
responsive
to dehydrationstress in Arabidopsis
thaliana. Mol. Gen. Genet. 238, 1725(1993)

77

GENOMICS

AND BIODIVERSITYMANAGEMENT:AND CHALLENGES

IACR-Long Ashton Research Station,
Key Words: Biodiversity;

Angela Karp
Universityof Bristol,UK

Management; Conservation;

OPPORTUNITIESLong Ashton, BS41 9AF,

Molecular tools; Genomics

INTRODUC TION
Over the past decade, DNA-based assays have revolutionised
our ability to characterise
biodiversity
at the genetic level. The large number of reports worldwide describing the use ofmolecular techniques for the characterisationof germplasm and natural populationsaretestimony to this success. However, using molecular tools to assist in the management of
biodiversity
requires more that just a descriptionof what is present. This paper briefly
considers the key issue currently facing biodiversity
management and the extent to which theavailable molecular techniques contribute to the addressing of these issues. Limitations
andgaps will then be examined and the potential contributionof new approaches emerging fromthe field ofgenomics will be discussed.
BIODIVERSITYMANAGEMENT ISSUES

Biodiversity
can be defined as the Earth's living resources, encompassing the number andvariety of all living organisms from terrestrial,
marine, aquatic and other ecosystems. The
fact that it embraces all living things is significant
in the context of molecular tools, which aremost often developed in model organisms and, thus, will need to be extended outwards towork for even those species which hitherto have not been subject to molecular studies.
Biodiversity
issues are equally broad and can be addressed at the species, population andwithin populationlevels (Karp et al 1997).
At the species level, the identificationoftaxonomic units and the determination of the uniqueness of species are essential information
for systematicstudies and conservation.
Molecular techniques
can contribute in theclassificationof organisms and the derivation of phylogenies.
Furthermore, they can help thedefinitionof distinctivenessof species and populations by ranking them according to theirevolutionary
history.
Molecular
markers can also help identify
hybridisationandpolyploidisation
events.
At the below species level the issues that need to be addressed differ, depending upon whether
ex situ or in situ management is being addressed.
Ex situ biodiversity
includes genebanks,zoos, botanical gardens and other collections.
Here, the key issues revolve around theacquisition,
maintenance, characterisation
and utilisationof genetic diversity.
In situbiodiversity
includes natural populations,as well as on-farm and managed or semi-managed
populations(e.g. forests).
Here, the main issues revolve around the identificationofpopulations
for conservation,
determining
the most effective
ways of managing the
populations in order to conserve diversity and sustainable utilisation.
An important adjunct to biodiversity
management at all levels is the ability
to identify
individuals
categoricallyand to assign parentage.
This enables relatedness
and family
structures to be determined and sources of gene flow identified and the amount estimated.

78
THE CURRENTTECHNIQUESCONTRIBUTION&

LIMITATIONSOF

MOLECULAR

Although it is not possible here to detail all the major issues that have to be faced, one aspectthat all levels of biodiversity
management clearly share in commonis their requirement for
information on genetic diversity.
It is here that molecular techniques can make a significant
contribution.
The first major attractionof DNA-based assays was the potentiallyinfinitenumber of character differences
they offer for the detection of diversity.
For biodiversity
management, however, it is actually the nature of the information that can be obtained that isof crucial importance to consider for molecular data to be useful in the decision-making
process.
The information that can currently be obtained on biodiversity
using molecular techniques canbe summarised as follows:(i) a quantificationof the diversity present; (ii) an understandingof how the diversity is distributed
among and within populations,in space and over time; (iii)
an insight into evolutionary history and genetic distance; (iv) the degree of relatedness amongindividuals
within a population;(v) whether individuals
are genetically
identical
or distinct;(vi) the amount of gene exchange amongindividuals
and populations.
Collectively
this information is extremely important for biodiversity
management. A large
numberof different molecular techniques are able to provide information of this kind, however,despite recent advances and our increasing knowledge of plant genomes, no single techniquecurrently provides all of the information listed above. More often than not, a combination oftechniques is needed to cover all the requirements of information that may be needed, which
increases the cost of applying a molecular approach. Alternatively,
a compromise has to bemade which reduces either the type of information that can be obtained and/or the number ofsamples that can be analysed. This places a limit on the potential gains from the applicationofmolecular techno logies.
A second limitationis that molecular markers are generally employed in an anonymous
fashion. This has invited criticism of the functional relevance of the diversity revealed using
marker approaches. As a result, although the contribution of molecular technique is making an
enormous impact on our ability to characterise
biodiversity,
evaluating the "significance"orassessing the "value" of the diversity
revealed and utilising
the data in decision-making
processes still remains difficult.
The main reason for this is the gap between our
understanding of diversity at the genetic (or molecular) and phenotypic (or functional)
levels.
The rapidly increasing information available from genome mapping and genomics projects isproviding an expanding resource, which may help to bridge this gap. Three of these advances
will be briefly discussed.
BRIDGING THE GAPUsing genome maps and closely

linkedmarkers

High-resolution
genetic maps, with genome coverage of one marker per cM, in many of the
major crops now enable markers of known location and distribution
to be chosen for use inbiodiversity
studies. Furthermore, synteny and comparative mapping increase opportunities
for importing locus identities
from one species to another. In lettuce, Sicard et al. (2000) usedmolecular markers to screen diversity
in genes known to confer resistance against downy
mildew caused by the fungus Bremia lactucae. The genes characterisedso far are clustered in79four linkage groups. Resistance gene candidates (RGCs) encoding nucleotide binding sites(NBS) and leucine rich repeat regions (LRR) have been identified
and one family of over 24
members is located in the major disease resistance cluster. A microsatellite
marker and two
primer pairs that amplify sequences encoding a region in the middle of the LRR region wereto screen accessions of known resistance phenotype of cultivatedlettuce (Lsativa),
the wild
lettuces, L. serriola, L.saligna and L. virosa and natural populations ofL. serriola. Very goodcorrelation was found between the markers and the allelic variation at the resistance gene loci.
Using sequence information

to target"functional"

diversity

For species that are not major crops alternative methods of using markers can be exploitedeven where genetic map information is limited.
The molecular technique of amplified
fragment length polymorphism (AFLP) (Vos et al. 1 995) involves first restriction
digestion of
genomic DNA followed by two rounds of PCR amplification.
Primers are designed on thebasis of the restriction
sites plus additional
nucleotides
that act as selective
bases. The
resultant multi-locus
profileis highly
reproducible
and extremely discriminative.
The
technique can also be adapted for use with primers that target specific sequences within the
genome, or sequence-specificamplification
polymorphism (SSAP) (Ellis et al 1998).
This
approach is currently being used to investigate
the feasibilityof screening for biodiversityinfunctionally
relevant regions of the genome in an EU-funded project (Contract No. BIO4 980332) in which different specific target genes are being investigatedby different participatinggroups. The gene families being studied include those encoding protein kinases, signaltransduction factors, disease resistance, phytohormones and cytoskeleton.
Genomics and Biodiversity:

ESTs,

DNA chips and microarrays

In DNA microarrays, different sequences are spotted on the glass slide and information isgained by determining at which of these sequences hybridisation
occurs. There are basically
two types of microarray:
expression arrays and arrays for identifying
and genotyping
mutations and polymorphisms.
Expression arrays are being used to monitor gene expression
levels in different
tissues and under different situations as a first step towards functional
characterisationof genes. Their main application
at present in is the functional assignment ofgenes. In contrast, genotyping arrays have the potential
to make a direct contribution
togenetic diversity studies in plants since they offer the possibilityof locating, identifying
andcataloguing genotypic differences in large sets of known DNAs simultaneously(Lipshutz
etal. 1999).
Although these approaches have yet to be applied to Biodiversity
studies, thefollowing areas look very promising and may change significantly
our strategies for using
molecular tools for Biodiversity
management in years to come.
Whole Genome Typing: Where microarrays have been assembled for the genomes of key
organisms it is possible to screen DNA fromother related species (target DNA) against the array(probe DNA) and identify gene differences. The approach is possible
for whole organellar
genomes (eg. mitochondrial
DNA) and for microbial genomes. It may be possible for higherorganisms for subsets of genes. Comparison can be made between whole genomes for theidentificationof interesting differences, such that genes present in the probe but absent/modified
in the target will be identified.
It has the disadvantage that any new genes present in the target
organism will be missed but it enables the process of whole genome comparisons to be started
Genotyping allelicvariation at large numbers of loci: As different
alleles of a gene are
identified
and sequenced, it will be possible to use arrays to screen for all known alleles at80
multiple loci simultaneously.
population studies.

This has the potential

to be an extremely

powerfulapproach for

Use of ESTs as single nuclear markers for genetic diversity
studies:
As more ESTs become
available
it will be possible
to use these as markers to identify
sequence polymorphisms.
In
the past, the limitationof this approach has been the paucity of sequences that diverge at rates
suitable for population
studies and thus the limited number of regions in the genome that canbe assessed. The huge number ofESTs being identified
open up the possibility
for generating
sequence data generated from multiple points in the genome.
Comparative analysis of developmental
genes: As gene sequences become available
it ispossible to determine to what extent the same genes have evolved in different organisms, andif so, to what extent their function differs. Comparative analysis of developmental
genes inthis context is particularlyinterestingfor Biodiversity
studies as it connects taxonomic
classification,phylogeny and Biodiversity
with molecular basis of morphology.
Identifying
regions of the genome under selection:
Instead of trying to identify
candidategenes for traits of adaptive significance,
if sufficient haplotype data become available
in theform of SNPs it may be possible to identify regions of the genome that have been subject toselection on the basis of information on recombination events, rather than using mutation dataon its own.
ACKNOWLEDGEMENTS
IACR receives grant-aided support from the Biotechnology
and BiologicalCouncil of England. All experiments complied with the current UK laws.

Sciences

Research

REFERENCES
Ellis,

T.H.N., S.J. Poyser, Ml Knox,A.V.Vershinin and MJ. Ambrose. 1 998. 7>7-copia class
retrotransposon insertion site polymorphism for linkage and diversity
analysis of pea.
Mol. Gea Genet. 260:9-9.
Karp, A., K.J. Edwards, M. Bruford, S. Funk, B. Vosman, O. Seberg, A. Kremer, P. Boursot, P.
Arctander, D. Tautz and G.M. Hewitt. 1997. Newer technologies
for biodiversity
evaluation: Opportunities
and challenges.
Nature Biotechnology.
1 5 :625-628.
Lipshutz,
R.J., P.A. Fodor, T.T. Gingeras and D.J. Lockhart. 1999. High density synthetic
oligonucleotide
arrays. Nature Genet. Suppl. 2 1 :20-24.
Sicard, D., S-S Woo, R. Arroya-Garcia, O. Ochoa, D. Nguyen, A. Korol, E. Nevos and R.W.
Michelmore. 1999.
Molecular diversity
at the major cluster of disease resistancegenes in cultivatedand wild Lactuca spp. 99:405-41 8.
Vos, P., R. Hogers, M. Bleeker, M. Rijans, T. Van De Lee, M. Homes, A. Frijters, J. Pot, J.
Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA
fingerprinting.
Nucl. Acids Res. 23 :4407-44 1 4.
Witsenboer, H., J. Vogel, and R.W.Michelmore. 1 997. Identification,
genetic localization
andallelic diversityof selectively
amplifiedmicrosatellite
polymorphic
loci in lettuce andwild relatives {Lactuca spp.). Genome 40: 923-936.

31

RNA editingin plant mitochondria,
cytoplasmic
and plant breeding

Simon Litvak1,

Armand Mouras2 and Alejandro

male sterility
Araya1

1 Laboratoire
REGER. UMR-5097. CNRS-Universite
Victor Segalen Bordeaux II. 146
rue Leo Saignat. 33076 Bordeaux cedex. France, (simon.litvak@reger.u-bordeaux2.fr)
2 Laboratoire
de Biologie Cellulaire
et Biotechnologie
Vegetale. Universite
Victor SegalenBordeaux 2. Avenue des Facultes. 33405 Talence. France.
Key words : RNA editing,mitochondria,transgenic

plants,

CMS, fertility

restoration

Introduction.
The term "RNA editing" was coined for the first time after the discovery of theextensive insertionand deletion of uridine residues in mitochondrial
RNAs of trypanosomes(Benne et al 1986). RNA editing has.been also found in mitochondria
and chloroplasts
from land
plants, in the mitochondriaof some fungi, in the nucleo-cytoplasmic
compartments of animalcells and in the genome of some human viruses like paramyxovirus, hepatitis
C virus and HIV1.The RNA editing process may involve base modifications
or base changes in RNA and theinsertion and/or deletion of nucleotides
in mRNA (for reviews see Araya et al. 1994; Schuster
and Brennicke 1994; Smith et al. 1997; Stuart et al. 1997). RNA editing in the mitochondria
ofhigher plants was described simultaneously
by three independent groups in 1989 by comparing
the mtDNA sequences and those of the corresponding
cDNAs (see references in Araya etal.1994).
Direct sequencing of the protein ATP9 from wheat mitochondria(Begu et al. 1990)
showed that edited mitochondrial
mRNAs are translated
in functional
proteins. An importantconsequence of the discovery of RNA editing was that the sequence of a given protein cannot be
deduced from that of the corresponding gene. RNA editing in higher plants organelles involves
the transitionof some C residues to U. Discovery of RNA editing in plants confirmed theuniversalityof the genetic code in plant mitochondria. RNA editing in higher plants can be foundin most mitochondrial
mRNAs. Editing
involves essentiallythe coding regions of plantmitochondrialtranscripts and less frequently non-coding regions like introns (Knoop et al. 1 99 1 ;Zanlungo et al. 1995). The rare editing events found in introns, tRNAs and rRNAs lead generally
to a correction ofmispaired
stem regions. When the C residue edited to U ofmRNAs is placed at
the 1st or the 2nd codon position, the change leads to an amino acid substitution
while, changes inthe third position do not modify the nature of the amino acid ("neutral" or "silent" editing).
There
is no current explanationof the prevalenceof silent editing.
The same transcript
may bedifferently
edited depending of the plant species. Thus, atp9 mRNAis edited at 8 sites in wheat,
10 sites in petunia and tobacco and only 4 sites in Oenothera. In all cases, the editing positions
are conserved. RNA editing is observed in all land plants with the exception of the moss
Marchantia polymorpha where all protein consensus sequences are already found at the genomic
level (Malek et al. 1996).
This led to the idea that bryophytes
had lost the RNA editingmachinery. However recent reports show that RNA editing is present in several bryophytes.
No
RNA editing is found in algae. In chloroplasts,
the lack of initiation
codons of some sequenced
genes led to the finding that rpsl2 from maize, psbL from tobacco and coxl (Kudla et al. 1992;
Kadowaki et al. 1995) possess an ACG (Ala) codon that after mRNA editing led to a AUG
codon. In mitochondrialand chloroplast
RNA editing the C-to-U changes lead to the creation of
new initiationand termination codons and to amino acid changes, improving the similarity
withthe functional protein consensus sequence. This situation may be considered as a "repair" process
giving a "sense" to the mRNA-encoded message. Our recent observation that RNA editing
operates in some HIV-1 mRNAs led to the proposal that, inversely, in this specificcase, RNA

82
editing may be a "defense" mechanism allowing the inactivationof a protein (Vpr) involved invirus infection and in some cellular functions (Bourara et al. 2000).
Mechanism of plant mitochondrial
RNA editing. We have developed an in vitro system using
a mitochondrial
lysate to study the biochemicalmechanism of RNA editingin wheatmitochondria(Araya et al. 1992). The unedited atp9 transcript (u-atp9) was double-labeledwith
3H at the base and 32P in the phosphodiester
linkage of cytidines.
After incubation with the wheat
lysate, RNA was digested with nuclease PI and submitted to 2D-TLC. Double labeled products
were found to comigrate with UMP and CMP standards. Our results indicate unambiguosly that
the RNA editing proceeds via the deaminationof C residues in plant mtRNAs (Blanc et al.
1995). Using a similar approach with etiolated
pea seedlings,
an independent group confirmed
the role of cytidine deaminationin plant mitochondrial
RNA editing (Yu and Schuster 1995).
The study of the RNA editing process in plant mitochondria
has allowed to find some rare U-toC changes in organellar transcripts(Malek et al. 1996). This reaction has been explained either
as the result of a reverse reaction of the cytidine deaminase or as a transamination step. When we
incubated the edited atp9 transcript
32P-labeledin the UMP residues with the mitochondrial
lysate we did not observe any radioactivity
associatedwith the C residues suggestingthat thedeamination reaction is not reversible under our assay conditions.
Thus, cytidine demination (C-to-U) in plant mitochondria(as describedabove) and in animals
for the editing of apoB have been shown. Adenosine deamination to inosine (A-to-I) operates in
the case ofglutamic
acid and serotonin receptors in the CNS (Bass, 1 997).
RNA editing of non-coding regions. The role of RNA editing in non-coding regions must be
considered with caution. Editing, upstream of the intiation
codon of Oenothera rpsl4 transcripts,
increase the complementarity
with the 3' end of 1 8S rRNA, suggesting that RNA editing mayhelp to create a ribosomal binding site. Similar events have been described in the case of wheat
mitochondrialtranscripts
coxll and orf25. It has been reported that about a dozen plantmitochondrial
genes are interrupted by type II introns. Several cases of RNA editing have been
described in the intronic region. In the case of type II introns the formation of a stem structure is
crucial for the splicing reaction (Farre & Araya 1999). In most intronic RNA editing events, Cto-U changes modify the secondary structure of the edited RNA as well as the group II intron
consensus sequence.
The biologicalimplicationof RNA editing. While in trypanosomes or Physarum mitochondria,
RNA editing seems to be essential for organelle function by creating the consensus translatablereading frames, the need for RNA editing in plant mitochondria
is less evident. The main
outcome of RNA editing in plant organelles would be the increase of the homology between theedited transcript
products (proteins,rRNAs, tRNA) and their counterpart found in organisms
other than plants. Translationof partial edited RNAs may generate variant proteins(Lu and
Hanson 1996)
increasing
the genetic repertoireof an small genome. RNA editing may beconsidered as a correction process leading to the synthesis of functional proteins. This is clear in
the case of wheat coxll transcript. The homologous coxll subunits from other species have a
Cys228 residue which is essential
for the activity of the protein. Only in the wheat coxll gene thisCys is absent and emerges only after mRNA edition (Covello and Gray 1990). Similarly,
editingof wheat atp9 and potato apocytochrome b (cob) increases the hydrophobic
character of these
proteins, thus favouring their integrationin the mitochondrial
membrane (Zanlungo et al. 1993).
Moreover, it has been shown that RNA editing allows the conservation of leucine-richregion in
atp9, atp6, nad3, nad4 and coxll of different species. In our laboratory
we have tested thefunction of editing by using transgenic plants. The rationale was to allow the accummulation ofan unedited protein inside mitochondria.
For this purpose, tobacco plants bearing the atp9 gene
fused to a mitochondrialtransit peptide were constructed. These transgenic plants showed
mitochondrial
dysfunction
leading to a male sterile phenotype (see below) indicatingthat RNA
editing is crucial for mitochondrial
functions (Hernould et al. 1 998).

83
The cytoplasmic
male sterile (CMS) phenotype
and the constructionof CMS plants using
an unedited
mitochondrial
gene and fertility
restoration.
Plant CMS can be defined as aphenotype produced by the incompatibility
between nuclear and mitochondrial
genomes. CMS
phenotypemay be relied to high recombination,a step which plays an important role inmodelling the plant mtDNA. Mitochondrial
gene recombination
may lead, in certain cases, to theformation of chimeric genes. The chimeric proteins may affect normal mitochondrial
functionand lead to the emergence of CMS. Male sterility
has a great importance in agriculture,since
hybrids seeds carrying propitious
agronomical
traits like heterosis
and hybrid vigor may beobtained. Several approaches have been used to obtain CMS by artificial
methods. Mariani et al(1990)
have expressed a bacterial
ribonuclease(RNase) called Barnase controlled
by a tissuespecific promoter in pollen producing tissues leading to the loss of pollen production.
The samegroup induced male-sterility,
restoration
by crossing
the male steriles
plants with plants
expressing an specific Barnase inhibitor.
We have used an approach to impair the mitochondrial
function and thus mimicking CMS. This approach involves the targetingof the \i-atp9 fused to asignal peptide allowing it to enter the mitochondria. After integrationof the unedited construct,in the nuclear genome, transcription
and cytosolic synthesisof «unedited» proteins, male sterile
plants were obtained (Hernould et al 1993). The male-sterile
tobacco plants obtained using theunedited mitochondrial
gene were restored to fertility
by crossing
with transgenic
plantsexpressing the antisense atp9 mRNA(Zabaleta et al. 1996).
Genetic and cytologicalanalysisof CMS transgenic
plants expressing
the engineered
unedited
atp9 gene. Flowers of tobacco expressing u-atp9 exhibited
either semi-fertileor malesterile phenotypes.
Studies on a populationof 25 plants of several progenies
showed that: the
CMS character was generally
inherited as a Mendelian trait for 1 or 2 loci, that u-atp9 is
involved in CMS phenotype and stably inherited in the progeny of the original transformants.
Molecular experiments performed in several generations confirmed this genetic analysis (Mouras
et al. 1999). Cytologicalstudies showed that anthers from tobacco flowers, transformed with thechimeric u-atp9 gene, present several abnormalities
leading to pollen abortion. The tapetum cells
showed signs of degeneration like loss of cristae and swelling. Mitochondrial
modifications
were
correlated with the presence of u-atp9 transcripts, translation
products and a significant
decreasein O2 cosumption in non-photosynthetic
tissues (Hernould et al. 1998).
Work is under progress to confirm and improve these results by using tissue specificpromoters controllingthe expression of unedited plant mitochondrial
genes.
Acknowledgments.
Have also participatedin this work : M. Hernould, V. Blanc, E. Zabaleta, JC
Farre, C. Calmels, E. Sargos, D. Begu, Suharsono, X. Jordana (Chile),
P. Figueroa (Chile),A.
Breiman (Israel),
I. Kurek (Israel).
Work in our laboratory was supported by the CNRS, theUniversity
Victor Segalen Bordeaux 2, the Human Science Frontiers Program Organization(HSFPO)(RG-437/94
M), the European Union (CIl*-CT93-0058),
the Conseil
Generald'Aquitaine,
the French Ministere de 1'Agriculture et de la Peche, the France-Israel
Program of
BiotechnologicalApplications
to Agriculture(AFIRST)and the France-Chile
Joint Research
Program (ECOS-Sud).
Several importantreferences that should have been mentioned in thiscommunication have been ommited because of the length alloted to this manuscript.
References.
Araya, A., Domec, C, Begu, D., Litvak, S. 1992. An in vitro system for the editing ofATPasesubunit 9 mRNA using wheat mitochondrial
extract. Proc Natl Acad Sci USA 89: 1040-1044
Araya, A., Begu, D and Litvak, S. 1994. RNA editing in plants. Physiol Plant, 91: 543-550.
-Bass, B. L. 1997. RNA editing and hypermutation
by adenosine deamination. Trends Biochem.
Set, 22: 157-162.
-Begu, D., Graves, P. V., Domec, C, Arselin, G., Litvak, S. and Araya, A. 1990. RNA editingof wheat mitochondrial
ATPase subunit 9: direct protein and CDNA sequencing. Plant Cell, 2:1283-1290.

84-Benne, R., Van den Burg, J., Brakenhoff,
J. P., Sloof, P., Van Boom, J. H. and Tromp. M. C.
1 986. Major transcriptof the frameshifted
coxll gene from trypanosome mitochondria
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four nucleotides
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in wheat mitochondria.
Plant Mol Biol, 40: 959-967.
-Hernould, M., Suharsono, Litvak, S., Araya, A., and Mouras, A. 1993. Male-sterility
inductionin transgenic
tobacco plants with an unedited atp9 mitochondrial
gene from wheat. Proc. Natl.
Acad Sci. USA., 90: 2370-2374.
Hernould, M., Suharsono, Zabaleta, E., Carde, J. P., Litvak, S., Araya, A. and Mouras, A. 1998.
"Impairment of tapetum and mitochondria
in engineered
male-sterile
tobacco plants. Plant Mol.
Biol. 36: 499-508.
-Kadowaki, K., Ozawa, K., Kazama, S., Kubo, N., Akihama, T. 1995. Creation of an initiation
codon by RNA editing in the cox I transcriptfrom tomato mitochondria.
Curr Genet 28:41 5-422-Kudla, J., Igloi, G. L., Metzlaff, M., Hagemann, R., Kossel, H. 1992. RNA editing in tobacco
chloroplasts
leads to the formation of a translatable
psbl mRNA by C to U substitution
within theinitiation
codon. EMBO J. 1 1:1099-1
103-Knoop, V., Schuster, W., Wissinger,
B. and Brennicke, A. 1991. 7><ms-splicing
integrates
anexon of 22 nucleotides
into the nad5 mRNA in higher plant mitochondria.
EMBO J., 10: 348393.
-Lu, B., and Hanson, M. R. 1996. Fully edited and partially
edited nad9 transcripts
differ in size
and are associatedwith polysomes in potato mitochondria.
Nucleic Acids Res. 24: 1369-1 374.
-Malek, O., Lattig, K., Hiesel, R., Brennicke, A. Knoop, V. 1996. RNA editingin bryophytes
and a molecular phylogenyof land plants. EMBOJ., 15: 1403-141
1.
-Mariani, C, de Beuckeleer,M., Truettner, J., Leemans, J., Goldberg,
R. B. 1990. Inductionofmale sterility
in plants by chimaeric ribonucleasegene. Nature. 341:131-14
1-Mouras, A., Suharsono, Hernould, M., Zabaleta,
E., and Araya, A. 1999. Inheritanceof andinduced male-sterile
trait in transgenic
plants expressing
and engineered unedited
atp9 mt gene.
Mol. Gen. Genet. 98: 614-621.
-Schuster, W., Brennicke, A. 1994. The plant mitochondrial
genome : structure,
information
content, RNA editing and gene transfer. Annu. Rev. Plant Physiol Plant Mol. Biol, 45: 61-78.
-Smith H C, Gott, J. M., Hanson M. R. 1997. A guide to RNA editing. RNA. 3: 1105-1123.
-Stuart, K., Allen, T. E., Heidmann, S., Seiwert,
S. D. 1997. RNA editingin kinetoplastid
protozoa. Microbiol
Mol Biol. Rev. 61: 105-120.
-Yu, W., Schuster, W. 1995. Evidence for a site-specificcytidinedeamination
reaction involvedin C to U RNA editing of plant mitochondria.
J. Biol Chem., 270: 18227-18233.
-Zabaleta,
E., Mouras, A., Hernould. M., Suharsono, Araya, A. 1996. Transgenic
male-sterile
plant induced by an unedited
atp9 gene is restored
to fertility
by inhibiting
its expression
withantisense RNA. Proc. Natl Acad. Sci. USA., 93: 1 1259-1 1263.
-Zanlungo,
S., Begu, D., Quinones,
V., Araya, A., Jordana,
X. 1993. RNA editingof
apocytochrome b transcripts
in mitochondria
from 2 genera of plants. Curr. Genet. 24: 344-348.
Zanlungo, S., Quinones, V., Moenne, A., Holuigue, L., Jordana. X. 1995. Splicing
and editingof rpslO transcripts
in potato mitochondria.
Curr. Genet. 27: 565-571.

85

Mitochondrialby mitochondrial

genome diversity
among plant species:
genome or nuclear genome?

is the gene encoded

KOH-ICHI KADOWAKI
National
Instituteof AgrobiologicalKannondai 2-1-2, Tsukuba, Ibaraki

Resources, Department
305-8602,
Japan

Keywords:

genome diversity,

evolution,

gene transfer,of Genetic

mitochondrial

ResourcesII,

genome, rice,

The endosymbiont
hypothesis
for mitochondrial
origin has generally
been
accepted(Gray 1992).
Recent studies
show that the gene content of the plantmitochondrial
genome is much larger than that of animals, insects, etc. (Brennicke
et al.
1993).
A striking
example exists in liverwort,
lower plant, in which 16 kinds ofribosomal protein genes have been identified,
respectively,
whereas none of these genes
are encoded in mammals and yeast (Oda et al. 1992).
Several plant mitochondrial
genomes have been shown to contain pseudogenes,
and the total gene content of themitochondrial
genome is not always.thesame among higher plant species including
even evolutionarily
relatedspecies.
These observations
suggest that gene-transfer
events from the mitochondrion
to the nucleus are carried out by an active process inplants.
The existenceof such a process has been firstly
establishedrecentlybyisolationof mitochondrial
genes transferredto the nuclear genome, such as coxll genes
from cowpea (Nugent and Palmer, 1991).
However, knowledge of gene transfer
followed by a gene activation
process is still very limited.
In order to investigate
content of ribosomal
protein genes in higher plantmitochondrial
genome, DNA gel blot analyses
were conducted.
Sixteenkinds ofribosomal protein gene from liverwort mitochondrial
genome were used as probe andrice and pea were selected as plant materials.
Apparent difference
between the twoplants was identified
in probe rps2, rpslO, rpsl3 and rpl2 (Fig. 1). Absence of signal
in mitochondrial
genome strongly suggest that functional
copy is present in nuclear
genome because the gene product is essential
for biogenesisof mitochondria.
Theseresults show that plant mitochondrial
genome has great genetic diversity
and will be agood material to know mitochondrial
genome evolution.
Here, we report two cases of gene-transfer
event and a process of acquisition
for atargetingsignal as well as a nuclear expression.
The rice mitochondrial
genome has
sequences homologous to the genes for ribosomal
protein Sll (rpsll)and S14 {rpsl4),

86
but both of them are interrupted
by internal stop codons.
Functional
rpsll and rpsl4
genes were isolated from rice nuclear genome separately,suggesting
gene-transfer
event
from the mitochondrion
to the nucleus during flowering plant evolution.
First case is analysisof rpsl4 gene (Kubo et al. 1999).
The nuclear rpsl4 geneencodes a long N-terminal
extension
showing significant
similarity
to a part ofmitochondrial
succinate dehydrogenasesubunit B (SDHB) protein from human and amalarial
parasite {Plasmodium falciparum).
Isolationof a functional
rice sdhB CDNA
and subsequent sequence comparison to the nuclear rpsl4 indicatesthat the 5' portionsof the two cDNAs are identical.
The sdhB genomic sequence shows that the
SDHB-coding
region is divided
into two exons. Surprisingly,
the RPS14-coding
region is located between the two exons. DNA gel blot analysis
indicatesthat both
sdhB and rpsl4 are present at a single locus in the rice nucleus.
These findings
strongly suggest that the two gene transcripts result from a single mRNA precursor byalternative
splicing(Fig. 2). Protein blot analysis
shows that the size of the mature
RPS14 is 16.5 kDa, suggesting
removal of the N-terminal
22.6-kDa peptide
region.
Consideringthat the rice mitochondrial
genome lacks the sdhB gene but contains therpsl^-relatedsequence, transfer of the sdhB gene seems to have occurred before thetransfer of the rpsl4 gene. The migration of the mitochondrial
rpsl4 sequence into thealready
existing
sdhB gene could bestow the capacity
for nuclear expression
andmitochondrial
targeting.
Second is case ofrpsll
gene (Kadowaki
et al. 1996).
The gene, sll-1, encodes
an N-terminal extension as well as organelle-derived
RPSll region.
Surprisingly,
theN-terminal region has high amino acid sequence similarity
to the presequenceof betasubunit of ATP synthase from plant mitochondria,
suggesting
common lineage of thepresequences.
The deduced N-terminal
region of sll-2,
a second nuclear-encodedhomologue to the rpsll, shows high sequence similarity
to the putative presequenceofcytochrome oxidase subunit Vb. The results that sharing of N-terminal region together
with its 5' flanking
non-translated
nucleotide
sequence for different
proteins
stronglysuggest an involvementof duplication/recombination
for targeting
signal acquisition
after gene migration.
A remnant of ancestral
rpsll
sequence, transcribedandsubjected
to RNA editing,is found in the mitochondrial
genome, indicatingthat
inactivationof mitochondrial
rpsll gene expression
was initiatedat the translational
level prior to terminationof transcription.
A mitochondrial
sequence that has migrated into a nucleus needs to acquire many
sequence elements (e.g. promoter, mitochondrial
targeting signal for protein import, andpolyadenylation
signal)for its functional
expression
because of the differenceof gene87
expression

systems

between

nucleusacquisitionof a mitochondrial

targeting1987).

Schatz1987;

Vassarotti

et al.

duplicationof targetinga targeting

signalexpression

system.

Further

involved

in genetranslocation

subsequent
mitochondrial

signaland mitochondrion.

found

Severalmechanisms

forsignalhave been proposedso far (Baker

and

The

alternative

event

as

in this study

and gene expression.
studiesis new examples

These

examplesare required

to identify

from

the

splicingof the acquisition

show the diversityofof genea number of mechanisms

mitochondrion

activation
duringendosymbiosis.
genome is interesting,important,as wellto theGenetic
and informative

nucleusanddiversity
amongin this point.

the
plant

Acknowledgment
I thankcollaborator

members of my laboratory,
especially
Dr. N. Kubo and Mr. K. Ozawa andProf. A. Hirai for their many contributions
to this work.

References
Baker, A. and Schatz,

G.

mouse dihydrofolate

reductaseprotein

into yeast

1987.

Sequencesfrom a prokaryotic

gene can restore

mitochondria.

the importgenome or theof a truncatedprecursor

Proc. Natl. Acad. Sci. USA, 84, 3117-3121.

Brennicke, A., Grohmann, L., Hiesel, R., Knoop, V. and Schuster, W. 1993.
The mitochondrial
genome on its way to the nucleus:
different
stagestransferGray,in higher

plants.

M. W. 1992.

Int.

FEBS
The

of geneLett. 325, 140-145.

endosymbiont

hypothesis

revisited.

Rev. Cytol.

141,

233-357.
Kadowaki,acquisition
existing

K., Kubo, N., Ozawa, K. and Hirai, A. 1996. Targeting
presequence
after mitochondrial
gene transfer to the nucleus occurs by duplicationoftargetingsignals.

Kubo, N., Harada, K., Hirai,
encoding
mitochondrial

EMBO J. 15, 6652-

6661.

A. and Kadowaki, K. 1999. A single nuclear transcript
RPS14 and SDHB of rice is processedby alternative

splicing:

common use of thesame mitochondrial

proteins.

Proc. Natl. Acad. Sci. USA,96:9207-9211.

targetingsignalfor different

Nugent, J. M. and Palmer, J. D. 1991. RNA-mediatedtransfer of the gene coxll fromthe mitochondrion
to the nucleus during flowering
plant evolution.
Cell 66, 473-481.
Oda, K.,Yamato,K., Ohta,

Gene organization
polymorpha
Vassarotti,

deducedmitochondrialA., Stroud,E., Nakamura,from theY, Takemura,complete

DNA.J. Mol. Biol.

R. and Douglas,

M. 1987.

M., Nozato,

sequence

N, et al. 1992.

of liverwort

Marchantia

mutationsat the amino

223, 1-7.
Independentterminus

of a protein

act as surrogate

signals

for mitochondrial

import.

EMBO J., 6,705-711.

Fig. 1.
probes.
Triangle

Summary of DNA blot analysis
Presence and absence of signal
shows pseudogene.

using liverwort mitochondrial
ribosomal geneare indicatedby circle and cross, respectively.

Fig. 2. Schematic representationof rice nuclear rpsl4 and sdhB genes.
Boxes and thin lines represent
protein coding sequence and non-translated
regions,
respectively.
The box withhorizontalstripes
represents
the
rpl5
gene.
rpsl^-homologous
regions are shown by black boxes.
sd/zi?-related
regions are shownby hatched boxes.
Putative mitochondrial
targetingsignals of sdhB genes from riceand human are indicatedby boxes with both horizontal
and vertical lines and the box
with vertical stripes, respectively.

89

Genetic diversityof endosperm storage nutrientsin rice

Hikaru SATOH1, Yuji UEMURA1, Tadao C. KATAYAMA2,
Satoshi MINAKUCHI1,
Toshihiro

KUMAMARUand Masahiro OGAWA3

1. Faculty
2. Facultyof Agriculture,of Agriculture,

Kyushu University, Fukuoka 8 12, Japan
Kagoshima University, Korimoto, Kagoshima 890, Japan

3. Department of Life Science, Yamaguchi Prefectural

University,

Sakurabatake,Yamaguchi 753, Japan
Rice is one of the principal

sources not only in carbohydrates

but also in protein in

Asian peoples as well as African peoples.
40 to 80 % of the daily caloric intake ofpeople in these countries is derived fromrice. Rice also contributes 20 to 40 % of theproteins in the Asian diet. Overall, the quality and the composition of endosperm
storage starch and storage proteins affect greatly on the cooking and processing qualities
of rice.
Therefore, the development of improved germplasm with superior nutrisional andcooking quality is one of the most important subjects on rice breeding. Although theartificial

mutation technique

is one of the useful methods to develop the novel genetic

resources, spontaneous mutations preserved in the local rice germplasm are the mostimportant genetic resources. A wide range of variations was observed not only for the
storage starch but also for the storage proteins among the local rice cultivars collected
fromdifferent countries, as well as for other agronomically important characters such as
yield, heading

date or disease tolerance.

Diversity in seed storage proteins
Nutritional values of rice are determined mainly by its storage proteinsin a grain.

The proteins

molecular

are composed of many kinds of polypeptides

having different

weight, electric charge or amino acid composition.
These storage proteins of rice are
accumulated in the subcellular
proteinaceous particles, such as protein bodies (PB) in
starchy endosperm and aleurone particles in aleulone layer.
Two or three types of PB
were observed in the starchy endosperm of rice seeds under an electron microscope(Tanaka et al. 1976;
1980;

Harris and Boulter 1977;

Oparka and Harris 1982).

Wu and Chen 1978;

Tanaka et al. (1980)

isolated

Bechteland Juliano

two types of protein

90bodiesin the starchy

endosperm of fice, PB-I and PB-II, which are characterizedeach otherby differencein the morphology,

the polypeptide

composition

digestibility

by pepsin.

PB-I is sphericalwithstructureproteins)of more than 4 major polypeptide

prolamins(alcoholwith different

solubleapparent

consisting

molecular

composed of several

components with different

by osmium tetraoxide(acid

solublebands ( a subunits)
kD globulin,collected

and theand contains
groupsmasses (MMr) from 10 to 16 kD, which were eachhomogeneouslyor alkaline

a lamelar

fromproteins)

isoelectricwith no lamellar
consisting

points.

structureof 37-39 kD acidic

and 22-23 kD basic sabunit polypeptide

is stained

and contains

glutelins

subunitprotein.

When the seed storage proteinsfrom different

countries(preserved), and 26

of local rice cultivars

in Kyushu University)

in proteinspolypeptide

bands ( j3 subunit

salt soluble

SDS-PAGE and IEF, a wide variation

PB-II

were analyzed by

was observed in both glutelins

andprolamins.
Glutelin: Glutelina and /3 subunits

in a japonica

rice cultivar

IR36 were separated

(Fig.

la).

The a bands were designated

37kD)

and a -4 (34 and 33kD),(22.5kD)

and j3 -3 (22kD).

IR36,

rice cultivar

Kinmaze and an indica

into four and three bands, respectively,
as a -l(39kD),

a -2(38kD),

whereas, j3 bands were designatedby SDS-PAGEa -3 (37.5

as ]3 -l(23kD),

storage proteinsof about 1400 rice cultivars

The remarkablevariationcorresponding

j3 -2

The a -3 band ofKinmaze was smaller in MMr than that ofwhile a -4 band of Kinmaze was larger in MMr than that of IR36.

cultivars.

andThe seedfrom Asia were analyzed by SDS-PAGE.

was found on the MMr of the a -3 and a -4 bands of theseSome of the cultivars

such as IR24

have two a -3 bands with MMrs

to 37.5 and 37kD.

In the & subunit,a few cultivars

have the j3 -4 band

(21kD).

In IEF analysis,
7.5 for adimentional

glutelins

were separated into eleven bands ranging from pH 6.2 tosubunit, and nine bands from pH 7.8 to 8.4 for # subunit.
The twoelectrophoresis(IEF/SDS-PAGE)
showed that the respective SDS-PAGE

bands consists of several IEF bands and almost all IEF bands were detected as single
spots. A wide range of variations was observed in IEF band pattern of both subunits
amongthe cultivars,
the staining
differedintensity

such as the number of bands, the presence or absence of bands, and(Fig.

among the cultivars.

lb).

In the j3 subunit,

A close relationshipthe pi and the number of bands
was not observed in the band patternƒÀ91
between the a andsubunits.

Prolamins:

In SDS-PAGE analysis,

IR36rice cultivar)(indica

16 kD polypeptide
analyzed,

prolamins

were separable

groups (Fig

1).

in Kinmaze (japonica

15.5kD

A large variationand 14kD polypeptide

was observed
intensity

among cultivarsof 1 5kD and 13kD bands.

bands was found in some of rice cultivars

collected

from Bangladesh.

Based on the SDS-PAGE profiles,

classified

into at least

Southeast

Asian countries showed a wide range of variation

7 types.

andinto four major bands, that is, 10, 13, 15 andin the presence or absence and the staining

Furthermore,rice cultivar)

Generally,

the cultivars

the cultivars

testedwerecollected

from South andin prolamin

polypeptides

ascompared with Japanese cultivars.
A wide range of variations
number and staining

intensity

in prolaminof bands differedmajor band varied from 6 to 15.
be classified

detectingprolamin

The range of variations

variations

as well as glutelins.

Seed storage proteinsof Oryza glaberrima

which is cultivatedOryza sativa L. Glutelin
the extremelyof glutelin

polypeptideof O. glaberrima

lower amount ofa -1 subunit.

of 0. glaberrima

Steud in addition

by SDS-PAGE and IEF (Fig.

to the a -1 subunit.

Similarly,

observed between O. glaberrima

and O. sativa L.

collected

starchwas also observed

from South Asian and East African countries,

dij estibi l ity.

in
rice,
2).

from those ofby the absence or

The a -3 subunit was also absent in some

into three major groups (Fig.

in endospermdifferent

Steud. was characterized

SDS-PAGE was also classifiable

Diversity

was muchseems to be quite effective

were clearlycanamong the materials

Steud., another kind of cultivatedin Western Africa, were analyzed

SDS-PAGE and IEF profiles

testedto African countries

The IEF analysis

The

The number ofshowed that the cultivars

Asian countries in addition

wider than that of Japanese cultivars.

by IEF analysis.

among the cultivars.

The IEF profiles

into more than 15 types.

from south and Southeast

was also detected

the prolamin

profile by

2A). The difference

among local

wasrice cultivars

for amylose content and alkali

Fig. l Erectrophoreticalanalyses

A.SDS-PAGE

b:IR36analysisof glutelins.

l:Kinmazc

2:HO380

9:IR24

10:HO413

Erectrophoretical

BIEF analysis
l:Kinmaze

3:HO528

1!:HO1233

analyses

A.SDS-PAGE analysisL.

of seed storagea: Kinamze
B.IEF

Fig.2

analysisof G.sativa

proteins

4:HO952
12:HO1415

of O.glaberrima

5:HO473

6:HO62t-t

7:ALO1001

13:HO639

14:HO1421

I5:HO1438

6:ALOI112

7:ALO1144

Steud.

of seed storage proteinsof glutelins.
2:IR36

3:ALO1120

4:ALO1108

5:ALO1018

8:IR36

93

Dragavtsev
N.LVavilov

Institute

V.A.

of Plant Industry(VIR)

Russia*Problems of genetic and molecular approaches to the hereditaryimprovement of the quantitative
characters of crops
In 1935 year N.I. Vavilov
inheritanceof quantitative
Mendelian conceptions".

wrote: "We shall not be suprised if a comprehensive
study ofcharacters will result in the radical revision of the simplified

Begining from 1984 in the Laboratory of Ecological Genetics of plant quantitative
characters(VIR) we studied the ways "product of gene (or metabolic way) - quantitative character" (1)in parallel with traditional
approach: study the ways "gene - product of gene". On the way (1)we found the phenomenon of redeterminationof genetical formula (spectrum of genes) ofquantitative
character, when the limiting factor of environment is change. This phenomenonquite usual and widespread for any polygenic
complex of characters and sometimes forclassical
major genes. For example: phytoantisipins
constantly
present in leaf cells of rust
resistant genotypes but it does not determine the genetic variance between resistant and notresistant varieties if the rust in absent. But on the background of rust phytoantisipins
begin
determine the genetic variance between varieties for many quantitative
characters
For some characters like intensivenessof transpiration
or intensivenessof photosynthesis
spectrum of genes, which determine polymorphism of these characters, change several times
perday.
Proceeding from the phenomenon of redeterminationof spectrum of genes we developed a
new theory of ecology-geneticalorganization of quantitative
characters. Fig. l demonstates theessense of this new theory. From Fig.l it follows that if a component character "number ofgrains per plant" (NG) is influenced by drought and a character "mass of one grain" (MG) by cold, then the genetic nature of a resulting character:the "mass of grains per plants"(RCh), cannot be described either by the lanquage of traditionalmendelian genetics, or by thelanguage of molecular genetics, or by the language of any branches of genetics existing atpresent.
The truth is in the fact that genetic variabilityof NG is determinated by polygenes for drought
resistans MG - by polygenes for cold hardiness and RCh by both clusters of polygenes
accompanied with the drift in time and multiplicative
effect (NG multiplied
by MG, gives
RCh). So, the real nature of the initiationand development of RCh can be described only by
the lanquage of the theory of ecology-geneticalcontrol.
Some consequences from this theory: nature of polygenes. In very short interval of time any
quantitative
character have typical monogenic determinationaccording to the principalof
Justus Libih. Polygenic picture - it is result of redeterminationof spectrums of genes (ormetabolic ways) in time by lim-factors of environment (or other). Ecology-geneticaltheorycontradict to MAS (Marker assistance to selection).
The labile genetic formula (spectrum ofgenes) of a complex character cannot correlate with any rigid structure of•E 190000,

StPetersburg,

BolshayaMorskaya street 44

Fig. Three models of organizationof quantitative

characters.

Nature of transgressions
and prediction of their appearance in particular hybrid populations.
Ifformation of productivity
takes place in a breeding zone against the background of one andthe same lim-factor (for example drought on virgin soils of Kazakhstan),
the transgression for
productivity
can be obtaind from the crosses of a drought resistant variety (producing
themaximumof dry biomass per unit area) and a variety carrying good polygenes of attraction(affecting
the distributionof products of photosynthesis
from straw and leaves into an ear). If
breeding is carried in Western Siberia (in the period NG initiation
it is drought, and in the
period of MG formation it is cold) the transgression
for productivity
will be obtaind as a resultof crossing a drought resistant variety with a cold hardy one (by combination of two systemsof adaptivity)
and the like.
Nature of ecologicallydependent heterosis and predictionof its manifestation
under theconditionsof ecologicalor competitive
limitationof growth processes. If drought resistantand cold hardy genotypes are crossed and then the parents and Fi plants are grown against thebackground, when NG is formed under drought, and MG - under cold conditions the Fi plants
will surely display
heterosis
dlue to the recombinationof genes spectrum within thecomponents of the module of RCh.
Nature of effects of a genotype - environment interaction (GxE) and prediction of this effectin different environments. If we planted two varieties (one - drought resistant another - cold
hardy) at two geographicallocations (drought and cold), we must have changing of grades of
productivity- typical phenomenon GxE interaction.
Nature of GxE interaction
it isredeterminationof the spectrum of polygenes under quantitative
charactersin result ofchanging of lim-factors of environment.

95

Possibilities
of prediction
the configurationof graphs of Hayman and the ranks of parametersof Hayman without accomplishing
the genetic analysis in the diallel
crosses. If we intend toinclude in the matrix of diallel
crosses Swedish (cold hardy) varieties
and the varieties
adapted to the Saratov Region (drought
resistance)
and to carry out the experiment in the
Tyumen Province of Siberia (drought in spring influences the character NG and early colds inautumn influence the character MG), then for sure we shall obtain the graph of Hayman for
the "mass of grains per plant" character with a strong effect of "overdominance" (theregression line will cross X-axis far away from the zero point of coordinates).
The most importautthan32.

consequencesfrom the new theory:commonnumber of its now more

Dragavtsev V.A. 1996.Literature.
Algorithms
of an ecologo-geneticaland methods of creating the varietiesof crop plants for yield
N.I.Vavilov Federal Center of PGR, St.-Petersburg.

survey of the genofond
resistanceand quality.

96

Integrationof genomics
Resource Center.

and biodiversity

researchat the Wheat Genetics

Bikram S. Gill *, Gina Brown-Guedira 2, and W. John Raupp 1.
Department of Plant Pathology!
and the USDA-ARS 2.
Throckmorton Hall, Kansas State University
Manhattan, KS 66506-5502,
USA.
Key words: germ plasm, Ae. tauschii,

pest resistance,

genetic

resources,

Triticeae

Conservation of the biodiversityof the wild relatives of crop plants and its efficientmanagement and utilizationin crop improvement is critical for food security of the world.
Beginning in the early 1980s, the Wheat Genetics Resource Center (WGRC) at Kansas State
University assembled a working collectionof wild relatives of wheat for gene and allele mining
and germ plasm development using the tools of genetics/breeding,cytogenetics,
and genomics.
Information on the collection,
includingevaluation for useful genes and their transfer to wheat
and list of germ plasm releases and research publications
is available
on the WGRC website(www.ksu.edu/wgrc/).
For this presentation,
we will focus on Aegilops tauschii, identified
asthe D-genome donor of bread wheat in the 1940s.
Genetic system: The first prerequisite
for the efficient utilizationof a wild species is its
genomic analysis and then developing a system for genetic analysis and genetic manipulation
by a system of wild species/crop
plant shuttle crosses. Aegilops tauschii is self pollinated,
and a single plant can produce several thousand seeds. The DNA content (4,500 Mb) is thelowest among the three genomes of wheat. The individual
chromosomes can be identifiedbyC-banding (Friebe et al. 1992) or FISH (Rayburn and Gill 1986).
The species is amenable todiploid
genetic analysis or diploid/polyploid
shuttle mapping with bread wheat (Kam-Morgan
et al. 1989).
A high-density,
genetic map has been constructed (Gill et al. 1991, Boyko et al.
1999).
A BAC library is available(Moullet et al. 1999).
Genetic resources: Kihara, Yamashita, and Tanaka led the first Kyoto University
expeditionin 1955 to Pakistan, Afghanistan,and Iran and collected
179 strains ofAe. tauschii from 106
different habitats (Kihara et al. 1965).
At present, the world collectionof Ae. tauschii
contains 1,285 accessions of which approximately
400 are maintained at the WGRC. The
world collection
does not fully cover the geographic
range of the species. To fully assess thegenetic variation of this species, additional
collections are needed to fill the gaps in thecollection.
Genetic diversity:
Kihara et al. (1965)
were the first to document the tremendous genetic
diversity
in Ae. tauschii.
Since then, the collection
has been extensively evaluated for variation
for pest resistance (Gill et al. 1986), RFLPs, and other agronomic traits (Lubbers et al. 1991,
Dvorak et al. 1 998, and references cited therein).
Geographicalregions of genetic diversity
forthe analyzed traits have been documented.
Gene and allele mining:turgidum and Ae. tauschii

The resynthesisof bread wheat from a cross between Triticum(McFadden and Sears 1 944), was followed by the production of

97

numerous synthetic wheats (Kihara et al. 1965).
A large number of disease and insect
resistance genes have been transferred from Ae. tauschii to bread wheat via direct crosses or
through the production of synthetic hexaploids(Gill and Raupp 1987). Several genes havebeen tagged with molecular markers (Ma et al. 1993, Huang 1998, Raupp et al. 2000). DNA
primer sequences for an RGA-like marker that detects all alleles at the Lr21 locus (Huang
1 998) and cloned puroindoline(the hardness locus) genes (Lillemo and Morris 2000) havebeen used for allele mining. There appear to be at least eight functional alleles at the Lr21
locus and almost a dozen at the hardness locus. Table 1 summarizes the current status of the
mapped Ae. taushii-denved
resistance genes for leaf rust and Hessian fly in wheat.
T a b le 1 . D e s ig n a te d g e n e s fo r re sista n c e to le a f ru s t (L r ) a n d H e ss ia n fly (H ) tr a n s fe r re d fro m
A e . ta u s c h ii in to b re a d w h e a t.
G en eG e r m p la s m

C h ro m o so m elo c a t io n

R efer en c e

L r2 1

T H * 6 / R L 5 4 0 6 (SH )ID S

R o w la n d a n d K e rb e r 1 9 7 4 .

L r2 2 a

T H * 6 / R L 5 4 04 (S H )2D S

R o w la n d a n d K e rb e r 1 9 7 4 .

L r3 2

T H * 7 / R L 5 4 9 7 (S H )3D S

K e rb e r 1 9 8 7

L r3 9T A 1 6 7 5 / 2 * W I ( 8 6 ‑ 3 2 0 ‑2 0 )2D S

R a u p p e t a l. 2 0 0 0

L r4 0(L r 2 1 a lle le ? )W G R C 2,W G R C 7
T A 16 4 9 / 2 * W I

ID S

H u an g 1 9 9 8

L r4 1W G R C l0
T A M lO 7 *3 / T A 2 4 6 0ID

C o x e t a l. 1 9 9 4

L r4 2W G R C l1
C T Y *3 / T A 2450

ID S

C o x e t a l. 1 9 9 4

L r43

W G R C 16
T M P 6 4 /3 / L S 8 0 1 0 ‑7 1 /T A 2 4 7 0 // T A M 2 0 0

7D S ?

C o x e t a l. 1 9 9 4

H I3

K S 8 1 H 16 4 0 H F
K U ‑2 2 1 ‑ 1 4 / E a g le // N E 7 3 6 4 0 /C h e n ey

6D L

G ill e t a l . 1 9 8 7

H 22

W G R C 1
T A 1 6 4 4 / N e w to n // W I

ID

R a u p p e t a l. 1 9 9 3

H 23

W G RC 3
T A 16 4 2 / 2 *W I

6D

R a u p p e t a l. 1 9 9 3

H 24

W G R C6
T A 2 4 5 2 / T A 1 6 4 2 / 2 * W I 73 /
N W T

3D

R au p p et al. 19 9 3

H 26

K a rl * 3 / T A 2 4 7 3

4D

C o x a n d H a tc h e tt 1 9 9 4

98
Genomics: Aegilops tauschii is not only the source of useful genes for wheat breeding but its
genetic system can be exploited
via diploid/polyploid
shuttle mapping for tagging and cloningof useful genes in the D genome of bread wheat. Several dozen resistance genes transferredfrom Ae. tauschii into wheat are good candidates
for this approach.
First, a closely identified
marker tighly linked to the target gene is identified
in wheat. This probe is then used to
identify
a BAC clone(s) from the Ae. tauschii library for chromosome landing. The highresolution
mapping populations
may be constructed in wheat or Ae. tauschii.
Mutants of thetargeted gene also may be isolated in the donor Ae. tauschii accession or the recipient
wheat
line for genetic complementation.
Recently, a large project under the auspices ofNSF plant
genome research program has been initiated,and our laboratory is collaboratingwith Jan
Dvorak, Hongbin Zhang, and Olin Anderson to develop a global BAC-contig map of the Ae.
tauschii genome anchored to the seven D-genome chromosomes of wheat. This map will be an
indispensable
resource for the map based cloning of genes in wheat.
References
Boyko,
Ziegle,linkage
Genet.

E.V., K.S. Gill, L.K. Mickelson-Young,,
S. Nasuda, WJ. Raupp, D.S. Hassawi, J.S.
A.K. Fritz, D. Namuth, N.L.V. Lapitan, and B.S. Gill. 1999. A high-density
genetic
map of Aegilops tauschii, the D-genome progenitor of bread wheat. Theor. Appl.
99:16-26.

Cox, T.S. and J.H. Hatchett.
1994. Resistancecommonwheat. Crop Sci. 34:958-960.

gene H26 transferredCox, T.S., WJ. Raupp, and B.S. Gill. 1994. Leaf rust-resistancetransferredfrom Triticum tauschii to commonwheat. Crop Sci.

from diploidgoatgrasstogenes Lr41, Lr42, and Lr43
34:339-343.

Dvorak, J., M-C. Luo, Z-L. Yang, and H.B. Zhang. 1998. The structure of the Aegilops
tauschii
gene pool and the evolution ofhexaploid
wheat. Theor. Appl. Genet. 97:657-670.
Friebe, B., Y. Mukai, and B.S. Gill. 1992. C-banding polymorphisms
Triticum tauschii {Aegilops squarrosa).
Genome 35: 1 92-1 99.

in several accessions

Gill, B.S., J.H. Hatchett, and WJ. Raupp. 1987. Chromosomal mapping
resistance gene HI3 in the D genome of wheat. J. Hered. 78:97-100.

of Hessian

of

fly

Gill, B.S., WJ. Raupp, H.C. Sharma, L.E. Browder, J.H. Hatchett, T.L. Harvey, J.G.
Moseman, and J.G. Waines. 1986. Resistance in Aegilops squarrosa to wheat leaf rust,
wheat powdery mildew, greenbug, and hessian fly. Plant Dis. 70:553-556.
Gill, B.S. and WJ. Raupp. 1987. Direct
hexaploid
wheat. Crop Sci. 27:445-450.

genetictransfers

from Aegilops

squarrosa

L. To

Gill, K.S., EX. Lubbers, B.S. Gill, WJ. Raupp, T.S. and Cox. 1991. A genetic linkage map of
Triticum tauschii (DD) and its relationshipto the D genome of bread wheat (AABBDD).
Genome 34:362-374.

99Huang, L. 1998.
wheat introgressed

Molecular markers linked
from Aegilops tauschii.

to the leaf rust resistnace genes Lr39 and Lr40 of
M.S. Thesis, Kansas State University.
89 pp.

Kam-Morgan, L.N.W., B.S. Gill, and S. Muthukrishnan.
1989. DNA restriction
fragment
length polymorphisms:
a strategy for genetic mapping of D genome of wheat. Genome
32:724-732.
Kerber,tauschii.

E.R. 1987. Resistance
Crop Sci. 27:204-206.

to leaf rust in wheat: Lr32, a third

gene derived

from Triticum

Kihara, H., K. Yamashita, and M. Tanaka. 1 965. Morphological,
physiological,
genetical
andcytologicalstudies in Aegilops and Triticum collectedfrom Pakistan, Afghanistan
and Iran.
Results of the Kyoto University
Scientific
Expedition
to the Karakoram and Hindukush,
1995, Vol. 1. Kyoto University,
Japan. 118 pp.
Lillemo, M. and C.F. Morris. 2000. A leucine to proline mutation in puriondolineb isfrequently
present in hard wheats from Northern Europe. Theor. Appl. Genet. 100:1 1001107.
Lubbers, EX., K.S. Gill, T.S. Cox, and B.S. Gill. 1991.
geographicallydiverse accessions of Triticum tauschii.

Variationof molecular
Genome 34:354-361.

McFadden, E.S. And E.R. Sears.
Soc. Amer. 13:26-27(Abstr.).

synthesis

1944.

The artificialof Triticum

markers among

Spelta.

Ma, Z-Q., B.S. Gill, M.E. Sorrells,and S.D. Tanksley. 1993. RFLP markers linked
Hessian fly resistancegenes in wheat {Triticum aestivum) from Triticum tauschii(Coss) Schmal. Theor. Appl. Genet. 85:750-754.

Rec. Gen.

to twoMoullet, O., H-B. Zhang, and E.S. Lagudah. 1999. Construction
and characterisationofalarge DNA insert library from the D genome of wheat. Theor. Appl. Genet. 99:305-313.
Raupp, W.J., A. Amri, J.H. Hatchett, B.S. Gill, D.L. Wilson, and T.S. Cox. 1993.
Chromosomal location of Hessian fly-resistancegenes H22, H23, and H24 derived from
Triticum tauschii in the D genome of wheat. J. Hered. 84(2):142-145.
Raupp, W.J., S. Singh, G.L. Brown-Guedira, and B.S. Gill. 2000. Cytogenetic
mapping of the leaf rust resistancegene Lr39 in wheat. Theor. Appl. Genet.

and molecular
In press.

Rayburn, L.A. and B.S. Gill. 1986. Isolation
ofa D-genome specificfrom Aegilops squarrosa.
Plant Mol. Bio. Rep. 4:102-109.

DNA sequence

Rowland , G.G. and E.R. Kerber. 1974. Telocentric
resistance to leaf rust and other agronomic characters
Genet. Cytol.
16:137-144.

repeated

mapping in hexaploid
wheat of genes forderived from Aegilops squarrosa.
Can J.

100

Expansion of available

genetic

resources for wheat improvement bysuper-wide hybridization
HisashiTsujimoto,KeiichiMochida,Masahiro

Research and Graduate School of Integrated
Althoughmany landraces

Kishiifor crop improvement,challenged

as to how to use this biodiversity

their

are maintained

use for breeding
for practical

Genetic resources of wheat are usually classified
relatednessof the genomes.

common wheat
processes.

(genome;

These genetic

in the secondaryfor Biologicalin the world as genetic

is still

limited.

We arecrop breeding.
into three categories

The genes in the species

which the genome constitution

Institute

Science, Yokohama City University)and wild species

resources(Kiharaand Aegilopsofis AA, DD, AABB or AABBDD can be introduced

to

AABBDD)throughresources constituteof Triticum

based on theordinary

the primary

meiotic

recombination

gene pool of wheat.

gene pool are those in A or D genome of polyploid

species

Genes
in whichgenomes other than A or D are also present, e. g., AAGG, DDMMor CCDD. To use
germplasm of these species

removal of the GG, CC and MM genome(s)The tertiary

gene pool includes

of wheat.

To transfer the genes to a wheat chromosome, special

to induce translocationdifficultit is to obtaingeneraare easilyof the genetic

hybrids

from thosemethods are necessary

resources for wheat is based on how easy or

and the progenies.

crossedwithand Elytrigia

after pollination

genes located on genomes that are different

or homoeologous recombination.

Another classification{^Haynardia),is necessary.

wheat;

Triticum,(=Thinopyrum).

and culture techniques

Speciesbelonging

Aegilops,

to following
Secale,five

Dasypyrum

Treatment of plant hormones to ovaries

of hybrid

for wheat breeding.

producehybrids

collectionsof barleyshould be re-evaluatedaddition,

perennial

Triticeae

speciesused as range crops should

be studied

fromviewpointof wheat genetic resources.

Maize, sorghum, pearl millet

and Job's

tears

that are far-related

species

we assume that it should

genetic

resources

betweenAt present

embryos expanded available

any two species

belongingto tribe

be possible

Triticeae.

Large

as genetic resources for wheat breeding.

from wheat may be used as genetic

resources

toIn

for wheat101

improvement because it is known that the male nucleus of the pollen
can fertilize
earlywith wheat egg nucleus,

embryogenesis.

resources.
Followings

Specialthoughof these species

their chromosomes are soon eliminatedtechniquehave to be developed

to utilize

are our recent approaches

to expand wheat genetic

diversity

in
these

by wideand super-wide hybridization.
1. Productionof Leymus racemosus chromosome addition

L racemosus (= Elymus giganteus)
speciesof Triticeae

species

grows in sandy soil,

long strait

spikes(> 30cm),

Fusarium scab.
Bulgaria.

We collected(CS),backcrossed

treatedthree L racemosus specificprobedracemosus enabledadditionwith thesein the Blackand rescued

seacoast

embryos.

We

monosomic additions

andeach L. racemosus chromosome, we

repetitive

DNA sequences.
sequences

Fluorescenceand also genomic

in situDNA of L

Now we have producednine

14 lines.

huashanica

chromosome additionin commonwheat

P. huashanica

is a diploid(genome;growing in rocky open slopes

Province,NN) species

China.

The shape of leaves

different

from those of wheat and of L. racemosus.

species

to commonwheat 'CS' and obtainedwith CS and one fertile

BlFl

speciesare veryWe crossed the pollenof thisThe Fl was backcrossed

We are nowisolating

plantscarryingfrom the B2F1 and B1F2 generations.

in the genera Lolium, Festuca, Bromus and Avena to wheat

Lolium, Festuca, Bromus and Avena are not includedinand spikes

the Fl hybrid.

plant was obtained.

one or a pair of chromosomes of P. huashanica
3. Crossingin'Chinese

2. Psathyrostachys

Mt. Huashan in Shaanxi

andand pests including

the hybrid

and selectedThis

leavesto the commonwheat cultivar

To identify
repetitive

speciesthe chromosomes to be identified.

lines out of a possible

large thickof thisthe Fl with commonwheat pollen

clonedproduces

Europe.

stresses

2,4-D to the ovary,in the progeny.

NNXX) and perennial

to many abioticof this species

additionshybridization

by rhizomes,

the seeds

disomic(genome;Asia and North and Easternand is resistant

We crossed the pollen

Spring'is a tetraploidthat grows in Central
propagateslines in common wheat

in Triticeae

but are relatively

102
closely

related

their

basic

Festucato Triticeae

genera.

The chromosomes of these genera are large andchromosome number is 7.
ovinata,

Bromus catharticus,

We crossed
Avena sativa,

commonwheat.

However, no embryos appeared

were occasionallyformed in the ovule.

4. Introduction

is known to fertilize

thougheliminatedmultiflorwn,

Avena species

unidentified

tolumps of cells

the wheat genome. If it is possible,
wheat breeding.

Considering

pollination,translocation24hpollination.

obtained

wheat

haploids

withdetermine

what events take place

maize

after

betweenwheat and maize
It is importanttoand to know what is the cause forThus we observed the fertilization

wheat egg and maize sperm nucleus.

for

However, we could not

chromosome segments.

after pollination

maize-chromosome elimination.

resourcesof maize chromosomes occurs a few

to induce

chromosome by X-ray irradiation

wheat

maize chromosome segments into

we could regard maize as the genetic

that the elimination

we triedin early embryogenesis,

from the embryos, forming

The main goal of our study is to introduce

afterand diploidthe wheat egg, althoughmaize chromosomes are preferentiallydaysof Loliumof maize chromosomes to wheat

Maize pollen
haploids.

pollenprocess

We first confirmed fertilization

betweenof the female andmale nuclei

by fluorescencein situ hybridization

to the sectioned

embryosac

cells

embedded

with

7100

we developed

the

for

Technovit

resin.

Later

methodwhole-mount FISH and observed the dynamics of maize chromosomes in wheat cells in
detailusing a confocal

laser microscope.

Crops had been improved
progressed

remarkably

establishingmethods

during their long history
progressedof their cultivation.

Genetics

hastoin the20 century

and contributed

greatlyof crop improvement.

At present,

we can introduce

any genes

modifiedare grown

clonedin vitro to most crop species.

Many geneticallywidelyand circulatedcommercially.

However, we should not forget that genes used inare not totally

synthesized

the transformation
organism(s)with some modifications.

importanceof genetic

resources

artificiallyConsequently,

but are isolated
we should

even if gene transformation

for crop improvement in the future.

cultivars

from ankeep in mind the

becomes the main method

103

Development of novel wheat starches
Makoto YAMAMORI
Tohoku National Agricultural

Experimentby the use of germplasm
Station,Morioka,

Key words: Wheat •EStarch •EAmylose •EAmylopectin

Iwate 020-0824,

diversity
Japan•EGermplasm

Introduction
The major component of wheat grain endosperm is starch, accounting for about two-thirds ofthe weight offlour,
as it does in other cereal grains, such as rice and maize. Thus, starchshould influence
the physico-chemical
properties
and end-use of wheat flour.
Starch iscomposed of glucose polymers and can be divided into two kinds, amylopectin
and amylose.
Amylopectin
is a branched glucose polymer while amylose is a much less branchedoressentiallylinear polymer.
To characterize
starch, its amylose content (%) is often used. Amylose content is roughly
about 25% in normal cereal starch.
Compared to rice and maize, the amylose content ofhexaploid
wheat (Triticum aestivum L.) starch is relatively
constant.
For example, in rice andmaize starches from grain endosperm, an amylose-free phenotype (called 'waxy' or 'glutinous')
and a high amylose one exist, but both were absent in wheat. In this paper, I will describe
developmentof two new starches in wheat, 'waxy' and 'high amylose'.
Analysisofgermplasm diversity
in proteins(enzymes)
for starch synthesis
enabled their production.
These novel starches are expected to be utilized for food and various industries.
Finding of deficiency for Wx proteins (GBSS I) responsible
for amylose production
In Japan, domestic wheat is mainly used for 'udon' noodles.
Therefore, breeding new wheat
cultivars
with good noodle making quality is important to Japanese wheat breeders.
On theother hand, wheat imported from Australia is known to be superior to Japanese wheat in somepoints, such as glutinosityof cooked noodles, because Australian wheat is more glutinous thanstandard Japanese varieties,
and the Japanese tend to prefer to some glutinosity.
Since low
amylose rice is more glutinous when cooked, the amylose content of Japanese wheat wasmeasured by Hoshino et al. (1985)
and Kuroda et al. (1989).
They found a correlation
between amylose content and glutinosityof cooked noodles, and that two wheats, Kanto 79
and Kanto 107 (progeny of Kanto 79), contained comparatively
little amylose (22%) in 191
cultivars (average ca. 28%).
This finding prompted us to analyze the Wx (waxy) protein in
the Kanto lines.
Since the Wx protein (granule-boundstarch synthase I; GBSS I) is responsible
for amylose
production,
we predicted that the Wx protein may have mutated in Kanto 79 and 107. The
results of SDS-polyacryl
amide gel electrophoresis(SDS-PAGE)
showed that the amount of
Wxprotein in the Kanto lines decreased because they produced a thinner band ofWx protein
on electrophoretic
gel than did the control wheat (Yamamori et al. 1992).
Thus, the Kantolines were subjected
to two dimensional(2D) gel electrophoresis
which can more minutely
analyze proteins on gel than SDS-PAGE.
The 2D electrophoresis(Nakamura et al. 1993a)
separated wheat Wxproteins into three spot groups (Fig. 1). These were called Wx-Al, WxBl and Wx-Dl proteins derived from three genomes of wheat, A, B and D, respectively.
In
the Kanto lines, both Wx-Al and -Bl proteins were absent, only Wx-Dl was present(Nakamura et al. 1993b).
Therefore, we concluded that the lack of the two Wx proteins was
the cause for reduced Wx protein level and low amylose in the two Kanto lines.

Fig.

1 SDS-PAGE

and 2D electrophoretic

profilesof the Wx protein

and SGPs in hexaploid

wheat
Productionof waxy (amylose free) wheat
In Kanto 79 and 107, only the Wx-Dl protein synthesizes
amylose.
Therefore, if the Wx-Dl
protein in the Kanto lines could be nullified
by some method, we would obtain waxy (amylosefree) starch of wheat. One method we used was to find a wheat without the Wx-Dl protein in
the world germplasm (Yamamori et al. 1994), cross it to Kanto 107, then obtain waxy characterin the progeny (Nakamura et al. 1995).
Another way was to induce artificial
mutation for
Wx-Dl protein in Kanto lines.
Yasui et al. (1997)
succeeded in obtaining
waxy starch bymutagen (EMS) treatment.
Weexamined 1,960 accessions for the presence and absence ofWx-Al and 1,551 for thoseofWx-Bl and Wx-Dl. One Chinese wheat, Bai Huo, lacked the Wx-Dl protein and about
10% of accessions examined were deficient in either the Wx-Al or Wx-Bl protein (Table 1).
From the cross between Kanto 107 and Bai Huo, a waxy wheat was produced at the expected
frequency of three independent
genes, 1/64.
The waxy wheat starch turned reddish-brownwith iodine staining, which is indicativeof a waxy phenotype.
Table

1

Wx protein and SGP-1 deficiency

in wheat germplasm

From a different cross, Kiribuchi-Otobe
et al. (1997)
also produced a waxy wheat. Waxy
wheat bred in Japan shows novel propertiesof starch of flour, including
high starch crystallinity
in X-ray diffraction,a high gelatinisation
enthalpy in DSC (differential
scanning calorimetry)
thermogram, unique pasting property in RVA (Rapid Visco Analyzer)
analysis,and lower
milling yield (Yasui et al. 1996; Hayakawa et al. 1997; Kiribuchi-Otobe
et al. 1997; etc.).

Genetic

eliminationof a starch

granuleprotein,

SGP-1, produced

apparent

high amylose

105
starch in wheat
Starch granule proteins (SGPs)
are bound to starch, even after flour is washed by a buffer
containing
a detergent(SDS).
SGPs can be dissolved
and analyzed by SDS-PAGE whenstarch is boiled and gelled in a buffer containing
SDS. Besides the Wx protein, wheat starch
has three SGPs, i.e., SGP-1, SGP-2 and SGP-3 (Yamamori and Endo 1996).
SGPs areassumed to be entrapped within starch granules as starch grows. Recent molecular studies (Liet al. 1999) show SGP-1 is starch synthase II (SS II) for producing
linear glucose polymer(probably)
ofamylopectin.
SGP-2 is branching enzyme II (BE II) and SGP-3 is SS I.
SDS-PAGE separated SGP-1 into three bands, i.e., SGP-A1 from the A genome, -Bl fromthe B genome and -Dl from the D genome (Fig. 1). Thus, we could examine the loss of three
SGP-1 classes by SDS-PAGE (Yamamori and Endo 1996).
In the 1,9.60 accessionsof wheat,
we found three lacked SGP-A1, one lacked SGP-B1 and one was missing SGP-D1 (Table 1).
Like the Wx-Dl protein, these three kinds of SGP-1 deficiencies
were infrequent.
Thus, fromthe cross among the three accessions,a wheat carrying no SGP-1 was bred in order to elucidate
the effect of complete SGP-1 deficiency on starch (Yamamori et al. 2000).
The starch fromthis SGP-1 null wheat generated a higher blue value and showed an apparently
higher amylose
content by both colorimetricand amperometrictitration
methods(Table2).
Light
microscopic observation
indicatedthat starch granules were deformed and their hila seemed to
crack in SGP-1 null starch.
These facts indicate that SGP-1 is necessary for normal starchformation.
Table 2

Starch properties

of SGP-1 null (highamylose)and control wheats

A high amylose starch of wheat showed novel starch properties
In a high amylose mutant of maize, increased long chains of amylopectin enhance both apparent
amylose content and blue value.
Thus, the amylopectin structure of the high amylose wheat
was examined by high-performancesize-exclusion
chromatography(HPSEC)and highperformance anion exchange chromatography
with a pulsed amperometric detector (HPAECPAD) (Yamamori et al. 2000).
HPSEC detected an increase in high molecular weight fraction(Fr. I) and an abnormal shoulder in low molecular weight fraction (Frill).
Since Fr. I isbelieved to consist ofamylose and a long chain of amylopectin, apparent high amylose would be
due to the increased Fr. I. Since Fr. Ill contains shorter chains of amylopectin,a shoulder in
Fr. Ill means a varied structure of the amylopectin.
HPAEC-PAD detected chains of degrees
ofpolymerization(DP) 6-10 increased and DP 1 1-25 decreased in the amylopectinof the highamylose starch.
These results demonstrate
an abnormal structure of amylopectin,
especiallyshort chains.
Since amylopectin
forms the crystalline
structure of starch, variant amylopectinin highamylose starch might influence it. Crystallinityof the high amylose starch was examined by
DSC, X-ray diffraction
and a polarized
microscope.
DSC analysis did not detect a major
endothermal
peak deriving from amylopectin
disruption.
Major peaks caused by starch
crystallinity
did not appear in the X-ray diffractogram(Table 2). Further, microscopy did not
find a polarizedcross on starch granules.
The absence of the major peaks in the DSC
thermogram and X-ray diffractogram,and lack of polarizedcross, mean that the high amylose

106

starch lacks crystallinity.
The starch swelling power of the high amylose was less than that of normal amylose starch.
This indicatesa possibility
that the gelatinisation
property varies.
Conclusion
By geneticallyeliminating
enzymes for starch synthesis in wheat, we developed waxy and highamylose starches which had not existed before.
Three null alleles for Wx protein and SGP-1(SS II) from the A, B and D genomes were identified
by gel electrophoresis
in our study.
Ifsome method can be established
to recognize defects in three isoforms of a starch synthetic
enzyme from A, B, D genomes at molecular levels (protein,mRNA or DNA), wheat withmutant starch could be produced by combining three defects and nullifying
the enzyme. Whiletransgenic
wheat with antisense
inhibition
for starch synthesis
is effective
for producingamodified starch, our study shows that the diversity buried in genetic resources can also generate
novelty in wheat.
Acknowledgment:

Wheat accessions

were provided

by NIAR and wheat breedinglaboratories

of MAFF.
References
Hayakawa, K, K Tanaka,T. Nakarnura, S. Endo and T. Hoshino. 1997. Quality diaracteristics of waxyhexaploid
wheat (TriticumaestivumL): properties ofstarch gelatinization and retrogradation Cereal. Chem 74: 576-580
Hoshino, T., K Fukunaga and S. Oda 1985. Mutual relation among phyacoKterical properties of wheat flour forJapanese noodle quality in domestic wheat cultivars and lines. Jpn J. Breed. 35 (SuppL 2)256-257
Kiribuchi-Otobe, C, T. Nagairrine, T Yanagisawa, M Ohnishi and L YamaguchL 1997. Production of hexaploid
wheats with waxyendosperm character. Cereal Chem 74:72-74
Kuroda, A, S. Oda, S. Myagawaand H Seko. 1989. A method of measuring amylose content and its variation in
Japanese wheat cultivars and Kanto breeding lines. Jpn J. Breed. 39 (SuppL 2): 142-143
U Z, X Chu,G. Mouille, L. Yan, B. Kosar-Haiiemi, S. Hey, J. Napier, P. Shewry, B. Clarke, R. Appels, MK
Morell and S. Rahman 1999. The localization and expression of the class II starch synthases of wheat. Plant
Phyaol. 120: 1 147-1 156
Nakamura,T, M Yamamori,
H HBranoand S. Hdaka 1993a Identification ofthree Wxproteins in wheat (Triticum
aestivumL). Biochem Genet. 3 1 :75-86
Nakamura,T, M Yamamori,H Hirano and S. Hdaka 1993b. Decrease of waxy (Wx) protein in two common
wheat cultivars with low amylose content. Plant Breed. 1 1 1 :99-105
Nakamura, T, M Yamamori,H Hirano, S. Hdaka and T. Nagamine. 1995. Production of waxy (amylose free)
wheats.Mol Gea Genet. 248:253-259
Yamamori,M andT.R Endo. 1 996. Variation of starch granule proteins and chromosomemapping of their coding
genes in commonwheat. Theor. AppL Genet. 93 :275-28 1
Yamamori,M, S.Fujita, K Hayakawa, J. Matsuki and T. Yasui 2000. Genetic elimination ofa starch granule protein,
SGP-1, ofwheat generates an altered starch with apparent high amylose. Theor. AppL Genet. 101 :21-29
Yamamori,M, T. Nakamura,TR Endo and T. Nagamine. 1994. Waxy protein deficiency and chromosomal
location ofcoding genes in commonwheat.Theor. Appl Genet. 93 :275-28 1
Yamamori,M,T.Nakamuraand A Kuroda 1992. Variations in the content of starch-granule bound protean among
several Japanese cultivars ofcommonwheat (Triticum aestivumL). Euphytica 64 :2 1 5-2 19
Yasui, T, J. Matsuki, T. Sasaki and M Yamamori.1996. Amylose and lipid contents, amylopectin structure, andgelatinisation properties ofwaxy wheat (Triticum aestivumL) starch. J. Cereal ScL 24: 13 1- 137
Yasui T., J. Matsuki, T. Sasaki and M Yamamori.1997. Waxy endosperm mutants of bread wheat (Triticum
aestivumL.) and their starch properties. Breeding So. 47: 161-163

107

Rapid
and Simple
RAPD Marker

Methodfor Purity

Soontae KWONand Sei-myoung OH
Dept. of Applied Life Science, Andong National
Andong, Kyungpook 760-749, Korea.
skwon@andong. ac.kr,
Key words: inbreds,

hybrids,

Testof RadishHybrid

Seeds

Using

University,

RAPD marker, self-incompatibility

Abstract
In order to develop a rapid, simple, and economical method for testing the purity ofradish hybrid seeds using a procedure based on the polymerase chain reaction (PCR), eighty
random primers were screened with the genomic DNA of inbred parent lines and their Fl
hybrids.
Two primers, so called OPA02 and OPA19, generate reproducible
unique PCR
patterns which is distinguishedamong inbred parent lines as well as their hybrids. The
differencesin PCR patterns can be used for the purity test of radish hybrid seeds byidentificationof each parent lines and hybrids
without the need for DNA sequence
information.
Furthermore, RAPD-PCR method requires only minute quantitiesof plant
sample and DNA, and therefore can be used in situations in which the amount of plant sample
is limitingin the early stage of seed germination. Wedemonstrates the use of RAPD markers
to identify each of inbred parent lines and hybrids by rapid and simple method.
Introduction
The mdish(Raphanus
sativus L.) is one of the major vegetable crops in terms of the
production and field area in Korea. Although it differs with respect to the size, shape, colorand consistency dependingon the species, the chromosome number in all radishes
isidentical(n=9).
The origin of the radish is not fully understood, but it is supposed that theradish is native to central Asia, China or India because of the occurrence of many genotypes
from wild types to cultivated types. Based on the cropping season, the radishes cultivatingin
Korea are separated into three major groups such as spring, summer and autumn radishes
whose field area were 18,545, 3,426 and 15,13lha
in 1999, respectively.
Radish roots aremainly consumed not only as fresh vegetables but also as ingredientsof 'Kimchi' which isone of most important processed vegetables in Korea.
Commercial seeds of the radish are Fl hybrids which are produced by crossing selfincompatible
parent lines. Sexual incompatibility
is a widespread phenomenon in flowering
plant which restricts
inbreeding
within populations
and also seems to contributeto thereinforcement of species differentiation.
The occurrence of self-incompatibility
in Cruciferae
has been well known and contribute to effective breeding strategy and production of highquality and yield of hybrid radish roots. With the increasing emphasis placed on uniformity invegetable production, there has been a trend towards the use of Fl hybrids. By crossing two
geneticallyuniform, but distinct,
parent lines, Fl hybrids
are produced possessing
theattributesof a high level of uniformity and increased vigor in terms of yield, quality, shapeand texture as well as resistance to various environmental stresses. The success of this method
for producing commercial cultivars depends on being able to maintain uniform parent lines
and ensuring that the Fl seeds results from a direct cross between the two selected parents
only. The parent lines are normally self-incompatible,
thus making cross-pollination
essential
for Fl production.
In practice, the self-incompatibility
mechanism may not be entirely successful and a
low level of self-pollination
may occur resulting in the production of a small percentage of

108
siblingor selfing production.
During the cultivationof commercial seed radish, the resulting'sibs' or 'selfs' are readily identifiablebecause of their weakness in vigour as compare to
hybrids. However, in commercial point of view, hybrid seeds mixed with selfing or siblingseeds results in the reduction of uniformity and quality of the seeds compared with Fl hybrid
seed production.
In case of autumn cropping, Fl hybrid seeds are harvested on June 15 and marketed on
July 15 by seed companies, and then seeded by farmers on August 15 in Korea. The period oftime between Fl seed harvesting
and marketing, the seed company must go through processof drying, threshing,seed cleaning, coating, grading, packing and transporting.
Due to thetime restriction,
the seed company cannot test the purity of Fl hybrid seeds by the method of
growing the seedlings before marketing. To solve this problem, we tried to employ rapid andsimple method for testing the purity ofFl hybrid seeds using RAPD primer.
Materials and Methods
Inbreds and hybrids used
Radish inbred lines used were ER-101 and ER-450. These inbreds were maintained as
pure stocks by subjecting
to continuous selfing for about fifteen years and used as parent lines
for producing Fl hybrid seeds by crossing method of self-incompatibility.
The hybrid seeds
derived from two inbreds have been produced and commercialized
by the seed company formore than 10 years.
DNAisolation and PCR
Seeds were germinated on water moistened filter paper in 9cm petridish at 28°C. Fiveday-old single seedlingless than 50mg was collected and frozen in liquid nitrogen and thenground with a mortar and pestle. Ground tissue was combined with CTAB buffer, and DNA
was extracted as described in Bohem et al.(1999).
PCR reaction mix was 5 /^ 10 x buffer,
O.lmM each of dNTPs, 0.25p M random 10-mer primer(from Operon Technologies),
lOng
template DNA and 0.3units Tag DNA polymerase in 50 l& total volume. This mixture was
exposed to 45 cycles of 1 min at 95°C, 1 min at 38°C and 1 min at 72°C using the DNA
Thermal Cycler from MJ Research. PCR products were electrophoresisedin 1.2% agarose gel
and visualized UV light by EtBr staining.
Primer Selection andpurity test
In order to select marker primers which can be used for the purity test of ¥{ hybrid
seeds, genomic DNA of parent lines and their hybrids were PCR analyzed using 80 ten-base
RAPD primers. Two primers, OPA02 and OPA19, were selected based on the clarity andspecificityof PCR products amplified by the genomic DNA of parents and their hybrids.
A randomly selected set of 100 commercial hybrid seeds were incubated in 15cm
petridish by soaking with distilledwater at 25°C for 3-5 days. Each single seedling wascollected
and subjected to extraction of the genomic DNA for PCR using selected marker
primers. Based on PCR patterns of the hybrids,
seeds were separated into the crossing(hybrid)lines and the self mg(inbreeding)lines. The purity of hybrid seeds is calculated
asnumber of crossing lines per those of total seeds tested.

Results

and Discussion
The PCR patterns of the radish genomic DNA amplified by the RAPD primers showedvarious profiles depending on the radish genomes as well as primers tested(Fig.l).
Eightyprimers screened are classified
into four groups based on the banding patterns of PCR

products. First group is the primers showing no band in both of the parent lines, second is
those showing same bands between two parent lines(Fig.l
Type I), third is different
bands
among individuals
within populationsin the same parent line as well as different
bands
between parent lines(Fig.1
Type II), and forth is same bands among individuals
withinpopulationsin the same line but clearly different
between two parent lines, ER-101 and ER405(Fig.1
Type III). For the purpose of purity test, only the forth group can be used as
marker primer because the banding patterns of the PCR products are specific to each ofparent lines as well as their hybrids.

Fig. 1. Types ofPCR patterns amplified by random primer with radish genomic DNA.
Three individual
seeds from each of inbred parents, ER-101 and ER-4053 wereanalyzed by RAPD primers. M: marker DNA 123-ladder.
In addition to specificity
between parent lines? marker primer should possess anotherrequirement. The PCR patterns amplified by marker primer not only•Eshow no fluctuations
among individuals
within populationsin the same parent line but also among individuals
inhybrids.
Each parent line must possess at least one unique PCR band distinguishabletoanother parent line.

Fig, 2* Purity test ofFl hybrids using RAPD primer, OPA02.
M: marker DNA ladder, A: inbred parent ER-101, B: inbred parent ER-405.
Inbred lines mixed with commercial hybrid seeds are marked with A or B.

noFigure 2 show the PCR patterns amplified by OPA02 primer with the genomic DNA ofeach parent lines and 80 randomly selected hybrid seeds from commercial seed pack. As
indicatedin the PCR profiles, six inbred lines such as four ER-101(marked
with A) and two
ER-405(marked
with B) were clearly identified
in the group of eighty hybrid seeds(Fig.
2). Itmeans that the purityof the commercial
hybridseeds tested is 92.5%. This result
demonstrates the utility of RAPD markers for testing the purity of Fl hybrid seeds.
Recently, techniques
based on PCR have been used to detect polymorphisms
in various
plants, animal, and bacterial
species(Welsh
et al. 1991; Huff et al. 1993). RAPD markers cangenerate fingerprintsof DNA product, some of which are polymorphismsthat are present in
one parent and absent another. Polymorphisms
detectedby RAPD are inheritedin a
Mendelian manner and can be generated for any species without DNA sequence analysis.
Detectionof polymorphisms
by using RAPD technology
is faster and less laborious
than by
RFLP. The main advantages of RAPD over traditional
RFLP technology
are increased speedof analysis and dramatic reduction in the amount of DNA required for analysis. Reduction in
the amount of DNA required for a reaction is particularly
important in speedingup early
screening.
In case of radish, RAPD analysis could be done 3-5 days after germination instead of23 weeks in RFLP or protein analysis. Using one 96-well PCR machine, a single person cancomplete the analysis of approximately
250 individuals
with a primer in ten days period, fromthe beginningof seed germination
to final results;
similar results with RFLP analysis would
take at least 3-4 weeks, and most likely more. However, the main disadvantage
of RAPD
method in relation to the purity test of Fl hybrid is that most of polymorphisms
detected areconsidered
as dominant marker, for example, present versus absence in each of the parents,
and therefore heterozygotes
can not be distinguishedaccurately. To solve this problem, RAPD
marker which can be used for purity test should be tested with many individualsof populationin the same parent line to get reproducible
result.
This experiment suggest that it is the most important to select specific RAPD markers
out of many random primers for rapid identificationof each inbred parent lines and theirhybrids, and for the purity test of F1 hybrids.
Acknowledgement
This work was supported by a grant from R&D Promotion Program, Andong National
University,
Korea. Wethank Hae-jin Hwang for providing inbred stock seeds.
References
Boehm, C. L., H. C. Harrison, G. Jung, and J. Nienhuis.
1999. Organizationof American andAsian ginseng germplasm using randomly amplified polymorphic
DNA(RAPD)
markers.
J. Amer. Soc. Hort. Sci. 124:252-256
Huff, O. R., R. Peakall, and P. E. Smouse. 1993. RAPD variation within and among natural
populationof outcrossing buffalograss.
Theor. Appl. Genet. 86:927-934
Welsh, J., R. J. Honeycutt, M. McClelland
and B.W.S. Sorbal. 1991. Parentage determinationin maize hybrids using the arbitrarily
primed polymerase chain reaction(AP-PCR).
Theor.
Appl. Genet. 82:473-476

Ill

The molecularbiologyof oil palm flowering

Harikrishna
Kulaveerasingam1,
Zaidah Rahmat1, Tan Siang Hee1, Lim Chin Ching2,'
Choi Mei Chooi1, Lee Yang Ping1 and Sh. Shahrul Rahiah Syed Alwee3
Genome Centre, Institute of Bioscience,' Universiti
Putra Malaysia, UPM-Serdang 43400,
Selangor, Malaysia. 2MalaysianPalm Oil Board, P.O. Box 10620, 50720, Kuala Lumpur,
Malaysia and 2PAMOL Plantations,
P.O. Box 1, 86009, Kluang, Johor, Malaysia

ABSTRAC T
The cloning of elite high yielding
oil palm can result in yield increases of up to 30%.
However,high incidences of floral abnormalities
have been observed with tissue cultured oilpalm clones. These floral abnormalities result in the feminisation of a ring of normally sessile
stamenoid tissue in female flowers converting them into carpel-like tissue. Little is known
about the molecular basis of either normal oil palm floral development or abnormal floral
development.
A preliminary study of oil palm floral development was conducted byexamining floral morphology using histology and SEM techniques. By this analysis oil palm
floral development could be separated into 8 key stages. It was observed that development of
inflorescences derived from abnormal oil palms did not differ from normal inflorescences
until stage 7 when supplementary carpel-likestructures surrounding the central carpel wasobserved.
Oil palm homologues of Arabidopsis SOC 1 and LEAFY were cloned byhybridisation
to homologous sequences from rice and Arabidopsis respectively.
UIP2 andRLK5 were cloned by a cold plaque hybridisation procedure. Expression profiles of thesegenes were examined using northern blots containing RNA extracted fromeach of the 8 key
stages from normal palms. In some cases gene expression was below the detection limits ofnorthern blotting. UIP2 and RLK5 homologues were preferentiallyexpressed in meristem
tissue and were either undetectable or expressed at lower abundance in floral tissue. In situ
hybridisationand semi-quantitative
RT-PCR analysis was undertaken to facilitatea higherresolution examination of gene expression differences between normal and abnormal oil palm
flowers. It was observed that LFY was expressed in normal stage 4 flowers and wasundetectablein abnormal flowers. However, SOC1 that acts up stream of LFY in
Arabidopsis showed an abnormal accumulation of SOC1 transcripts in abnormal flowers at alater stage of floral development (stage 5). In order to determine if molecular differences
between normal and abnormal flowers could be detected at an earlier stage of development anapproach using DNA macroarrays was pursued. These DNA macroarrays have allowed theidentificationof other genes that are differentiallyexpressed between normal and abnormal
palms. These genes are currently being characterised to enable a better understanding gene
expression differences associated with floral abnormalities to be obtained. It is hoped that
this will ultimately
lead to improved strategies
at predicting
and preventing floral
abnormalities fromoccurring.
INTRODUC TION
Flowers contain specialisedorgans and tissues that differ significantly
from those of
the vegetative organs of a plant. Commercially grown oil palm are hybrids that are derived
froma cross between Elaeis guineensis v. Dura and Elaeis guineensis v. pisifera.
The tissue
culture cloning of elite palms can result in 30% increase in yields that would normally take

112
more than 15 years conventional breeding to achieve. Unfortunately the clonal propagationof oil palm results in the production of abnormal mantled palms whose incidence can range
from 0-100% depending on the genotype and culture conditions used. In order to determine
the factors that influence flowering and mantled flower development in oil palm, thephysiology,
cytology and molecular biology of the flowering process had to be studied.
Mantled palms are characterisedby feminisation of the normally sessile male tissue in thefemale flower. In mantled flowers the stamenoid tissue forms a ring of supplementarycarpel-like structures fused to the central carpel. Often mantling results in bunch failure prior
to oil biosynthesis
and in most cases results in no oil yield at all.
The oil palm is normally monoecious with separate male and female inflorescences on
the same plant, but hermaphrodite flowers do occur occasionally (Hartley, 1988). The maleand female inflorescence is similar in gross architecture in having a central axis or peduncle
that bear flower-bearing
spikelets.
Oil palm flowering consists of varying cycles of either
male or female flowers that are influenced by environmental conditions (Corley, 1 976).
It is highly likely that the flowering process in oil palm will in general be similar withgrasses as well as with dicots such as Arabidopsis.
It has been well establishedthat the firststep of flower development is the switch from the vegetative phase, during which leaves andshoots are produced to the reproductive phase where flowers are initiated. It has also been
determined that once this switch has been made floral meristem-identity
genes promote theinitiationof floral development.
In Arabidopsis,these genes include LEAFY (LFY),APETALA1 (API), CAULIFLOWER (CAL), APETALA2 (AP2) and UNUSUAL FLORALORGANS (UFO) Levy and Dean (1998).
Many of these genes have been isolated andcharacterised from numerousplant species including cereals, and in many cases share a highlevel of homology. Therefore these genes could be used as a means to identify and isolate
orthologues from oil palm. Once floral meristems have formed homeotic genes such as
AGAMOUS(AG), APETALA3 (AP3), PISTDLLATA (PI) and AP2 interact to produce thewhorl structure of flowers. At this stage of development the basic ground structure of theflower is laid down and their further development results in functioning mature flowers.
Weused an evolutionary strategy to clone gene homologues of AG and LFY from oilpalm by using AGAMOUS-LIKE (AGL's) genes from rice and LFY from Arabidopsis.
Other genes of interest appeared to have diverged in sequence and a cold plaque hybridisation
strategy was employed as a gene discovery and isolation tool. These genes were then used to
examine gene expression in both normal and abnormal flowers by way of northern blots,
macroarrays and in situ hybridisation.
RESULTS
Structural
examination of oil palm floral development
Floral development can be separated into 8 phases (in female flowers) as follows:
stage 1 ; inflorescence meristem develops, stage 2; spikelet primordia develop from primaryaxis, stage 3; bracts develop around spikelet
and sex determination
occurs, stage 4;
emergence of flower primordia, stage 5; emergence of male flowers in triad of female
inflorescence, stage 6; formation of female flower and bracteole in female inflorescence,stage 7; development of carpels on female flower, development of supernumerary carpels on
abnormal flowers, stage 8; carpels have fully developed.
Gene expression analysis by reverse northern's
A 123 CDNA clones of above 500 bp in size isolated by cold plaque screening were
subjected to reverse northern analysis. The probe used was amplified from total RNA of oilpalm immature male, female flowers and young leaves by PCR and the clones were shown to113fall into five subpopulations
which, correspond to the abundance of the genes in the mRNA
population.
Sequencing was carried out on 68 clones and sequence comparisons were made using
the BLAST algorithm (Altschul
et al., 1997).
By sequence analysis three categories of clones
could be found among the subpopulations.
The first category, which represented about 40%of the clones sequenced, did not have any homology to any known sequences in the Genbank
database. The second category contains clones that show similarity
to known low abundance
genes e.g. UIP2 and RLK5. The third category contains clones that show similarity to higher
abundance genes e.g. cyclophilin,
elongation factor 1-a and aldolase (see Fig 1).
Northern analysis was performed on 10 clones represented genes with a wide range of
expression profiles. Generally, all the tested clones gave very weak signals or none at all.
This observation supported the fact that the clones tested are low abundance genes. The
hybridisation
patterns also showed that the highest hybridisation
signals came from themeristem and very young male inflorescence.
No signals were detected from 6 cm maleinflorescence,the developmental
stages chosen to construct the male flower CDNAlibrary.
This thus explains why these clones were not picked up during the differential
screening as
they are present at such low abundance at that developmental
stage. Northern blotting also
yielded very weak signals with SOC1 and LFY orthologues.
DNA macroarrays containing 123 CDNA clones isolated by cold plaque screening
were hybridized to labelled
CDNA from normal and abnormal flowers at the same stage ofdevelopment (stage 5). Several clones were expressed only in normal floral tissue and not in
abnormal tissue (Fig 2). One of these three clones encoded HSP80-2. A more in depth studyof the expression of this gene during normal and abnormal development is on going.
In situ hybridisation
This method was used to examine the temporal and spatial pattern of expression of
RLK5, UIP2, SOC1 and LFY. We were interested in determining at what stage of floral
development did changes in temporal and spatial expression patterns occur between normal
and mantled flowers. As we could only detect morphologicaldifferences
at stage 7 we
decided that the abnormality must have occurred prior to this stage. As a first step candidate
genes involved in early floral development were used on in situ 's to examine gene expression
pattern differences
between normal and mantled flowers. The genes RLK5 and UIP2 areexpressed in meristems, SOC1 acts up stream of LFY a meristem identity gene and theirinteraction integrates the floral evocation signal pathway genes with the autonomous pathway
in Arabidopsis(Samac et aly 2000).
G en e ra l G e n e F u n ctio n

P erc en ta g e/ n u m b e rs

E xa m p les o f ge n e s

H o u se k e ep in g g en e s

1 0 ( 14 .7 % )A ld o lase,

F lo ra l d ev e lo p m en tal g e n es

14 (2 0 .6 % )H eat sh o ck p ro te in
K in ase s/s ig n a llin g

3 (4 .4 % )
3 (4 .4 % )m o lec u le s
U nk n ow n
O th e rs

2 7 (3 9 .7 % )l l (16 .2 % )U b iq u itin ,cy clo ph ilin
M A D S b o x p ro te in , Z m H o x
2 , p o lle n c o at p ro te in
H S P 7 0 , H S P 8 1‑2 , H S P 9 0
R L K 5 , P u tativ ep roteink in as e
A p o p to sis p ro tein , T A P 1

Fig.l Sequence comparison of CDNA clones derived from cold plaque screening
6 male flower CDNAlibrary. A total of68 sequences analysed.

ofa stage 5-

A

B

Fig«2 The same DNA Macroaixay of floral genes hybridised
to normal (A) and abnormal(B) stage 5 female floral CDNA. Arrows indicate up-regulated genes in each population.

MEFEMENCES
Altschul,
S. F., Gish5 W., Miller, W., Myers, E. W. and LIpman, D. I (1990).
Basic Local
Alignment Search Tool Journal ofMolecular Biology 215 : 403 - 410.
Corley, RHV (1976).
Inflorescenceabortion and sex differentiation.
In: RHV. Corley, JJ
Hardon, BJ Wood (eds), Developments in Crop Science. 1, 37-56,
Hartley, CWS(1988).
The Oil Palm. 3rd Ed. Longman Scientific and Technological,
UnitedKingdom. Pp 31-70.
Levy YY, Dean C (1998).
The transition
to flowering. The Plant Cell, 10, 1973-1989.
Samac, A, H. Gnouchi, Gold, SE, DItta, GS, Schwarz-Sommer, Z, Yanofsky, MF, Coupland,
G (2000).
Distinct
roles of CONSTANS target genes in reproductive
developmentofArabidopsis.
Science, 288, 1613-1616.
ACKNOWLEDGEMENTS
We thank Dr. Detlaf Weigel (The Salk Institute)and Dr Enrico Pe (Universityof Milan) for
kindlyprovidingthe LFY and OSMADS cDNAs; Golden Hope Plantations
and Advanced
Agricultural
Research for oil palm samples, Dr. Marty Yanofsky (Universityof California,
San Diego) for helpful discussions
and the Ministry of Science, Malaysia and the MalaysianPalm Oil Board for providing
research funding.

115

On the synteny among grass genomes
Yoshiaki Nagamura
National Instituteof Agrobiological
Key words: synteny, colinearity,

Resources, Tsukuba, Ibaraki
grass genome, rice, sorghum

305-8602,

Japan

In the last decade, molecular genetic maps using DNA markers have been developedin many plants including the Gramineous cultivars such as rice, sorghum, maize, barley
and wheat. Moreover, genetic maps using commonsets of restriction
fragment length
polymorphism(RFLP)
markers have recently provided tools for comparison of thearrangement of low-copy number genes among various grass species. This has led tofinding extensive conservation of gene order, so called synteny, within homoeologous
chromosomal segments.
I report on the synteny among cereal genomes, in particularsynteny between rice andsorghum genomes revealed by comparative mapping using rice DNA markers.

Comparative

mapping among grass genomes

In comparative
mappingamong grass genomes, the first publications
thatdemonstrated
synteny among the cereals were by Ahn and Tanksley (1) for rice andmaize and Kurata et al. (7) for wheat and rice. Subsequently
it has been reported that thecolinealityof gene order can be found widely among grasses(2,3, 4, 5, 9, 10, 1 1).
On the basis of the above reports, Moore et al.(8) showed that all the genome maps
could be combined in a single synthesis.
A recent evaluationof the extent of synteny
among grass genomes reported by Devos and Gale(8, 12).

RFLP linkage

map of sorghum

The Rice Genome Research Program (RGP) in Japan has constructed a high density
rice genetic map with 2,275 DNA markers covering 1,521.6
cM using 186 F2 plantsfrom a single cross between the japonica variety Nipponbare
and the indica variety
Kasalath (6). Among the mapped markers, about 1 800 rice DNA probes were analyzed
for polymorphismin the two parental varieties,
Sorghum bicolor (Nagano232)
and S.
bicolor (F63A5).
As a result, about 60% of analyzed rice DNA probes clearly revealed
cross-hybridization
to the sorghum genome and about 30% of these probes revealed
polymorphism.
So far, we have constructed
an RFLP linkage map of sorghum with a
total of 602 DNA markers that consist of rice DNA clones and maize DNA clones (theUniversityof Missouri).
This sorghum map is based on a set of loci that cover nearly
the entire rice RFLP linkage map (6). In this map, the maize CDNA clones were mapped
to linkage groups that correspond to previously
reported sorghum genetic map and theten linkage groups were defined as linkage group A to J as described on the known map

(5, 9, 10, 1 1). The total

map distanceis 15285

2. 1 cM, determinedby dividing

Syntenic

between rice and sorghum genomes

regions

the total

cM. The average distancemap distance

betweenloci isby the number of loci.

The majority of the mapped loci clearly indicateda high degree of colinearity
betweenthe 12 rice chromosomes and the ten sorghum chromosomes as shown in figure 2.

Fig. 2. Syntenk
The linkage
are indicatedregions

between rice and sorghum genomes.

groups of sorghum are shown as A to J. The corresponding
as R with the chromosome number ( 1 to 12).

rice chromosomes

Sorghum linkage group A is homoeologous mainly to rice chromosome 8 and to a
portion of rice chromosome 9. Similarly,sorghum linkage group B is composed mainlyof loci from rice chromosomes 4 and a part of long arm in rice chromosome 2, group C
is composed mainly of loci from rice chromosome 3 and a part of rice chromosomes 7
and 10. Sorghum group D linkage group corresponds
solely to rice chromosome 2
whereas group E corresponds mainly to rice chromosome 5. Group F is composed
mainly of loci from rice chromosomes 7 and 9, and a part of the long arm in rice
chromosome 8, group G solely from rice chromosome 1, and group H from rice
chromosome 6. Although linkage group I is composed of loci from a part of distal
region of rice chromosome 12, large remained region on linkage group I could not befound clear syntenic region corresponded
to any rice chromosome. Linkage group J is
composed of loci from rice chromosome ll. These results show that there is anextensiveconservationof gene order in rice and sorghum genomes. Comparative
mapping has revealed a high degree of colinearity
between the 12 rice chromosomes
and 10 sorghum chromosomes and the ordering of DNA loci on rice and sorghum
chromosomes corresponds
well.
These data provide
valuable
informationinunderstanding
the evolution of grass species. At a practicallevel, comparative mapping
between these two genomes can be useful in map-based cloning of specific genes. Since

117rice is considereda model species for genome analysis among cereals, genes located insorghum might be isolated by map-based cloning of the homologue in rice, and then byhomology in sorghum.

References
1. Ahn S, Tanksley

SD: Comparative

Natl Acad Sci USA 90: 7980-7984
2. Antonio BA, Inoue T, Kajiya
Nakagahra

M, Sasaki

five populations

linkagemaps of the rice and maize genomes. Proc

(1993).
H, Nagamura Y. Kurata N, Minobe

T: Comparisonof geneticof rice. Genome 39: 946-956

3. Devos KM, Gale MD: Extended

distance

Y, Yano M,

and order of DNA markers in(1996).

genetic

maps of the homoeologous

group

3

chromosomes of wheat, rye and barley. Theor Appl Genet 85: 649-652
4. Devos KM, Wang ZM, Beals J, Sasaki T, Gale MD: Comparative(1993).
genetic

foxtail

Genet 96: 63-68

millet(Setaria

italica)and rice (Oryza

sativa). TheorApplmaps of(1998)
5.. Dufour P, Grivet L, D'Hont A, Deu M, Trouche G, Glaszmann, Hamon P:
Comparative genetic mapping between duplicatedsegments on maize chromosome 3
and 8 and homoeologus
1030 (1996).
6. Harushimaregionsin sorghum and sugarcane. Theor Appl Genet 92: 1024-

Y, Yano M, Shomura A, Sato M, Shimano T, Kuboki

Lin SY, Antonio

BA, Parco A, Kajiya

N, Khush GS, Sasaki
using a single

H, Huang N, Yamamoto K, Nagamura Y, Kurata

T: A high-density

F2 population.

Y, Yamamoto T,

Genetics

rice genetic
148: 479-494

linkage

map with 2275

markers(1998).

7. Kurata N, Moore G, Nagamura Y, Foote T, Yano M, Minobe
Conservation of genome structure between rice and wheat. Bio/Technology

Y, Gale M:
1 2: 276-278(1994).
8. Moore G, Devos KM, Wang Z, Gale MD: Grasses,
Bio 5: 737-739

line up and form a circle.

(1995).

9. Paterson AH, Lin Y-R, Li Z, Schertz KF, Doebley
JW, Irvine J]E: Convergent domesticationof cerealcorresponding

genetic

10.

MG, Lee M, Bramel-Cox

Pereiraloci.

Science269:

1714-171

JF, Pinson SRM, Liu S-C, Stansel
crops by independent
mutations at

8(1995)P, Woodman W, Doebley

Constructionof an RFLP map in sorghum and comparative

37: 236-243(1994)ll.

Whitkus

R, Doebley

maize. Genetics

132:

1119-1130

J, Lee M: Comparative

mapping

genome mappingJ, Whitkus
in maize.

R:

Genomeof sorghum

and(1992).

12. Devos KM, Gale MD: Comparative
15(1998).

Curr

genetics

in the grasses.

Plant.

Mol. Biol.

35:

3-

118

.

SNP, Genetic

Diversityand Genepoolsfor Germplasm

Enhancement

Kazuo N. Watanabe
Dept. Biotech.
Intra-locus

Sci.,variation,

Kinki

University,

Inter-locus

interaction,

Uchita,Wakayama,

649-6493,

polyploidy,

Intra-specificvariationJapan

UNIQUENESS OF PLANTS IN GENOMICS
Plant genetic resources have been provided life to human being and to the earth. They canprovide indefinite
values for mankind. This is not only for the conventional uses but for
application
to fundamental research in genomics. Uniqueness of plants compared withanimals and microorganisms in genomics would be laid in the followings:
1) Feasibilityofcreating large and/or specific populations at will; 2) specific genetics lines without major
concerns on regulatory matters and ethics; 3) application
study on many populations in nature
or in artificial
conditions;4) not like bacteria which cannot be addressed its ecologicaldistribution
easily, in contrast, particularly,
many agricultural and/or industrial plant species
are in databases and also often in genebanks with relevant data; 5) monitoring of populations
are easy compared with animals which can have movementand drifting from one nitch toothers; 6) variation in reproductive
systems, they are options for conservation andpreservation of germplasm; and 7) frequent occurrence of polyploidy
and aneuploidy innature and feasibility
in artificial creation.
ALLELIC POLYMORPHISM, NUCLEOTIDE SEQUENCE VARIATION AND SNPS IN A
LOCUS OF INTEREST
It is naturalthat intra-locus

variations

with various alleles

occur regularly

in plant genes.

However, it seems that at present, elucidationof the relationships
between allelic difference
and their nucleotide sequences is more than challenging
and cumbersome (Weiss andTerwillinger

2000).

The accumulation of infonnation

on sequence variationand testing

feedback from different cases should provide more precise tools in selecting specific traits oralleles in germplasm enhancement (Kasai et al. 2000). The occurrence of SNPs in specificloci could givQ a prediction of genetic change and facilitationin crop breeding by pin-pointinga specific nucleotide change for the alternation.
Adding more to the dimension, polyploidy
is unique feature of plant genomics.
Polyploids
are especiallyseen frequently in Angiosperm: it is estimated that dicots consist ofaround 200,000

species,and more than half of the number in monocots could be polyploid

or

119
originated from polyploidization.
Uniquenessgenetics is that they have built-in intra-locusof the polysomic polyploids
with respect to thevariation due to the increased copy number ofallelesand consequently
more possibilityof allelic interactions(Watanabe et al. 1 99 1). Built-in heterozygosity
in disomic polyploids
however, yet its unique genetic
enhanc ement.

feature should be elucidated

compared with diploids
is a traditional
concept,

for facilitated

UNDERSTANDING AND CAPTURING GENETIC DIVERSITY

uses in germplasm

ARE THE KEY FORCOMPREHENSIVE GERMPLASM ENHANCEMENT
Intra-specificgenetic

variation,

which creates genetic

diversity,

is fundamental

material forplant breeding. Increase in intra-locus variation by the enhancement of genetic diversity in theavailable
genetic resources, had been demonstrated drastic gain in the agronomic traits.
Adequate use of such genetic diversity
by participatory
plant breeding gained success incombating major constraints such on rice crops against rice blast (Wolfe 2000). Now
challenge shall be made how the genetic diversity can be effectively
captured and maintainedfor immediate needs. Component studies on the quantitative
traits into single factors could
make the clear understanding
and allelic combinations
and inter-locus
interactions
incorresponding
loci
McCouch 1997).
INTER-SPECIES

for the quantitative

DIVERSITY

traits(Nadeauand Frankel

2000,

Tanksleyand

IS UNEXPLORED IN CROP GENETIC RESOURCES

It is estimated that merely less than 1% of species out of 10 millions of species
is studied on the biodiversity
aspects (Wilson 2000). Even on the agricultural
around 30 taxa get a focus for the major investment,

and the related

in this planet
species, only

genera, that is wild

species, have been treated rather as orphans. In contrast, the genetic diversity in these relative
genera have been demonstrated substantial
additionsin germplasm enhancement such on
tuber-bearing Solarium (Watanabe et al. 1994). My example at present shall also give the ideathat incorporationof specificallelesfrom distantly

relatedwild species

could provide many

advantages into crop genetic improvement.
In order to understand the genetic diversity
and the holistic sense of species
variations, universal measurements such on genetic markers are required over different taxa ofplant species. In contrast, the majority of quarter million species in Angiosperm, do not have agenetic marker for genome mapping, consequently genome-wide information is not available
for the genetic
evolutionary

diversityin order to solving

aspects. A challenge

the questions

in conservation,from my research is given to this pitfall

utilization

andby taking a case on

120
rare and extinguishing
genetic

crop species.

Noweven the distantly related genera could be immediate sources for augmenting
diversityin the crop cultivars by overcoming the classical genepool barriers. Now

genepools can be put into one entity into a genie pool with the employment of the modern
biotechnology.
The variations
in alleles, in other word, SNPs responsible
for phenotypicalteration,systematic

reside in one genie pool, and configurationof genetic components should lead touses of "the modern genepool" consists of the diverse plant genetic resources ofthis planet. Also the prediction obtained from SNPs could be applied
the desirable alleles from the available genepools.

to recognize

and capture

CLOSING
Genomics and modern technology must be supportive on the past experience and knowledge
which are the theory and central dogma on the genetics. The respect to the classical theory
and findings is important as they remain correct, meanwhile we add new knowledge on to it
for better comprehension.
Science must be innovative, on the other hand, in a sense
retrospective
for a better scientificpast for the future. On this respect,and ethicalindigenousdevelopment, and there is learningknowledge and traditional
practicefrom therelated tothe plant genetic shall be considered as a component of conservation and uses of genetic
diversity
as bioprospecting
is also ultimately
importantaspect associatedwith thebiodiversity.
ACKNOWLEDGMENTS
Author thanks for the support from a grant from Japan Science Promotion Association,

JSPS-

RFTF96L00603

and acompetitive(Furusawa/Mise
grant (G026)

Project),

a gift grant from Sumitomo Foundation,from Kinki University.

REFEREN CES
Kasai, K., Y. Morikawa, V. A. Sorri, J. P. T. Valkonen, C. Gebhardt and K, N. Watanabe
2000. Development of SCAR markers to the PVY resistance gene Ryadg based on a common
feature of plant disease resistancegenes. Genome43: 1-8.

Nadeau, J. H. and W. N. Frankel 2000.

The roads from phenotypicvariation

to genediscovery:

mutagenesis

Tanksley,

S. D. and S. R. McCouch 1997. Seed banks and molecular maps: Unlocking

genetic

versus QTLs. Nature Genetics 25 :38 1-3 84.

121
potentialfrom the wild

Science

Watanabe,K., S.J. Peloquin,

polyploidization:

Somatic

Watanabe,K, M. Orrillo,

enhancement withtetraploid

Fl hybrids

Genet. 88(2):disomic

277:

1063-1066.

and T. Endo.

doubling

or 2n gametes?

S. Vega,

R. Masuelli

tetraploid

Solarium

betweenS. acaule

Genetic

significanceof mode of

Genome 34: 28-34.
and K. Ishiki.
acaule.II.

and tetrasomic

1994.

Potatogermplasm

Asessment

to breeding

value

tetraploid

potatoes.

of

Theor. Appl.

135-140.

Weiss, K.M. and J. D. Terwillinger
with SNPs? Nature Genetics
Wilson,

1991.

2000.

How many diseases

does it take to map a gene

26: 151-156.

E. O. 2000. A Global biodiversity

Wolfe, M. S. 2000. Crop strength

through

map. Science
diversity.

289:2279.

Nature 406: 681-682.

Diversityof Plant Genomes Revealed by Analysis
Gene Sequences and Microsatellites

ofRobert J Henry, Centre for Plant Conservation Genetics
Southern Cross University, PO Box 157, Lismore, NSW, 2480, AUSTRALIA
Molecular analysis is allowing the genetic relationships
between plants to be better defined.
Sequencingof plant genes provides a key tool for phylogenetic
analysis.
The analysis of
microsatellite
loci represents
a specificopportunity
revealing
genetic relationships
andpopulation
diversity.
Recent analysis (Soltis
et al, 1999) confirms the view (Figure 1) thatflowering
plants cannot be simply divided into monocotyledonsand dicotyledons(Henry etal,1997).

Fig 1. Relationships

Analysisof Ribosomal

between higher

plants(Henryet al. 1997;

Soltiset al, 1999)

Genes

Ribosomal genes in plants have been widely used for phylogenetic
analysis.
The spacer
between 5S ribosomal genes have proven very useful for species level distinction.(Koet al,
1996).
The 18S ribosomal RNA gene sequence and the internal transcribedspaces between
the ribosomal genes have been widely used to determine phylogenetic
relationships.
Thesesequences allow analysis
at different
levels.
The ITS sequences are especially
useful for
analysis of species divergence within genera(Abedinia
et al., 1998).
The sequence of the 18S
ribosomal
genes have been used in establishing
relationships
within the gymnosperms(Graham et al, 1996) This analysis reveals the very high levels of genetic diversity
withinsome gymnosperm species.
Examples of recent applicationsof these methods to explore
diversity
in different plant groups will be presented.
Microsatellite

analysis

Microsatellite
or simple sequence repeat (SSR) loci are especially
useful for exploring genetic
diversity
within plant species because of their relatively
high rate of evolution.
The extent towhich microsatellite
loci are conserved across taxa is a very useful indicatorof the genetic
diversity within and between the groups being analysed. The extent of sequence conservationin microsatellite
loci has been investigatedin many species.
Microsatellites
found in

expressed sequenced tags including
those found within coding regions are more highly
conserved allowing wider transferabilityof these molecular markers between different plant
species (Table 1). The type of informationthat can be gained by these analysis will beillustrated
by presentationof recent data from several different plant groups.
Table 1

Plant microsatellite

analysisand cross-species

transfer

Species

Sources of SSR

Transfer to otherSpecies

Reference

BarleyEST

Wheat

Henry et a., unpublishedS ugarc ane

Genomic/ES T

Saccharum species

Cordeiro

Grape

EST

Vitis genus

Scott et al., 2000

Tea Tree

Genomic

Myrtaceae

Rossetto

et al., 2000

Pinus

Various

Pinus species

Shepherd

et al., unpublishedet aL, 2000

Cereals
Wheat and barley microsatellites

from genomic clones and ESTs have been compared.

The ITEC database has been analysed for microsatellite
loci.
More than 700 loci wereidentified
in the first 18,000 sequences (Holton et al, unpublished).
These loci have provenpolymorphic with the barley loci likely to be of value in wheat analysis.
Rice microsatellites
are now available in large numbers from the genome sequence. The
complete microsatellite
compliment of a higher plant genome could not be analysed in thisspecies.
Sorghum microsatellite
sugarcane.

loci

can be detectedusing

primers

from related

speciessuch asSugarcane
Sugarcane has a highly complex genome. Microsatellite
markers from sugarcane are useful inthe analysisof the Saccharum officinarum and Saccharum spontaneum progenitor
species(Cordeiro
et al, 2000). EST derived microsatellites
are much more easily transferred
to progenitor
species (Cordeiro et a/., unpublished).

124
GrapesGrape(Vitis vinifera)
microsatellites
were identifed
from ESTs (Ablett
et al,
Microsatelites
from grape ESTs were readily transferred
to other Vitis species (Scott
2000).
These loci were more transferable
than those from genomic libraries (Rossetto
unpublished).

2000).
et al.,.
et al,

Pines
Microsatellites
related species

in Pinus species have been successfullytransferredsuch as the hard pines (Shepherd
et al, unpublished).

betweenmore closely

Eucalypts
Microsatellites
derived from the Australian tea tree (Melaleuca alternifolia)(Rossetto
et al,
1999a) have been found to transfer widely within the Myrtaceae family (Rossetto et al, 2000)
to species including
Eucalypts.
Most variation in this species was with rather than between
populations(Rossetto
et al, 1999b).
Mangroves
The mangrove species Avicennia marina, was found to have very high levels of genetic
diversity
as measured by microsatellite
polymorphism(Maguire
et al, 2000a).
Most variation
was between rather than within populations(Maguire et al, 2000b)
Conclusions
The transferabilityof microsatellites
from ESTs is much higher than that for microsatellites
from genomic clones. The ease of transfer within different
groups reflects
differencesinsequence divergence and genetic distances.
Microsatellite
transfer efficiency
can thus be used
as a measure of genetic diversity
within and between plant taxa.
Acknowledgements
Supportof the Cooperative
Research Centre for Molecular
Plant Breeding
Cooperative Research Centre for Sustainable
Production Forestry is acknowledged.

and the125

References
Abedinia,
parviflora
406.

M., R. Henry and S.C. Clark. 1998. Distribution
and phylogenyof Potamophila
R.Br. a wild relative of rice from eastern Australia.
Genet. Res. Crop Res. 45:399-

Ablett, E., G. Seaton, K. Scott, D. Shelton, M. Graham, P. Baverstock,
S. Lee and R. Henry.
2000. Analysis
of grape ESTs: global gene expression patterns in leaf and berry. Plant Sci. inpress.
Cordeiro,sugarcaneG., G. Taylor and R. Henry. 2000. Characterisation(Saccharum sp.) a highly polyploid
species. Plant

of microsatellite
Sci. 155: 161-168.

Graham, G.C., R.J. Henry, I.D. Godwin and D.G. Nikles. 1996.
pine (Araucaria
cunninghamii).
Aust. Syst. Bot. 9:893-902.
Henry, R.J.
Dicotyledons:markers from

Phylogenetic

positionof hoopand P.J. Harris.
1997. MolecularDistinction
between Monocotyledons
more than a simple dichotomy.
Plant Mol. Biol. Rep. l5:216-218.

Ko, H.L. and R.J. Henry.
identificationin admixtures.

1996. Specific
5S Ribosomal
RNA primers
Plant Mol. Biol. Rep. 14(l):33-43.

for plant

Maguire, T., K. Edwards, P. Saenger and R. Henry. 2000a. Characterisation
microsatellite
loci in a mangrove species Avicennia marina (Forsk.)
Vierh.
Theor. Appl. Genet. 101:279-285.

andspecies

and analysis(Avicenniaceae).

of

Maguire, T., P. Saenger, P. Baverstock
and R. Henry. 2000b. Microsatellite
analaysisofgenetic structure in mangrove species Avicennia marina (Forsk) Vierh. (Avicenniaceae).
Mol.
Ecol. in press.
Rossetto,
M., A. McLauchlan, F.C.L. Harriss,
R.J. Henry, P. Baverstock,
Maguire and KJ. Edwards. 1999a. Abundance and polymorphismof microsatellite
tea tree (Melaleuca
alternifolia- Myrtaceae).
Theor. Appl. Genet. 98: 1091-1098.

S.L. Lee, T.
markers in

Rossetto,
M., R.W. Slade, P.R. Baverstock,variation
and assessment of genetic structure
Mol. Ecol. (submitted).

Microsatellite- Myrtaceae.

R.J. Henry and L.S. Lee. 1999b.
in tea tree (Melaleuca
alternifolia)

Scott, K.D., P. Eggler, G. Seaton, M. Rossetto, E.M. Ablett, L.S. Lee and R.J. Henry. 2000.
Analysis of SSRs derived from grape ESTs. Theor. Appl. Genet. 100:723-726.
Soltis,P., D. Soltis and M. Chase. 1999. Angiosperm
as a tool for comparative
biology.
Natur. J. 1:42-44.

phylogeny

inferredfrom multiple

genes

126

"Genome fusion for crop improvement",
Daisuke Shibata

a new style of breeding(Kazusa DNA Research Institute1)

Key words; Transformation-competent Artificial
quantitative
trait loci (QTL), genome, Arabidopsis

chromosome (TAC),
thaliana.

Plant transformation,

Introduction
Plant breeding is the technology that human has been used since the beginning of agriculture.
Selectionof useful traits from natural variations
and introductionof these traits into
cultivatedplants have produced a large collectionof agricultural
materials.
Breeding
programs depend on sexual crosses of closely related plants, otherwise, an agricultural
important trait of a plant may not be transferred directly to other plants that can not becrossed to the plant. Cell fusion of two organisms and subsequent regeneration could be amean for introducing the whole genetic information into the other. However, it has beenrealized that cell fusion of two distinct cells results in partial incorporation of either genome
into the other genome, and even if succeeded, the efficiency of regeneration from the fusedcell into the whole body is very low. Furthermore, such regeneration is limited in cell fusionof closely related species. As a new technology, genetic engineering has shown the power oftransferring useful genes into plants. However, the technology available at the present time islimited for introduction of only several genes. Our final goal is to introducing important traits,
which are regulated with a complex set of genes, into other crop species by genetictrans formation.
We developed a vector for plant transformation with large DNA fragments andpropose a new meanfor introducing useful traits from a plant into another plant using thevector system. I conceptualize the process as a new style of breeding, "GenomeFusion forcrop improvement".
Genomefusion for crop improvement
The vector that we developed for plant transformation with large DNA fragments is called
Transformation-competentBacterial Artificial
Chromosome (TAC) (Liu et al., 1 999, Shibata& Liu, 2000). A genomic DNA library whose member clones carry approximately
100 kb ofgenomic DNA fragments of a plant species can be prepared using the TAC vector. The large
DNA fragments are transferred directly into other plant species via Agrobacterium. The
whole genome of a plant species, from which a TAC library is prepared, could becontiguously covered along with all chromosomes with thousands of TAC clones as physicalcontigs (see below such case in Arabidopsis).
Recent progress of genome analysis of crop
species could make a preparation of such contigs easier. It is possible to transfer the whole

1 532-3

Yana, Kisarazu,

Chiba 292-0812,

JAPAN

127
members of the TAC contigs into other plant species for preparing a population of transgenic
plants, whatever it is laborious.
The TAC transgenic population shares a conceptual similarity
with a population ofnear isogenic lines (NILs) of a plant species with DNA segments of a wild relative. Recently
a newbreeding program has been proposed for introducing useful traits from a wild relative
into a present cultivated variety. In a previous breeding program a useful trait found in a wild
relative or an unfavorable variety has introduced in a present cultivated variety by crossing
them and subsequent backcrossing with the present variety until a near isogenic line (NIL) is
obtained. On the other hand, in a new program, a population ofNILs in which any segment in
the wild relative can be found in 100 - 200 lines are produced from a backcrossed population
using DNA marker assisted genotyping without tracing traits. Useful traits, most of which arefound as quantitative
trait loci (QTLs),
are identified
in the final NIL population.
This
breeding program is called the advanced backcross QTL method (Tanksley & McCouch,1997).
The NIL population
is similar to the TAC transgenic populationin terms ofpresentationof a genome in other plant population.
A TAC transgenic plant carries anadditional
copy of a genomic fragment. On the other hand, a NIL line has an introgressedsegment. The critical difference between the two breeding strategies is that the TAC-based
strategy can be applied to any donor genome, if the recipient plant is transformable with thevector.
The presentationof a genome in other plant populationis the initial
step forintroducing useful traits found in the donor genome in the recipient genome. If useful QTLs
are known to exist in the cross of the recipient and donor species, those QTLs should be
identified
in the TAC transgenic population as similarly to the advanced backcross lines.
Although epistaticinteractionof two or more loci is not detected in the population,
QTL
analysis of F2 or recombinant inbred (RI) lines could suggest epistatic loci. The information
will be used for identificationof lines showing epistatic interactionin the population. Oncelines carrying QTLs be identified, these lines will be crossed sexually for integration of these
traits in a recipient crop. If the donor genome species is incompetent for sexual cross to therecipient species, QTL information is not available. However, searching the TAC transgeniclines could identify lines that exhibit useful traits. The TAC fragments that are identified
tocarry useful genes can be used for testing the usefulness of these genes in other crops.
'Genome fusion for crop improvement' is defined as the process in which two genomes are
fused to a degree, to which useful traits are introduced from other genome.
A model system for genome fusion
We have been testing the conceptualized
strategy"Genome fusion for crop
improvement" using a model plant, Arabidopsis thaliana, as much genome information isavailable in the plant. To test the usefulness of the TAC vector system for stable introduction

128of large DNA fragments into plants, an Arabidopsis
mutant, sgrl, was used forcomplementation tests. The sgrl locus was physicallycovered with several TAC clones thatcarried approximately
80kb DNA fragments. Introductionof these TAC clones demonstrated
that the mutant phenotype was recovered to the wild type, showing the usefulnessoftransformation with large DNA fragments (Liu et al., 1999). Furthermore, the filamentous
flower mutation of Arabidopsis
was also recovered by introducing
a 80kb TAC clone,following the isolationof the FILAMENTOUS FLOWER gene (Sawa et al., 1999). Stable
integrationof several DNA fragments by the TAC system in the Arabidopsis
genome has
been shown in our laboratory.
Given that the Arabidopsis
genome is completely sequenced before the end of 2000,
sequencing of the insert ends of all TAC clones of the Arabidopsis
library is currently
underway at Kazusa DNA Research Institute in order to construct a TAC-based physical mapof the genome. Approximately
1,500 TAC clones carrying 80 kb genomic DNA fragments
may cover the whole genome of the Arabidopsis.
My laboratory is now working to
introducing the selected TAC clones into other accession and crop species.
Acknowledgements
I thank Dr. Satoshi Tabata of Kazusa DNA Research Institutecollaboration
with end-sequencing of the Arabidopsis
TAC clones.

(Chiba,Japan)

for

References
Liu, Y-G , Y. Shirano, H. Fukaki, Y. Yanai, M. Tasaka, S. Tabata and D. Shibata. 1999.
Complementation of plant mtants with large genomic DNA fragments by atransformation-competentartificial
chromosome. Proc. Natl. Acad. Sci. U. S. A. 96:
6535-6540
Sawa, S. , K. Watanabe, K. Goto, Y.-G. Liu, D. Shibata, Ei. Kanaya, E. H. Morita, K. Okada.
1 999. FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis,
encodes a protein with a zinc finger and HMG-related domains. Genes Dev. 1 3 : 1 079-1 088[see erratum: 13, 2337]
Shibata, D. and Y-G. Liu. 2000. Agrobacterium-mediatedDNAfragments. Trends in Plant Science 5: 354-357

plant transformation

Tanksley, S. D. and S. R..McCouch. Seed banks and molecular maps: unlocking
potential form the wild. Science 277, 1063-1066

with large

genetic

Proceedingsof poster presentations

In alphabeticalorder by author

129

Genetic diversity
and phylogenetic
relationshipmelons, based on isozyme and microsatellitein east and south Asian
polymorphisms.

Yukari Akashi1}, Tadayuki Wako2), Masaharu Masuda1} and Kenji Kato1}
1) Faculty of Agriculture,
Okayama University,
Okayama 700-8530, Japan
2) National Research Institute of Vegetables ornamental Plants and Tea, 360 Kusawa,Ano, Mie 514-2392, Japan
Key words: Cucumis melo, CAPS, microsatellite,

isozyme, phylogenetic

relationship

INTRODUCTION
East and south Asian melons are known as important genetic resources, having
resistantgenes for various kinds of diseases.
Irrespectiveof their importance andgenetic diversity,
classificationof east and south Asian melons is not consistent.
They are divided
into two varieties,
makuwa (dessert type) and conomon (picklingtype),and cultivatedas different
crops in Japan and China.
Both varietiesaresupposed to have originatedin India, the secondary center of diversityof cultivatedmelon, and were introduced into China from west by the 'Silk Road' (Kitamura
1950).
Some reports also suggested that they had been originatedfrom weed melon (var.
agrestis)
in China (Walters1989).
In this study, therefore,
isozyme, CAPS andmicrosatellite
variations
among east and south Asian melons were analyzed.
MATERIAL AND METHODS
Isozyme analysis:
Melon accessions examined were seventy-eight
landraces (C.
melo) from India, Myanmar, Laos and China, thirty-one accessions of vars. makuwa
and conomonfrom China, Korea and Japan, eight accessions of var. agrestis from India,
Korea and Japan.
In this study, Indian melon accessions were divided into fivegeographicalpopulations(West, Center, North, South and East), based on annual
precipitationin each area.
Cotyledon and root were sampled from two seedlings ofeach accession
for analysisof 6-phoshogluconate
dehydrogenase(6-PGDH),phosphoglucoisomerase(PGI), glutamic-oxaloacetictransaminase (GOT), peroxidase(POX) and acid phosphatase(APS).
DNA analysis:
DNA was extracted from the seedling of melon accessions, andmicrosatellite
and CAPS polymorphisms
were analyzed in two ACC synthase genes(CMe-ACSl , CMe-ACS2) and two ACC oxidase genes (CMe-ACOl, CMe-ACO3).
RESULTS AND DISCUSSION
Since most of Indian and Myanmar melons have not been classified
into varieties,
seed length was measured for all accessions, which exhibiteda continuous variation (413nn) among Indian cultivatedmelons.
Seed length of vars. makuwa and conomon
was shorter than 9mm, and was clearly different from those of vars. reticulatus,
cantaloupensis
and inodorus.
In the present study, therefore,
Indian melons weredivided into two groups, large (^ 9mm) and small (< 9mm) seed types.
Gene diversity
calculated
from allelic frequency at nine isozyme loci and four
ethylene-related
genes indicatedthat Indian melon was rich in genetic variation,
which
decreased from India toward east (Table 1).
Ap-31 and Pgd-ll were frequent in India
and Myanmar, while most of the melons in Laos, China, Korea and Japan carried PdgIs and Ap-33 except Chinese inodorus.
As to Ap-33, the frequency was more than
50 % in small seed melons in north and east India, though it was less than 10 % in otherparts of India.
Weed melon (van agrestis)in Japan and Korea proved to carry thesame isozyme alleles as vars. makuwa and conomon. On the contrary, Indian agrestis
was different from those, and carried the same alleles as Indian small seed melon.
The analysis of ethylene-related
genes, for seventy melon accessions showed thatvars. makuwaand conomoncarried the same alleles (Acsl-cl:
S/+, Acol-msl: (AT) 10),and nearly monomorphic (Table 2).
These alleles were frequently
found in central
and east India, indicatingthat vars. makuwa and conomon related with small seedmelons in north and east India, as also indicated by isozyme analysis.
Small seed type melons seemed to be originatedin central India and were selectedunder wet conditionin east, since they were frequent in central and east India andgenerally
more tolerant to wet stress (data not shown).
Because of heavy rain in east
India and Myanmar, only wet tolerant melons should be selected and establishedspecialpopulationsin Laos and China, from which vars. makuwa and conomonseemed to be
derived.
Therefore, further study is necessary to clarify the origin and differentiationof vars. makuwaand conomon.
Table 1. Allelicand polymorphic

frequency at three isozyme loci, Ap-3 (acid phosphatase),Px-2 (peroxidase)index within melon accession groups in east and south Asia.

Table 2. Allelic frequency at two ethylene-related
accessions in India, Mvanmar and China.

and Pgd-1 (6-phoshogluconate

dehydrogenase),

genes, CMe-ACSl and CMe-ACOl , in small seed type of melon

ACKNOWLEDGMENTS
Wewould like to thank Dr. K.R. Reitsma, Iowa State University,
Kyoto University,
for kindly supplying
the seeds.

and Dr. E. Nawata,

REFERENCE
Kitamura,S (1950)
Notes on Cucumis of Far East. Acta Phytotax. Geobot. 14: 41-44
Walters,TW (1989)
Historical
overview of domesticatedplants in China with special
emphasis on the Cucurbitaceae.
Econ. Bot. 43: 297-313.

131

DNA sequence data and phylogeny:
Vigna subgenus Ceratotropis a case study
Koji Doi, Akito

Kaga, Norihiko

Tomooka and Duncan A Vaughan

NIAR, Tsukuba, Japan
Introduction
The subgenus Ceratotropis(genus Vigna, Fabaceae) consists of about twenty species ofwhich 8 are cultivatedfor food or forage (Tomooka et al., 2000). The wild species havepotentially
useful traits for breeding.
Knowledge of the phylogenetic
relationships
amongtaxa is helpful
to developing
strategies
for conservation
and use of wild species. The
objectiveof this study was to analyse a core set of germplasm from the subgenus
Ceratotropis based on sequence data of the internal transcribedspacer region (ITS) of nuclear
rDNA.
Materials
and Methods
Total genomic DNA was extracted from leaves of 25 accessions of different
taxa in the
MAFF genebank system. PCR amplificationof ITS rDNA and direct sequencing
were
performed by standard procedures. Phylogenetic
analysis was carried out using two methods;
neighbor-joining
method (NJ), and maximum likelihood
method (ML).
For the NJ method,
DNADIST and NEIGHBOR programs in PHYLIP ver. 3.57c were employed.
The sequence
data set was also subjected to fastDNAml for ML analysis. Bootstrap test with 1000 repeats
was performed for each analysis.
Results and Discussion
The data set analysed consistedof 670bp sequences for each sample for phylogenetic
analysis. Trees by NJ (Fig. 1) and ML (not shown) have similar topology.
The results agree
closely with classificationof this subgenus based on morphology, biochemical
and molecularanalyses (Konarev et al.; Tomooka et al. 2000 and in preparation).
The subgenus has been
divided into 3 sections,sections Angulares, Radiatae and Aconitifoliae.
These three sections
are clearly separated. Only one species V trinervia that is considereda member of section
Angulares appears intermediate
among the three sections and this may be indicativethat thisspecies can be a genetic bridge among three sections.
The general topologyof the phylogenetic
tree (Fig. 1) suggeststhat taxa in section
Angulares are closely related. This is reflectedin the terminology
for groups within thissection the V. minima complex and the V hirtella
complex. The V minima complex,
consisting
here of V. minima, V. riukiuensis
and V nakashimae, is a clearly defined group.
However, accessions assigned to V hirtella do not form a distinct phylogenetic
group. Section
Angulares appears to be a recently evolved branch of the subgenus Ceratotropis
that iscurrently in the process of dynamic evolution.
While the ITS sequence can be a useful tool for phylogenetic
studies, analysis of several
molecular markers and their comparison are necessary to draw conclusions that do not havestatisticalbias. Consequently
we are analyzing the CPDNA atpB-rbcL spacer region.
References
Konarev, A., N. Tomooka & D.A.Vaughan. 2000. Proteinase inhibitor
polymorphismin thegenus Vigna subgenus Ceratotropis and its biosystematic
implications.
Euphytica(in press)
Tomooka, N., Y. Egawa and A. Kaga. 2000. Biosystematics
and genetic resources of thegenus Vigna subgenus Ceratotropis. Pages 37-62 in Proceedingsof the MAFF International
Workshop on Genetic Resources. Wild Legumes. MAFF, NIAR, Tsukuba, Japan.

Fig.

1. Neighbor-joining

1000

cyclesangularis

tree based

on rDNA ITS sequence.

of the test are shown,var. nipponensis,

aco: acontifolia,

exi: exilis,

radiataglabra,var. radiata,

sub: suhramaniana,
umbellata

rad-s:

rp-r: reflexo-pilosa(wild)

tril:

radiatavar. sublobata,var. reflexo-pilosa,trilobata,trin:

trinervia,

valuesan-a: angularis

gra: grandiflora,

mungo var. mungo, mu-s: mungo var. silvestris,

Bootstrap

var. angularis,

Mr: aff. hirtella,

nak: nakashimae,
riu: riukiuensis,
spn: current

nep: nepalensis,
rp-g:

an-n:

min: minima, mu-m:
rad-r:

reflexo-pilosa

unnamed species,

um-c: umbellata

above 500 on(cultigen),var.

sti: stipulacea,
um-w:

133

Phylogenetic
Grassland.

Analysisof Genus Zoysia

for Improvementof Indigenous

Grazing

EBINA Masumi, *ABE Atsuko, KOBAYASHI Makoto, **KASUGA Shigemitsu,
Hiroshi and NAKAGAWAHitoshi
keywords:

AFLP, cultivar

registration,

genetic

resources,

phylogenetics,

ARAYA

Zoysiagrass

Introduction
Zoysia spp are distributed
widely in eastern Asia, and western Oceania.
In Japan, three
species, i.e. Z. japonica, Z. matrella and Z.tenuifolia
are cultivatedas grazing and turf grassfrom old days.
Especially,
Z. japonica (Japanese lawngrass)that is considered
to beindigenous to Japan, play important role as dominant species in grazing grassland that consist of
many wild native plants, from Touhoku (mid-northern)
District to Kyushu(western)District
Since almost all of the grassland is located in mountainous area, Zoysia japonica that can be
maintained without power farming system is requisite species.
In spite of its record on human use, breeding of Zoysiagrasshas started quite recently.
National Grassland Research Institute (NGRI)
conducts the collection of Zoysia ecotypes allaround Japan, and evaluates their characteristics.
This presentation will report the two major fruits of the research activity on diverse genetic
resources, i. e. information derived by phylogenetic analysis,
and a new cultivar selected fromecotypes.
Material and Methods
Five species including
wild relatives were collected mainly in Okinawa islands where is locatedin subtropics.
Authors were interested in diversityof indigenous wild species and variationin their phenotype.
Since classificationand identification
were difficult because theirmorphologicalcharacteristics
were plastic affectedby environmental
variation, DNA
fingerprinting
by AFLP was considered to be conclusive factor.
AFLP analysis and 5S
rDNA sequencing analysis were applied to examine five species described above and other two
species.
A series of experiment from DNA extraction to sequencing analysis was conductedusing the tools listed below (Table 1).
Table 1

List of the tools used.

items

tool sDNAextraction

DNAZOL kit (GIBCO BRL)

AFLP

ABI system, ABB73XL, GeneScan (ABI)

phylogenetic

analysis

Diversity

Database using UPGMA

5S rDNA cloning

TaKaRa EX-Taq TOFO TA-cloning version F (Invitrogen)sequence

Big Dye terminator (ABI)

Sequencing

analysia

DNAsis (Hitachi)

National Grassland Research Institute:
768 Senbonmatsu, Nishinasuno, Tochigi,
329-2793
JAPAN* Shimane Prefectural LivestockExperiment Station:
3755 Koshicho, Izumo, Shimane,693-0031 JAPAN** Nagano Prefecrural Livestock Experiment Station:
10 Kataoka, Shiojiri,
Nagano, 399-071 1 JAPAN.

About 1000 accession of Zoysia ecotypes collected from about 100 sites were evaluated theircharacteristics
in the experiment field in NGRI, according to the Descriptors for Characterizationand Evaluation in Plant Genetic Resources.
Result and Discussion
Clearlypolymorphic
bands
were detected in only a few
primer combination of AFLP
to analyze genetic divergence.
But 5S rDNA sequencing domain did not have critical differences.
Five out of sevenspecies showed clearly divided
cluster (Fig. 1).
AlthoughZ.
sinica var. sinica and Z.
macrostachya collected in thesame island were similar insome morphologicaltraits,they were obviously separated
into different
cluster.
It issuggestedthat these species
rarely cross each other in natural habitat
In contrast, thefact that Z. japonica and Z.
matrella were given the samecluster separating sub-cluster isconsidered to be due to highpossibilityof crossing in natural habitat
Zoysiagrassisperennialplant and usually
propagatesvegetativelyinnatural habitat
Vegetative
propagation is likely to advanFig. 1 Phylogenetic
tree obtained by UPGMA method
tageous in subtropics,
but itand Jaccard coeficient.
possesses
selfcompatible*1 and *2 indicates the plants were collected in Okinawa
fertileflow eringand Yaeyamaislands, respectively.
notwithstanding.
This factreveals potential of large genetic diversity in indigenous Zoysiagrass population.
Among genetic resources evaluated, an ecotype "Nankoku9" collected in Kouchi prefecture
showed high performance to elongate stolon comparing to standard cultivars "Meyer","Emerald" and "Myako" (Fig.2).
After clonal selection and detail examination for cultivar
registration,
the line was named"Asagake" and is undergoinginspection by plant breeder's
right
office.
"Asagake" is expected tocontribute toward improvement of
pasture establishment
by its capacity to cover soil rapidly.
Furthersurvey is being conducted to findsome excellent genetic resources
available for cross breeding.
Thus, diversity of genetic resourcesin genus Zoysia, suggests indigenous ecotypes can be treasure
Fig.2 Varietal differenceof stolon elongation.
house of breeding materials.

135

Screeningof Allelopathic

activity

from Rice Cultivars.

Yoshiharu

FUJII*1and David

R. GEALY*2*1 : National
Instituteof Agro-Environmental
Sciences, Tsukuba, Japan*2: USDA-ARS, Dale Bumpers National Rice Research Center, AR, USA
Long-term goals of the development of allelopathic
rice cultivars include improved
economics of weed control and maintenance of high quality ground water by supplanting
heavy herbicide usage on rice with the use of weed suppressive rice cultivars (Fujii,1994).
Experiments were conducted with rice using an agar diffusion Plant box method (Fujii, 1 999a,
1999b).
Apparent allelopathic
activity previously
was observed in the second year of a
two-year field trial in transplanted,
water-seeded rice. In this study, the red Shrine rice
called Awa Akamai reduced total weed biomass about 90% compared to the commercial
Japanese cultivar, Nippon Bare. The mechanisms responsible
for the weed suppression in
AwaAkamai may be valuable for the improvement of weed suppression in commercial
cultivarsof rice.
Suppressionof barnyardgrassand broad leaf paddy weeds have beenshown in the field in Tsukuba with AwaAkamai and other red rice cultivars.
A plant box method used for assaying activityof diffusable
phytotoxic
metabolites(potentiallyallelopathic
compounds) in agar in the laboratory
is summarized briefly as
follows.
Rice plants were grown in sand culture in pots for four to eight weeks. Roots
were washed with tap water and then with deionized water to remove sand. Roots of threerice plants were placed gently into a nylon-mesh-bounded cylinder (3.2 cm diameter by 6.6
cmtall) which was placed upright in one corner ofa 70 by 70 cm wide by 100 cm-deep clear
plastic box. Plant stems were taped to the corner of the box to stabilize the plants in the box.
Hot liquid agar (0.75%; 250 ml per box) was cooled to 40 C and poured into the boxes,
covering all root material, but not covering stems. Boxes containing agar and rice plants
were cooled on ice until agar had solidified(about 30 min.).
Thirty-one lettuce seeds wereinserted partiallyinto the surface of the agar in an equidistantgrid pattern. Seeds were about
half way into the agar with the germ (pointed)
end down using porcelain forceps.
Boxes
were sealed around the rice stems and over the box surface with plastic wrap to minimize
water loss, and placed in a fluorescent lighted incubator for 5 days (25 C, 14-hr days: 20 C,
10-hr, nights).
Lettuce seedlings were then removed and root and shoot lengths determined
to the nearest mm. Regression analysis was used to estimate lettuce root length at zero
distance from rice roots (y-intercept),and slope of the inhibition
curve.
In plant box tests, roots of several rice lines, including
Awa Akamai was highly
inhibitory
to the elongation of lettuce roots. Nihonbare (a non-suppressive cultivar in fieldtests in Tsukuba) was usually less inhibitory
to lettuce root elongation than was AwaAkamai.
In plant box experiments in which lettuce seed planting was delayed two or three days,
allowing
allelochemicals
to diffuse greater distances,apparent allelopathic
activitywas

136
greaterfollowingin the delayed
suppressiveplanting
cultivar,

Presently,
similarlongerdelays.

Interestingly,

Nihonbarestudies.

These results

seem at odds with the hypothesis

Nihonbare,

it is not clear whether

producesallelochemicals

barnyardgrassof lettuce

in such assays.

In other

was standardized

againstknown alkaloid

allelochemicals

lycorine(Wallerroot allelopathic& Fujii, 1999).
activity
againstwas only

10% of those
about

and also improved

Experiments
lettuce seedlings

used in the standard

60% of that

numbers of experimentsin the standard

and greater

the precision

replicationin detecting

lettuceroots dramaticallyin low quantities

that the nonor of low activity.

or other weeds of rice would exhibit

to thatwere about

inhibitedstudies,

allelopathic

activitysuch as berberine

sensitivity
in rice rootshydrochloride

andalso were conducted that demonstrated
riceusing quantities
of rice roots and agar thatroot box method.
method.

Theseof treatments
small differences

Durationassaysof these

allowedto be completed

assaysfor greater
more cheaply,

between treatments.

References(1)

Fujii,

Y. 1994.

The allelopathic

effectof some rice varieties,

pp. 160-165.

in Integrated

Management of Paddy and Aquatic Weeds in Asaia, FFTC Book Series, No. 45(2)Fujii,(3)

Recent Advances in allelopathy,
1 :25-28
Fujii, Y. 1999b. Allelopathyof velvetbean:
candidateY. 1999a.

Discriminationof allelopathic

and proof methods for allelopathy,

substances,pp.

Determination
33-47

and identificationin BiologicallyAgrochemicals. H. Cutler and S. Cutler (ed.) CRC Press.
(4) Waller G.R. and Y.Fujii,
1999. Biochemical
analysis
Microorganisms
Ecology. Inderjit,

greenhouse and field test.

Activeof allelopathic

and Soil Secondary Metabolites,
pp. 75-98. in Principles
K.M.M.Dakshini, and C.L. Foy (ed.) CRC Press.

of L-DOPA as a
Naturalcompound;and Practices

Products:
Plants,in Plant

137

Structuralvariation of waxy gene and differentiation
Setaria italica (L.) P. Beauv,

in foxtail

millet,Fukunaga, Kenji.^, Makoto Kawase2) and Kenji Kato^
1) Laboratory of Plant Breeding, Faculty of Agriculture,
Okayama University, Tsushima-naka,
Okayama 700-8530,
Japan 2) Laboratoryof Plant Biotechnology,
Department of Crop
Science, Shikoku National Agricultural
Experiment Station, Sen'yu-cho 1, Zentsuji, Kagawa
765-8508,Japan
Key words:
crop evolution, foxtail millet, geographicaldistribution,
insertion
sequence,
waxygene
INTRODUCTION
The waxy gene encodes granule-bound starch synthase (GBSS), which is a key enzyme inamylose synthesis of plants, and controls amylose contents in the endosperm.
Foxtail millet, Setaria italica (L.) P.Beauv., has been utilizedin various ways peculiar toeach area of Europe and Asia (Sakamoto
1987).
Landraces of foxtail millet with waxy
endosperm were cultivatedmainly in continental
Southeast Asia, and eastern Asia (Nakayama
et al. 1998).
Two dominant alleles (non-waxy and low amylose content) and a waxy allele
are known in foxtail millet.
In this paper we analyzed structuralvariation
in waxy gene to investigate
whetherlandraces with waxy endosperm originatedmonophyleticallyor polyphyleticallyand toclarify difference between three types (non-waxy, low amylose and waxy).
MATERIALS AND METHODS
Materials used
For determining the sequence of waxy gene, a non-waxystrain from Afghanistan (Code.
No.77) was used. For analysis of genomic Southern hybridization,
67 strains (see Table 1)
were analyzed.
Determinationof the sequence of CDNA and genomic DNA of waxy gene using PCR
methods
For amplificationof CDNA, mRNA was extracted from immature foxtail millet seedaccording to the method by Wang et al. (1995)
and poly (A)+ RNA was purifiedbyoligo(dT)-cellulose.
CDNA was constructedby Marathon CDNA amplification
kit(Clontech).
RT-, 5'- and 3'- RACE and genomic DNA PCR were carried out using primers
as shown in Fig.l.
Sequencing was carried out by Shimadzu DSQ-1000L sequencer.
Genomic Southern analysis
To find insertion/deletion
variation in waxy gene, genomic Southern hybridization
was
carried out according to Southern (1975).
Genomic DNA was digested by EcoRl.
Three
PCR products were used as probes as shown in Fig.l.
Hybridizationand detection wereconducted using ECL hybridizationand detection kit (Amersham-Pharmacia).
RESULTS AND DISCUSSION
Structure of waxy gene of foxtail millet
Genomic structure of waxy gene was summarized in Fig.l.
consisted of 14 exons, among which the first exon is untranslated.

Waxy gene of foxtail

milletProbes used exlex2
M1R3
M5R10
Fig. 1. Structure of waxygene and insertion sequences offoxtail millet and primers and probes used.
TTourist, •E SINE, -^^å MP^^-farge insertions 1 for types III and IV, 2 for V and 3 for type IIV.

138
SouthernanalysisTable 1. RFLP variation of waxy gene and its
Seven phenotypes
were found in theassociation with wanness and geographicaloriginanalysis of genomic Southern hybridizationof foxtail millet landraces.
as shown in Table 1. Interestingly,
RFLP
types correspondedwith the phenotypesofwaxiness. Non-waxy type correspondedwith RFLP types I and II, low amylose typewith types III and VI, and waxy type withtypes
IV, V and VII.
Geographicaldistributionof each RFLP type of the waxy
landraces was different as shown in Table 1.
Type IV was distributedin Japan andTaiwan, type V in Korea and type VII in
Myanmar. Insertions of large sequences inwaxygene gave rise to RFLP types III, IV, V
and VII as shown in Fig. 1, and presence of
these insertions was confirmed by restriction
analysisusing other restriction
enzymes(data not shown).
Small insertions
in types
II and VI were identified
as Tourist in the
intron 1 and SINE-likesequence in the
intron 12, respectively,by PCR andsequencing analyses (Fig. l).
Origin of waxy type in foxtail millet
All of three RFLP types of waxy foxtail millet had insertion sequences, larger than 5 kb inwaxy gene. Waxy phenotype can be attributed
to these large insertions,as found in maize(Veragona et al. 1992), which alter expression pattern of waxy gene. Based on the structural
analysis, it was concluded that waxy types of foxtail millet originatedpolyphyletically.
ACKNOWLEDGEMENTS
Thanks are due to Dr. Y. Sano, Hokkaido University,
for providing
some information for
amplificationof waxy gene. Weappreciate Dr. Y. Kubo, Okayama University,
for his technicaladvice and suggestions.
REFERENC ES
Fukunaga, K., E. Domon, M. Kawase (1997)
Ribosomal DNA variation in foxtail millet and asurvey of variation from Europe and Asia. Theor. Appl. Genet. 95:751-756
Nakayama, H., M. Afzal, K. Okuno (1998)
Intraspecificdifferentiationand geographicaldistributionof Wx alleles for low amylose content in endosperm of foxtail millet, Setaria
italica (L.) P. Beauv. Euphytica 102: 289-293
Sakamoto, S. (1987)
Originand dispersalof common millet and foxtail
millet.
JARQ
21:84-89
Southern, E.M. (1975)
Detectionof specificsequences among DNA fragments separated bygel electrophoresis.
J. Mol. Biol. 98:503-517
Varagona, M.J., M. Pufugganan, S.R. Wessler (1992)
Alternative
splicing
inducedbyinsertion of retrotransposons
into the maize waxy gene. The Plant Cell 4: 811-820
Wang, Z.-Y., R-Q. Zheng, G.-Z.Shen, J.-P. Gao, D. P. Snustad, M. G. Li, J.-L. Zhang andM.-M.Hong (1995)
The amylose content
in rice endosperm is related
to thepost-transcriptional
regulationof the waxy gene. Plant J. 7: 613-622

139Core collections,

DNA markers and bio-informatics:
new tools for "mining"
resources held in gene banks - Sorghum as an example

plant

genetic

C. Grenier1'2, PJ. Bramel1, P. Hamon3, J. Chantereau2,
M. Deu2, M. Noirot3, V.G. Reddy1, S.
Kresovich4,
K.E. Prasada Rao5, V. Mahalakshmi1,
J.H. Crouch1, R. Ortiz1^CRISAT, Patancheru,
502 324 AP, India (r.ortiz(a)cqiar.orqh
2CIRAD, Montpellier,France; 3Univ.
Montpellier
III/IRD;
Montpellier,France; 4Comell Univ., Ithaca, USA; 5Academy of Biological
Sciences,
Visakhpatnan,
AP, India
The large collectionof sorghum [Sorghum bicolor (L.) Moench] landraces
held at the International
Crops Research Institutefor the Semi-Arid Tropics (ICRISAT),
representsa challenge
for themaintenanceof both the accessions
and the information
documented for this germplasm..Accessibility
and knowledge of this collection
are essential
factors for an efficient use of sorghum genetic resources
by both breeders and farmers. This large size of the sorghum landrace collection
maintainedby
ICRISAT led to the establishmentof a core collection.
Core collections.
A core collectionrefers to a limited
set of accessions derived from an existing
germplasm collection,chosen to represent the genetic spectrum in the whole collection.
The corecontains the maximum diversity
availablein the collection.
Accessions
in the core are chosen primarily
to be representativeof the whole collection,
to be ecologicallyor geneticallydistinctfrom one another,and to maximize genetic diversity.
The core collection
serves as an entry point to the whole collection.
Identificationof core collections
also simplifies
the process of multiplicationand distribution
topotentialusers by substantiallyreducing the number of accessions
that need to be maintainedin
active collections.
Core collections
can be formed using characterization
information
availableongermplasm accessions,which can further determine
cluster groups of similar accessions
together.
A
series of investigations,
reported below, were undertaken to define sorghum core collections
and tocompare each of their sampling
procedures.
During this research,
an assessment was also made,accordingto geographic
and taxonomic classification,
to determine the diversity
pattern maintained
inthe landrace collection
and its adequacy.
This assessment
indicatesthat the sorghum collection
held intrust at ICRISAT was underrepresented
for the race bicolor and for accessions
from China.
Furthermore, this assessment
highlighted
redundancies
especiallyin races caudatum and durra, as
well as in the germplasm acquired from East Africa and South Asia.
Morpho-agronomic and passport data. Different
sampling
strategies,
either random or nonrandom, were proposed to obtain core subsets of reduced size. Three subsets were establishedfollowinga random sampling
within a stratified
collection(logarithmic
strategy:L), a sample based
upon morpho-agronomic
diversity(principal
component score strategy:
PCS), and a sample based
upon an empiricalknowledgeof sorghum (taxonomic
strategy:T). Comparisonsof these samples formorpho-agronomic
characterizationand passport information
were assessed to determine
their impact
on phenotypic
diversity(Grenier
et al. 2000a).
For their overall diversity,
the three subsets did not
differ, as shown with the two-dimensional
representationof the morpho-agronomic
diversityand theShannon-Weaver diversity
indices. When comparisons
for morpho-agronomic
and passport data wereconsidered/
the PCS subset looked similar to the entire landrace collection.
The L subset showed
differences
for characters
associatedwith the photoperiod
reaction that was consideredin thestratificationof the collection.
The T subset was the most distinct
from the entire landrace collectionas
it over-represented
the landraces
selected by farmers for specific uses and covered the widest range
of geographicaladaptationand morpho-agronomic
traits.
Molecular markers. Microsatellite
markers (SSR) are used in crops to differentiate
among genotypes
and as tools in marker-aided
selectionand gene introgression.
Early research with the sorghum world
collection
showed significant
variationin the five microsatellite
markers examined:
2.4 was theaverage number of alleles
per locus within accessions
and 19.2. in the whole sample of 25 accessions(Dje et al. 2000). This sorghum collection
seems to be highly structured
geneticallywith about 70% ofgenetic diversity
accounted for among accessions.
In contrast,
morphologicallydefined races ofsorghum, or the geographic
origins accounted for below 15% of the total genetic diversity.
This resultshows that microsatellites
are useful in the identificationof individual
accessions
with a high relative
contribution
to the overall allelicdiversityof the collection.
Hence, microsatellite
markers were used toquantify
genetic diversity
in each of the sampling
procedures(PCS, L, T) using polymorphism
at 15
microsatellite
loci (Grenier
et al. 2000b).
The landracesof each subset were genotyped
with three140
multiplex
polymerase
chain reactionsof five fluorescentprimer-pairs
each with semi-automatedallelesizing. The average allelicrichnessfor each subset was equivalent(16.1,
16.3 and 15.4 allelesperlocus for the subsets PCS, L, and T, respectively).
The average genetic diversity
was also comparable
for the three subsets(0.81,
0.77 and 0.80 for the subsets
PCS, L, and T, respectively).
Allelic
frequency
distribution
for each subset was compared with a %2 test but a few significant
differenceswere observed. A high percentage
of rare alleles(71-76%of 206 total rare alleles)
was maintainedinthe three subsets.
The global
molecular
diversity
retainedin each subset was not affectedby thephenotypic
sampling.
Bioinformatics.
Researchers
worldwide
have developed
DNA marker-based
maps of sorghum.
Information
gatheredby these research teams on sorghum maps has been included
by ICRISAT in the
WWW.Informationon all linkage
groups from a specificsource or on a particular
linkagegroup from a
specific
source are displayedwith details
at http://qrep.icnsat.cqiar.org/rflp/sorqhum
maps.htm
Comparative
genome mapping of related
plant species
has shown that the organizationof genes ishighlyconserved
during the evolutionof members oftaxonomic
families.
This has lead to theidentificationof genome co-linearity
between the well-sequenced
model crops and their related
species (e.g. Arabidopsis
for dicots and rice for monocots).
Co-linearity
overrides
the differencesinchromosome number and genome size. The rice genome project has constructeda RFLP linkagemapwith 2275 DNA markers (Harushima
et al. 1998).
By studying
the synteny between the rice andsorghum genomes a consensus linkage
map consistingof 607 DNA markers covering
1285 cM wasdeveloped.
A high degree of co-linearity
exists between the 10 sorghum linkage
groups and the 12
rice linkage
groups (Nagamura
et al. 1998).
An interactive
representationof available
data from thisrice-sorghum
genome synteny project (http://qrep.icrisat.cgiar.org/svnteny/intro.htm)
was developed
at ICRISAT. Likewise,
all sequencesin GenBank were screened for the presence of SSR. These
sequences have been collated
in a databasewith direct linkage
to primer design software. There are
21,800 sequences available
for sorghum, and 622 SSR were detected
recently(http://qrep.icrisat.cqiar.orq/homepaqe/genomics/output.asp).
Information
on publicly
available
micro-satellite
markers in is being compiled
in a separate
databaseto be used for removing overlap
with GenBank sequences prior to selectionof sequences
for designprimers.
Outlook. ICRISAT and its research
partners
have designed
experiments
across a range ofenvironments
to simulatedifferent
types of drought
stress encounteredby rice and sorghum. We
should expect in the mid-term to identify
and characterize
useful genomic regions conferring
drought
tolerance
to cereals. The common regions relevant
to drought tolerance
will be further
saturatedandannotated.
Additionallyappropriate
test materials
will be chosen to assess the relevanceof thesegenomic regions in each of the targeted
cereal crops in managed stress environments.
In the longterm this approachshould lead to the isolationand characterizationof candidate
genes for droughttolerance
because the orderingof DNA loci between rice and sorghum chromosomes corresponds
well.
This gene order conservation
between both genomes permits that genes in sorghum are isolatedbymap-based cloningat the homologue in rice, and then by homology in sorghum.
Referen ces
Dje, Y., M. Heuertz, C. Lefebvre and X. Vekemans. 2000. Assessment of genetic diversity
within and among
germplasm accessions in cultivatedsorghum using microsatellite
markers. Theoreticaland Applied
Genetics 100:918-925.
Grenier, C, PJ. Bramel-Cox, M. Noirot, K. E. Prasada Rao, and P. Hamon. 2000a. Assessment of genetic
diversityin three subsets constituted
from the ICRISAT sorghum collection
using random vs nonrandom sampling procedures A. Using morpho-agronomicaland passport data. Theoreticaland Applied
Genetics 101: 190-196.
Grenier, C, M. Deu, S. Kresovich, PJ. Bramel-Cox, and P. Hamon. 2000b. Assessment of genetic diversity
in three subsets constituted
from the ICRISAT sorghum collection
using random vs non-random
sampling
procedures
B. Using molecular markers. Theoreticaland Applied Genetics 101: 197-202.
Harushima, Y., M. Yano, A. Shomura, M. Sata, T. Shimano, Y. Kuboki, T. Tamamoto, S.Y. Lin, B.A. Antonio,
A. Parco, H. Kaniya, N. Huang, K. Yamamato, Y. Nagamura, N. Kurata, G.S. Khush and T. Sasaki. 1998.
A high density rice genetic linkage map with 2275 markers using a single F2 population.
Genetics 148:
479-494.
Nagamura, Y., T. Tanaka, H. Nozawa, H. Kaidai, S. Kasuga and T. Sasaki. 1998. Syntenic regions between
rice and sorghum genomes. In: Proceedingsof the International
Workshop on Utilizationof Transgenic
Plants and Genome Analysis in Forage Crops. National Grassland
Research Institute(Nishinasuno,
Tochigi,
Japan) Report 9:97-103.

141

Applicationof heavy-ion bombardment to mutate wheat and barley

Ichiro Honda,1 Michihiro
Wada,1 Tomoko Abe,2 Tadao Asami,2 and Shigeo Yoshida2
lNational Agriculture
Research Center, Ministry of Agriculture,
Forestry and Fisheries,
3-1 -1 Kannondai, Tsukuba 305-8666,
Japan, 2The Institute of Physical and Chemical
Research (RIKEN), 2-1 Hirosawa, Wako 321-0198,
Japan
Key words: heavy-ion bombardment, barley,

wheat, mutant

Recently, heavy-ion bombardment (HIB) to plant is investigatedto obtain a certain
mutant. Abe et al. extensively
studied the applicationof this method to tobacco plant using
accelerated heavy-ion beams from a ring cyclotron, and they established
it as the effective
method to obtain a mutant (Abe et al., 2000). Applicationof this method to other plantsuch as Arabidopsis(Shinozaki
et al., 1997) and roses (Kitakura et al., 2000) were alsostudied, however, those to agricultural
important crops such as wheat, barley, and rice havenot been reported. In this study, the HIB application
to wheat and barley for obtaining acertain mutant were examined.
Materials and Methods
In the experiment in barley (Hrodeum vulgare L.), recent Japanese breeding line ofbarley, Kantohadaka 77 (six-rowed, naked cariopsis, uzu, waxy endosperm) was used as aplant material. Both imbibed (12 hr, 20°C) and dry seeds were irradiatedwith 5-400 grayof dose by heavy-ion beam (l5N; 1 35Mev/u)
accelerated by RIKEN ring cyclotron andcultivatedin 1997-1998.
Their growth were observed and individual
M2 seeds were
harvested.
After braking dormancy by 1 % H2O2 treatments, individual
10-20 seeds were
sown, and an occurrence of albino plants was examined. Remaining M2 seeds were mixed
by their absorbed doses, aliquots of them were cultivatedin 1 998- 1999, and early headingor short culm plants were selected as putative mutants. Some of these lines were crossed
to Kantohadaka 77 and/or Misatogolden(two-rowed, Japanese barley cultivar).
In 1 9992000, M3, F] and F2 plants were cultivated and their phenotypes were examined.
In the experiment in wheat (Triticum aestivum L.), recent Japanese bread wheat cultivar,
Bandowase was used as a plant material. Both imbibed (24 hr, 4°C) and dry seeds were
irradiatedaccording to the method described above. Treated seeds were sown on the trayof vermiculite,
cultivated to their 2 leaf stages, and their growth were examined.
Results and Discussion
In the barley experiment in 1 997-1998,
the cultivation
condition was not good forbarley, only 21 % plants was survived even in control (without HIB) experiment. In HIBover 20 and 200 Gy to imbibed and dry seed of barley, respectively,
their growths wereseverely inhibited,
and then their M2 seeds were not obtained.
Occurrences of albino plantof individual
M2 seeds were observed in 5 Gy to imbibed seed, 10 and 20 Gy to dry seedtreatments. These results suggested that a certain mutation can be induced by HIB tobarley and optimal doses of irradiation
are estimated about 5 Gy in imbibed and 50 Gy in
dry seed treatment, respectively.
Occurrences of albino plants in M2 seeds were reinvestigatedin 1999-2000
HIB as well as NaN3 treatments to Misatogolden.
Among each
300 M2 line, albino plants were appeared in 4, 10 and 21 M2 lines of 5 Gy imbibed, 50 Gy
dry HIB seeds, and NaN3 (8 mM) treatment, respectively.
These results suggest that the
mutation can be induced even in this line by HIB. However, this method may be lesseffective than NaN3 treatment to mutate barley. These results were summarized in Table 1.
In wheat experiment, as shown in Table 2, the growth of over 50 Gy imbibed and 200
Gy dry HIB seeds were inhibitedseverely. The optimal doses in wheat may be 10-20 and
1 00 Gy treatment to imbibed and dry seeds, respectively.

In 1998-1999,
M2 seeds of barley Kantohadaka 77 were cultivated,
1 1 early heading
and 15 short culm plants were selected as putative mutants. Some of these lines werecrossed to Kantohadaka 77 and/or Misatogolden.
In 1999-2000,
M3, Fl5 and F2 seeds were
cultivated,
and their phenotypes
were examined. All the 1 1 putative early heading lines
didn't head earlier than normal line. Among 1 5 putative short culm lines, severe dwarf line
(d3, selected from 100 Gy dry HIB seeds), and two semi-dwarf line (d4 and d5, selectedfrom 50 Gy dry and 5 Gy imbibed HIB seeds, respectively)
whose leaf expansion areimperfect, were selected as homozygous phenotypes for short culm. Phenotypes of F]
between them and Kantohadaka 77, and Misatogolden
were as same as those ofKantohadaka 77, and ¥{ between Kantohadaka 77 and Misatogolden,
respectively.
Asshown in Table 3, all their F2 were segregated into mutant and normal with the 1 :3 ratio,
expecting that all of these 3 line is single gene recessive mutant. As shown in Figure 1,
culm lengths of F2 between Kantohadaka 77 and d3 mutant were clearly segregated.
These results clearly demonstrate that HIB is effective to mutate barley. Using thisunique method, a novel mutant of crop may be developed in the near future.
Table

1. Effectof heavy-ion bombardment

Table 3, Segregation

on barley

Table 2. Effectof heavv-ion

bombardment

on wheatofF? plants

Fig. 1. Segregationof F2 beteween Kantohadaka
and d3 mutant

77

References
Abe, T., Miyagai,M., and Yoshida, S. 2000. Effectiveplant mutation method using heavy-ion
beams (IV). RIKEN Accel. Prog. Rep. 33:140
Kitakura, T., Hara, Y., Abe, T., Miyoshi, K., and Yoshida, S. 2000. Effects of heavy-ion
beam irradiation
on shoot elongationin roses. RIKEN Accel. Prog. Rep. 33: 141
Shinozaki,N., Tanaka, A., Yokota, Y., Watanabe, H., Tano, S., Kanai, T., and Kitayama, S.
1997.
Effects of Ne ion beams on survival of Arabidopsis
thaliana.
RIKEN Accel. Prog. Rep.
30:130143
Development
Eiichiof DNA marker linked

to fruitskin color in JapaneseInoue1, Katsuki Amano2, Masakazu Kasumi3, Fumio Sakuma4, Hiromichi

pear {Pyruspyrifolia

Nakai)

Hara2 and Teruo Tsukihashi2

1 Gene Research Center, Ibaraki University, Ami-machi, Ibaraki 300-0393
2 School of Agriculture, Ibaraki University, Ami-machi, Ibaraki 300-0393
3Plant Biotechnology Institute, Ibaraki Agricultural Center, Iwama-machi, Ibaraki 3 19-0292
4Horticultural Institute, Ibaraki Agricultural Center, Iwama-machi, Ibaraki 3 1 9-0292
In the breedingof fruit tree, the shortening
the selection
period is an important purpose, because long years
and considerable
cost and labor are needed for the selectionof fruit character.
In the case of Japanese pear
(Pyrus pyrifolia Nakai), the selectionof fruit characters such as the fruit skin color is impossible
for some years
until developing
fruit.
Recently, the juvenile
selectionusing the DNA marker has been used practicallyin themajor crops.
If the marker-assisted
selection(MAS) were practicallyused in the fruit breeding,it seems to beable to rapidly
improve the breeding
efficiency.
The objectiveof this study was developmentof the DNA
marker for fruit character and application
to the MAS in the Japanese pear. In this poster, two experiments fordeveloping
DNA marker linking to the gene controllingthe fruit skin color were reported.
Screeningof RAPD markers linked to the fruit skin color by using bulked

DNA analysis

Two Fj progenies from 'Kosui' x 'Kinchaku' and 'Niitaka'x 'Chikusui'segregating
for fruits skin color wereused in this experiment (Table 1). Total DNA was extracted from leaves by modified CTAB method.
On eachcross combinations,
two kinds of DNA samples were bulked from the Fi individuals
with red-skinned
fruit andblue-skinned
fruit respectively.
Random 10-mer primers (Operon Technologies,Inc.) were independently
used
for RAPD analysis.
PCR was conducted by thermal cycler programmed for one cycle of 10 min at 95°C, 40
cycles of 1 min at 93°C, 2 min at 40°C and 2 min at 72°C, and one cycle of5 min at 72°C as final extension.
Amplification
products were separated on 1.5% agarose gels and stained by ethidium bromide.
RAPDs wereobserved and recorded under UV light.
Total 180 random primers were screened against the four DNA bulks
from two progeny.
An average of4.5 bands were amplified per primer (Total 818 bands).
Three bands wereassociatedwith red skin color and five fragments were associated with blue skin color.
A fragment generated
by OPH19 was selected by the linkage analysis
in all Fj progeny (Fig. 1).
The linkage value between thismarker and blue skinned phenotype was about 8% (Table 2).
This marker seemed to be useful for the selectionof the blue skinned individuals.
Identificationof bud mutation at fruit skin color by AFLP analysis

To detect polymorphisms
on the gene controllingthe fruit skin color directly,
amplifiedfragment length
polymorphism(AFLP) analysis was conducted among six bud mutation varieties on the fruit skin color and their
original
varieties(Table 3).
The AFLP was detected using Plant Mapping Kit of PE Applied
Biosystems.
Total genomic DNA was cut by two restriction
endonucleases(EcoRl and MseX). Double-stranded
adapters
were ligatedto the ends of the restriction
fragments.
Using the constructs
as template,pre-selective
amplification
was performed by the specificprimers for the adapter sequence.
Then, selective
amplification
was also performed by the selective
primer sets with fluorescentdye-labelon the EcoRl side.
Theamplification
fragments were observed and analyzed by the ABI PRISM310
genetic analyzer.
Total 774
fragments were generated by using ten selective
primer sets. On 22 fragments, the polymorphism
was observed
between bud mutation and original variety (Table 3).
Four kinds of fragments could distinguishmore than one
bud mutation variety.
A fragment that generated by ACT-CAA primer set was commonin the three varieties
with blue skinned fruit. This fragment may be used in order to select the individuals
with blue skinned fruits.
Above two experiments were possible
to utilize
for the marker development even in plant species, which has
highlyheterozygosis
genome, without requiring
genetic linkage
map and informationof DNA sequence.
Therefore, it seems to be an effective technique for conveniently
developing
selection marker in the fruit tree.
At present, we are developing
the sequence tagged site (STS) marker.

F1

Table 1. Segregation
amongprogeny
DNA analysis

of fruit skin color
used for bulked

Fig.1.
Separationof amplification
products
generatedby primer OPH19
from four kindsof bulk
DNA (M; ZHindIII+^X174/HaeIII,
7R; No.7/Red-bulk,
7B; No.7/Blue-bulk,
19R;
No.l9/Red-bulk,
19B;
No.19/Blue-buIk,
White
arrow head; RAPD marker 'OPH194255).

Table

2* Segregation

Table 3* Amplified

fragment

analysislengthof a RAPD marker 'OPH19425'

polymorphism(AFLP)of original

and fruits

variety

skin

color

and bud mutation

variety

145

Intra-specificgenetic differentiationin East Asian Vigna species
Ceratotropis)
revealed by AFLP markers.

(subgenus

A. Kaga1, K. Doi1, M.S.Yoon2, N. Tomooka1 and DA.Vaughan1
1 NIAR, Tsukuba, Japan, 2NIAST, Suwon, Korea
Within the genus Vigna subgenus Ceratotropis the V. minima complex species V.
nakashimae and V. riukiuensis are endemic to East Asia. These two species can act as bridging
species for gene transfer between the cultigens
V. angularis and V. umbellata that exhibit
compatibility
barriers and V. riukiuensis has been suggested as gene donor to V. angularis forheat tolerance. The objectivesof this research were to measure the inter and intra-species
diversity
in taxa of Vigna subgenus Ceratotropis from East Asia.
Materials
and methods: In total 36 samples of V minima complex taxa, two samples of V.
umbellata and V. angularis and 5 samples of V. reflexo-pilosa were analysed (Fig. 1). Total
genomic DNA was extracted and AFLP fingerprinting
was carried out by standard procedures.
Jaccard's coefficient
was calculated and similarity matrices were also subjected to principle
component analysis (PCA). Shannon' s information index for phenotypic diversity
and genetic
diversity
based on inferred nucleotide
diversity were calculated. A neighbor-joining
tree wasconstructed on the basis of the pairwise distance by PHYLBP ver. 3.57.
Results and discussion:
A total of 1,611 fragments were generated by 12 primer pairs andscored fromgels. The number of polymorphic fragments identified
within species ranged from
35 for V. reflexo-pilosa
to 299 for V. minima. A total of 801 polymorphic
fragments weredetected within species compared to 643 among species.
Relationships
among species: A matrix of inferred nucleotide diversity
from AFLP data wasprepared and used to create a phenogram that shows phylogenetic relationships
among accessions(Fig. 2). This analysis revels a clear separation of each species. Vigna minima, V.nakashimae andV. riukiuensis
are clearly separated from each other. Vigna minima has an average genetic
distance of 0.026 from V.nakashimae and V. riukiuensis. Vigna nakashimae and V. riukiuensis
have a genetic distance of 0.01 16 from each other. A three dimensional PCA plot that accounts
for 69% of the variation reveals that each taxon is clearly separated(results
not shown). These
results support the view that these are three distinct taxa.
Variation within species: Vigna riukiuensis has a restricted distribution
in Taiwan and Okinawa
prefecture, Japan, and the 16 populations analysed cover much of the species range. Clear genetic
differences
between V. riukiuensis
populations
from Miyako and Yaeyama archipelagos(Iriomote, Ishigaki and Yonaguni) have been revealed (Table 1). One population fromHateruma
is intermediate.
The genetic distance between Miyako and Hateruma populations
and those of
Iriomote, Ishigaki and Yonaguni is 0.0039. This suggests that there is a floristic barrier between
Ishigaki and Miyako islands.
The 18 populationsof V. nakashimae analysed come from much of the eastern part ofthis species distribution.
The genetic distance between Korean and Japanese populations of V.
nakashimae (0.0036)
and Yaeyama and Miyako archipelago
populationsof V. riukiuensis(0.0039)are very similar (Table 1). This may reflect the fact that these two species have evolved
over a similar period of time in the areas studied.
Reference
Yoon, M.S., K. Doi, A. Kaga, N. Tomooka and D.A.Vaughan. 2000. Analysis of the genetic
diversity in the Vigna minima complex and related species in East Asia. J. Plant Sci. (Inpress)

Table 1. Phenetic and genetic
accessions from different regions

distancesand Shannon's

diversity

index(Ha)among V. minima

Fig. l(left).
Collection
sites of populationsof V. nakashimae, V. riukiuensis
and V.
reflexo-pilosa
used in this study.

Fig. 2(above). Neighbor-joining
tree of all diploid
Vigna species analysed based on a matrix ofinferred nucleotide
diversity.
Number at branch indicates probability
supportingthat branch.
Branch length corresponds to nucleotide diversity.
ang= V. angularis; nip= V. angularis var.
nipponensis: nak= V.nakashimae: min= V. minima; riu= V. riukiuensis.

147

Morphological
Hideyuki

and Biochemical

Variationsin Radiation

Mutants of Rice

Kajiwara* , Takayo Kaneko* , Minoru Nisimura* ** National** National

Institute
Institutefor Agrobiologicalfor Agrobiological

Keywords: leaf color, mutant, proteomics,

Resources, Tsukuba, Ibaraki 305-8602 Japan
Resources, Oomiya, Naka, Ibaraki 3 1 9-2293 Japan
radiation,

two-dimensional

electrophoresis

I ntroducti on
Mutants of rice had been isolated by gamma-irradiation
for the plant breeding in gamma
field of National Instituteof Agrobiological
Resources (NIAR).
Thousands of mutants whichwere isolated from cultivars showed the changes in morphology. However, their biochemicaland genetical characters are not yet understood.
Recently, genome analyses of higher organism including rice have been progressed andthe proteomics, one of the major post-genome studies, have started in all over the world. We
started the proteome analysis of rice for the understandingof the unknown functions of the
newly identified
genes using radiation mutants of rice. In this paper, we show the preliminary
results by rice mutant analysis and have described the perspective of proteome analysis using
radiation mutants.
Materials and Methods
Mutants of rice (Oryza sativa L.) had been isolated by gamma-irradiationin gamma fieldat NIAR, Oomiya, Japan. Radiation mutants from Nihonmasari and Norin 8 were cultivatedin the field at NIAR, Tsukuba, Japan, 2000. The length of culms and panicles were measured
to know the basic morphologicalvariations.
Young leaves (the third leaves and others) wereisolatedand proteins
were analyzed by the two-dimensional
polyacrylamidegelelectrophoresis(2D-PAGE).
2D-PAGE analyses were performed based on the OFarrells
procedure.
However, several points were changed to improve the sensitivities
andseparations.
To prevent the overlappingof the protein spots, proteins were fractionatedinto
two categories, without detergent and with detergents.
Sequentially,
proteins were extracted
by using detergents conditions after the non-detergent condition.
Contaminants which wouldinterfere the isoelectric
focusing of proteins such as salts and organic compounds were
removed by acetone precipitations.
Cross-linking
reagents and ratio in acrylamide gel werechanged to improve the separation and to reduce the background of silver staining.
After theelectrophoresis,
proteins
were visualized
by Coomassie brilliant
blue staining or silver
staining which has no oxidative process. The proteins were digested by trypsin in gel andanalyzed by a mass spectrometry.
Results and Discussion
The length of culms and panicles,

the days to heading

after seeding

were measured in148
radiation
mutants, 122 derived from Nihonmasari and 158 derived from Norin 8. Based on
those data obtained by field studies, the radiation mutants were found to be different
fromstandard cultivars in basic characters.
Mainly mutants in color of leaf were analyzed by novel 2D-PAGE. All of proteinsextracted by lysis buffer including
several detergents
showed thousands of spots in gel. It
was hard to compare the electrophoresis
pattern because of too many spots. Therefore, we
fractionatedproteins into two categories, which were then extracted by with and without
detergents.
We focused on the comparative analysis in the soluble fraction which wereextracted without detergent.
The protein fraction should have the enzymes related to dark
reaction, as mutants in leaf color are supposed to have mutations related to chloroplastorphoto synthesis.
Several strains showed some differencesfrom the standard cultivars.
Some spots had
increased and decreased contents of proteins and some strains showed changes of isoelectric
points.
Summarized the differencesin spots between standards and mutants, mutation ofprotein which were caused by the substitution,
the deletion of amino acid, or the posttranslational
modifications
were not observed every spots in 2D-PAGE profile. The changesof the electrophoretic
behavior were classified
into some categories.
The proteins found by
this study would be the candidates of the proteins which should be related to the leaf color
and/or the mutated gene.
The differences in mutants could be visualized
by Coomassie brilliant
blue staining andmore than one microgram of proteins were hopefully
expected to be existed
in thepolyacrylamidegel. According to our experience of primary structure analysis of proteins,there should be enough contents of proteins in polyacrylamide
gel for the analysis by a mass
spectrometry, though a protein sequencer could not analyze one spot and it is impossible
toreact the Edman degradationin N-terminallymodified proteins.
Trypsin was applied to the
spots and the analysis have in process to identify
the mutant gene or protein expression
affected by the mutations in leaf color. Some photosynthesis
related proteins were identified
by the analysis of the mass spectrometry.
Acknowl edgement
This work has been supported in part by a grant of rice genome project.
We alsoacknowledge to Prof. H. Hirano at Kihara Institute for BiologicalResearch, Yokohama City
University.

149

The search
Kouichifor and
Kashiwaba,

analysisof sources

resistance

Norihiko
Tomooka,
NIAR, Tsukuba,

Akito
Japan

to legumeKaga,storage

Duncanpests.

VaughanLegume storage pests are a major source of crop loss. The objectivesof the research
reported here are to find new sources of resistanceto the major insect pests of legumes,
Callosobruchus
spp. the bean weevils, to isolate the chemicals
involved in resistanceandincorporate the sources of resistance into breeding lines.
Materials

and Methods
63 Vigna subgenus Ceratotropis
accessions
that constitutea species core germplasm
collection
have been evaluatedfor resistanceto the bean weevil species
Callosobruchus
maculatus and C. chinensis. Resistance was first tested using whole seeds (Fig. 1) then again
using artificial
seeds made from ground seed flour for each accession (Fig. 2-4). Detailsofmethodology
for evaluatinginsect resistanceare provided in Tomooka et al. (2000).
The
bioactive chemicals responsible
for resistance in Vigna umbellata were concentrated in n-butanol
after extraction in 85% methanol. Then they were fractioned
using a silica gel column eluted
with AcOEt-MeOH (2:8).
The bioactive
fraction obtainedby column chromatographywasfurther purified on a LH-20 column and then by HPLC (Simazu-lOA).
Results

and discussion
About 25% of the species core germplasm collection
evaluated were found to have a highlevel of resistance to both C. maculatus and C. chinesis this compares to about 1% or less forevaluation of different varietiesof the cultigen, mung bean (V. radiata)(Tomooka et al., 2000).
Based on the results of evaluation the most promising source of resistance to Callosobruchus
spp. in cultivatedspecies of the subgenus Ceratotropis
appears to be the rice bean, Vigna
umbellata (Table 1). Wild species exhibiting
resistance have the disadvantagethat the chemicals
responsible
for resistance need to be tested carefully
for their safety in human food. Transfer ofresistance from one cultigen to another does not pose this problem.
The bioactive
chemicals responsible
for resistance
to C. chinensis in V. umbellata havebeen isolated. In the artificial
seed test, seeds consistingof 3% chemical extract and 97% azuki
bean flour, larva emerged from eggs but died before the second instar (Fig. 4). The chemical
structures of these compounds are now being elucidated.
Direct transfer of bruchid resistance from V. umbellata to V. angularis, azuki bean, is
problematicdue to incompatibility
barriers. Consequently,
V. nakashimae and V. riukiuensis
arebeing used as a bridging species to facilitate
gene transfer. At the same time the F2 populationofthe cross between V. umbellata and V. nakashimae is being used as a mapping population
todetermine the location of the gene(s) involved in resistance to both Callosobruchus
chinensis andC. maculates.
Reference
Tomooka, N., K. Kashiwaba, D.A.Vaughan, M.Ishimoto and Y. Egawa. 2000. The effectivenessof evaluatingwild species:
searching for sources of resistance to bruchid beetle in thegenus Vigna subgenus Ceratotropis. Euphytica 1 15:27-41.

Table 1. Levels of resistanceof accessions
species against Callosobruchus
chinensis
on 3 replications.

Fig
Fig
Fig
Fig

of cultivatedVigna subgenus Ceratotropis
and C. maculatus ( Bruchid beetles ), based

1. Rice bean( Menaga ) seed 40 days after C.chinensis
eggs had been laid.
2. Artificial
beans made from rice bean flour 40 days after C. chinensis
eggs had been laid.
3. Artificial
beans( control ) made from azuki bean flour 28 days after C. chinensis
eggs had been laid.
4. Azuki been flour mixed with extract from Menaga seeds 40 days after C. chinensis eggs had been laid.
These artificial beans contains 3% freeze-driedmaterial after 85%MeOH-nBuOH extraction.

Okayama

151

Molecular analysis
and its possible
Facultyof genetic diversity
in east Asian wheat landraces,
association
with adaptation to abiotic stresses

Kenji Kato and Kazunobu Matsumotoof Agriculture,
University,
Okayama 700-8530,

Key words: ABA responsive

gene, adaptation,

CAPS, genetic

differentiation,

Japan
wheat

INTRODUCTION
Hybrid weakness, such as necrosis, grass clump dwarfness and partial pollen
sterility,
is often seen in Fx hybrids between commonwheat cultivars.
Geographicaldistributionof such complementary
genes suggested
the occurrence of genetic
differentiation
between western and eastern wheat cultivars (Tsunewaki 1970).
Ecogeographicalconditionis largely
diversified
within China, being generally
colder and less precipitation
in west and north China.
Iwaki et al. (2000)
revealed
ecologicaldifferentiation
between west and north China and east and south China, by
the analysis of genes for vernalization
response.
Analysis of adaptively
neutral genes,
such as Igcl for gametocidal inhibitor(Tsujimoto
et al. 1998), isozyme genes (Kato etal. 1998) and so on, indicated genetic differentiation
between two areas.
In the present study, RAPD as adaptively
neutral polymorphism
and CAPS for ABA
responsive
Em genes as adaptively
importantpolymorphism
were analyzed
tocharacterize genetic diversity
in east Asian wheat.
MATERIALS & METHODS
A total of 56 wheat landraces (Triticum aestivum) covering 10 localities
from China
to Japan (Fig. 1) were studied for DNA polymorphism.
RAPD analysis : Total DNA was extracted from etiolatedshoots of 10 days oldseedlingby CTAB method, and used as a template for PCR amplification.
Twelverandom primers (12mer,
Bex), which successfully
detectedpolymorphismin thepreliminary
experiment, were used for RAPD analysis.
CAPS polymorphism: A specific primer set was designed to amplify a specificregion (1.5 - 2.2kbp) ofEm (Litts et al 1991) and EmH2A (Futers et al 1993) genes
which code Em protein and are responsive to ABA. Amplified product was digested
by several restriction
enzymes for CAPS analysis (Fig. 2).
Nucleotide
sequence of
amplifiedproducts was determined by using the Dye-Primer method.
RESULTS & DISCUSSION
Fourteen polymorphic
markers were obtained by RAPD analysis of 56 wheat
landraces.
In four areas of west and north China (Tibet,
Northeastarea, Gansu &Ningxia, and Xinjiang),
the number of polymorphic
markers and polymorphic
index
were more than 8 and 0.107, respectively,
indicating
the existence of large genetic
variation.
In six areas of east and south China, Korea, and Japan, genetic variation was
smaller as indicatedby two indices which were less than 8 and 0.114, respectively,
being speciallysmaller in spring type landraces.
Frequency of ll markers was
significantly
different
between spring wheat area (west and north China) and winterwheat area (east and south China, Korea and Japan).
Cluster analysis based on genetic
distance grouped ten areas into two clusters (Fig. 1). Cluster I consisted of areas ineast and south China, Korea and Japan, while areas in west and north China weregrouped into cluster II.

The analysis
of esterase
isozyme revealed genetic
similarity
in areas fromTranscaucasus to west and north China (Washio & Kato 1996), indicatingthat wheat
spread eastwards without severe selection in these semi-arid areas. On the other hand,
wheat landraces in east and south China is geneticallydifferentiatedfrom those in west
and north China.
Spring wheat is mainly cultivatedin west and north China where
winter is severely cold and rainfall
is less during wheat season. On the contrary,
winter wheat is mainly cultivatedin east and south China with relatively
warm and wet
climate.
It was therefore suggested that bottle neck effect due to severe selection foradaptively
importantcharacters
caused drastic change of genetic structure of wheat
populations
in east and south China.
CAPS polymorphism
at Em gene was detected by the digestion
with four restriction
enzymes, Taq I, Dra I, HinfI and Hinc II, and at EmH2A gene with Taq I. Sequence
analysis of Em gene for typical three cultivars
revealed that all of the site changes
occurred in 5f upstream or 5' untranslated
regions (Fig. 2).
Nucleotide
substitutionat
ABA responsive elements (ABRE and G-box) was also detected in type B landraces.
As to EmH2A gene, Taq I site change in 31 untranslated
region was confirmed.
CAPS polymorphism
at Em gene was surveyed for 56 wheat landraces, and they
were classified
into three types as shown in Fig. 2. In winter type wheat, frequency of
three types is not different between areas. On the contrary, the frequency is different inspring type wheat, and B type instead of A type is frequently
found in east and south
China and Japan. As to EmH2A gene, 'V type seemed frequently found in spring typewheat, though geographicaldifference was not clear.

Fig. 1. Genetic relationshipbetweenlocal wheat populations,based onRAPD polymorphism.

Fig.2. Structure and restriction
map of Em and EmH2Agenes, and primer
bindingsites.
Asterisks
indicate polymorphic
restriction
sites, whileother sites are not indicated.
+,-; Presence and absence of restriction
site.

AC KNOWLEDGMENTS
study was partly supported
by a Grant-in-Aid(No.08660008)
from theof Education,
Science, Sports and Culture of Japan.
REFERENCES
Futers TS, S Onde, M Turet and AC Cuming (1993)
Plant Mol BioL, 23: 1067-1072.
Iwaki K, K Nakagawa, H Kuno and K Kato (2000)
Euphytica
111;137-143.
Kato K, K Matsumoto and T Washio (1998)
Proc. 9th Inter. Wheat Genet. Symp. Vol. 2;
240-243.
Litts JC, GW Colwell, RL Chakerian and RS Quatrano (1991)
DNA Seq., 1: 263-274.
Tsujimoto H, T Yamada and T Sasakuma (1998)
Breed. Sci., 48: 287-291.
Tsunewaki K (1970)
Seiken Ziho, 22: 67-75.
Washio T and K Kato (1996)
Breed. Sci., 46(Suppl.
2): 329.
This
Ministry

153

Molecular cloning and structuralcharacterization
kinase gene from rice (Oryza sativa L.)of a receptor-likeprotein

Motoshige
Kawata1, Tetsuo Oikawa1, Yoko Matsumura1, Fumiyoshi
Kawasaki2 , Fumio Takaiwa2 and Shigeru Kuroda1

Fukumoto1,'Hokuriku
2NationalJapan

943-0 1 93, Japan
Ibaraki 305-8602,

National
Institute

Agricultural
Experiment Station, Inada, Joetsu, Niigata
of Agrobiological
Resources, Kannondai, Tsukuba,

Key words Oryza sativa,

receptor-likeproteinkinase,Shinjicell differentiation

Receptor protein kinases mediate cellular signaling
processes in diverse biologicalsystems. The few plant receptor-likeprotein kinase that have been studied to date waspostulated
to mediated self-recognition,differentiation,and disease resistance (Song et al.
1995; Takahashi et al 2000). In rice, the biological
function of most of these genes remains
unclear. We have used RT PCR and 3fRACE to identify diverse receptor-likeprotein kinase
genes expressed in the callus, shoot, root and immature seed of rice {Oryza sativa L.). The RT
PCR primers encoded the conserved regions among the catalytic
domain for phospholylation.
These primers amplified, six transcripts
from immature seed, two from callus and shoot, andone from root. The eleven RT PCR products were sequenced to permit design of genespecific forward primers for use with anchored oligo dT primers in 3'RACE. Sequencing of
the 3'RACE products indicatedthat every transcripts
encoded receptor-likeprotein kinase.
From the results of the homology search based upon the database, seven of eleven transcripts
proved to be an new protein kinases. Sequence of the D309 transcripts
showed highhomology to the maize CR4 gene, which is related to the cell differentiaitonof aleurone layerof the maize seeds (Becraft et al 1996).
To isolate a genomic clone for a receptor-likeprotein kinase having high homology tomaize CR4 gene, the 5.5kb Hindill DNA fragment was subcloned in plasmid vector from rice
BAC library (Nakamura et al. 1997). The complete DNA sequence contains 373bp of a 5'
untranslated region, 2,706bp of an open reading frame, and 346bp of a 3f untranslated
region.
The open reading frame encodes a 902 amino acid peptide with a calculated molecular was of
97,376 kD (Fig. l). The protein for this open reading frame was designated OsCR4. The open
reading frame we assigned is likely the complete open reading frame. Several topologicalfeatures of the predicted
OsCR4 protein was noted. 1) Leu rich repeats (LRRs) domain(amino acids 32-330)
followed by Cys clusters (amino acids 352-406)
were existed, both ofwhich have been suggested
to be involved in receptor-ligand
interactions(Kobe and
Deisenhofer1994).
2) The hydropathy
analysis revealed that the protein contains highlyhydrophobic
regions (amino acids 420-441)
which is immediately
followed by several
positively
charged amino acids (amino acids 412-418)
3) cytoplasmic
domain possessing all
the hallmarksof catalytic
domains of eukaryotic protein kinases (amino acids 408-901)(Fig.2).
These structuralfeatures of extracellular
ligand-binding
domain, the membranespanning hydrophobic
segment and the highly consensus kinase domain strongly suggest thatthe encoded protein function as a receptor protein kinase.
DNA blot analysis suggest that the japonica and indica rice varieties contain a single
copy of the OsCR4 gene showing no polymorphisms.
On wild rice species,
DNA

154
polymorphisms
were shown in O.australiensis
and O.officinalis.
O.rufipogon,
which isbelieved in the ancestry of the O. sativa showed the same patterns with the O. sativa. These
results indicatedthat the analysis of DNA polymorphims by using OsCR4 gene was preciselyreflected
the revolutionallyrelationshipamong Oryza species;
Acknowledgements
technical
assistance.
Agriculture,
Forestryof Japan.

The authors thank Mr. Satoru Momose and Ms. Masako Ikeda forThis work was supported in part by a project grant from the Ministry ofand Fisheries
and from the Ministry of Education, Science and Culture

Refernces
Becraft P.W., P.S. Stinard and D.R. McCarty. 1996. CRINKLY4: a TNFR-likereceptor kinase involved in maize epidermal differentiation.
Science 273: 1406-1409
Kobe B. and J. Deisenhofer
1994. The leucine-richrepeat; a versatile binding motif. Trends
Biochem. Sci. 19:415-421
Nakamura S, S. Asakawa, N. Ohmido, K. Fukui, N. Shimizu and S. Kawasaki. 1997. Construction of an 800-kb contig in the near-centromericregion of the rice blast resistancegene Pi-ta2 using a highly representative
rice BAC library.
Mol. Gen. Genet. 254 (6):611-20Song W.Y., G.L. Wang, L.L. Chen, H.S. Kim, L.Y. Pi, T. Holsten, J. Gardner, B. Wang,
W.X.Zhai, L.H. Zhu, C. Fauquet and P. Ronald 1995. A receptor kinase-likeproteinencoded by the rice disease resistance gene, Xa21. Science 270: 1 804-6
Takasaki T., K. Hatakeyama, G. Suzuki, M. Watanabe, A. Isogai and K. Hinata. 2000. The S
receptor kinase determines self-incompatibilityin Brassica stigma. Nature 403 :9 1 3-6

Fig.l
Genomic organizationof OsCR4 gene.
positionsof the three exons, and overlappedof the open reading
frame.

Fig.2 Topological
OsCR4 gene.

featureof the deduced

Shaded box indicate
box depict the positionamino acidsequenceof the

155

Gene Organizationof Sugar

Tomohiko Kubo, Satsuki Nishizawa,
Estiatiand Tetsuo Mikami

Beet

Mitochondrial

Akira Sugawara,

NorikoGenomeItchoda,Laboratoryof Genetic Engineering,
Graduate School
Hokkaido University,
Sapporo 060-8589,
Japan

of Agriculture,

Key words : Sugar

Gene- tRNA

Beet- Mitochondria-

Mitochondrial

Amy

Introduction
Mitochondrial
genomes of flowering
plants are very interesting
because of
their unique gene-expression
system, extensive
genome rearrangements
and some important
breeding
traits such as cytoplasmic
male sterility.
However, since the report of Unseld et a/. (1 997), Arabidopsis
has been
the only flowering
plant that the completesequence of mitochondrial
genome is available.
Here, we describe the nucleotide
sequence of sugar
beet mitochondrial
genome, the second completedsequence of themitochondrial
genome of flowering
plant.
Sequence
analysis
The entire genome size of the sugar beet mitochondria
is 368,799
bp, with
an overall G+C content 43.9 %. Homology searches to match the entire
genomic sequence with known sequences in the public DNA data basedetected
29 protein-coding
genes, 5 rRNA genes, and 25 tRNA genes,
which altogether
account for 1 1.3 % of the genome. Althoughmost of thegenes identified
here are also found in Arabidopsis
mitochondria,
a few
differences
are noted in the gene content between sugar beet andArabidopsis.
The sugar beet mtDNA also contains repeatedDNA
segments, the size of which varies from 50 to 22,739
bp. The repeatedsequence elements cover 1 0.3 % of the genome. It is well documented
thatplant mtDNAs contain many sequences of foreign origin. We identified
chloroplast
DNA (CPDNA) and nuclear-likesequences of 2.1 % and 3.3 % of
the genome, respectively.
Sugar beet mtDNA has at least 20 group II introns
in seven proteincoding genes. A total of six trans-splicing
introns and 1 4 c/s-splicing
introns are found. The sum of all intron lengths
represents
7.0 % of the
genome. Two introns seem to be lost from rps3 and nad4 genes during
the course of the evolutionof sugar beet lineage.
A total of 559 ORFs were identified,none of which, however, exhibited

156

apparent homology to any reported genes (and to any of the Arabidopsis
ORFs) or carried significant
proteinmotifs. Three and six-tenth
percent of
the sugar beet mitochondrial
genome sequence is homologous
tomitochondria!
plasmid DNAs. We also found the sequences with homology to
the 5'- or 3'-untranslated
region and the intergenic
sequence of
mitochondrial
genes in the other plant species;
they do not exceed 6.8 % of
the sugar beet genome. The major portion (55.6å å å %) of the genome has no
similarity
to any nucleotide
or protein sequencesin the public data bases.
Furthermore,when the sugar beet mtDNA sequences are compared tothose of Arabidopsis,the shared sequencesby the two plant species total
78,057bp, representing
21.2 % of the sugar beet genome.
Novel tRNA gene in sugar beet mitochondrial
genomeOne of the notable findingis that sugar beet mitochondrial
genomecontains two trnC-GCA genes: one is the usual, 'native'class trnC-GCA(trnC7-GCA)
and the other is the novel one that has not been documentedyet (trnC2-GCA).
The trnC7-GCA is a pseudogene
because nocorrespondingtRNA molecule could not be detected.
On the other hand,
trnC2-GCA is activelytranscribedand seems to be processed
into maturetRNA molecule. It is thus concluded
that trnC2-GCA is the onlytRNAcys(GCA)
gene that functions
in sugar beet mitochondria.
Acknowledgements
This work was supported
in part by Grants in Aid for Scientific
Researchfrom the Ministryof Education,
Science and Culture, Japan, and a Grant
from the Program for Promotionof Basic Research Activities
forInnovative
Biosciences,
Japan. A. E. is the recipientof a Japanese
Government (Monbusho)
Scholarship.
References
Unseld,M., Marienfeld,

J. R., Brandt,

P. and Brennicke,

mitochondrial
genome of Arabidopsis
366,924
nucleotides.
Nature Genet.

thaliana
1 5: 57-61.

A. 1997.

contains

The

57 genesin157

The relationshipmitochondrial
Shiho

between intron splicing
and RNA editingcox2 gene under low temperature.

Kurihara-Yonemoto

1, Hirokazu

Handa

Hokkaido

National

Laboratoryof Plant Genecology,
Sapporo, 062-8555,
Japan

Agricultural

Experimentof wheat

Station

There are few data for the mitochondrial-encoded
gene expression in higher plants undervarious stress conditions,especially
under cold condition,compared to the case of nuclear
genes. We investigatedthe effects of low temperature on the expressions of mitochondrialencoded genes in wheat. During the course of our study, we found that the transcriptional
pattern of cox2 gene (encoding the subunit 2 of cytochrome c oxidase) was affected by the
low temperature.
Northern analysis indicatedthat the precursor transcript
before intron
splicing
was increased by cold, although the level of mature message was not changed.
Mitochondrial-encoded
genes in higher plants are regulated through several posttranscriptional
events, such as RNA editing, intron splicing,etc. Wheat cox2 gene has a intron(Bonen et al 1984), which belongs to group II intron. One of important features for splicing
of group II intron is a specific binding between exon and intron sequences, which assists theformation of their unique tertiary structure. An RNA editing event is reported on the intron
binding site 1 (IBS1)of cox2 first exon, one of such specific binding sites. We found that cold
treatment severely suppressed the RNA editing on the IBS I of precursor transcript.
These results suggest that low temperature could affect the intron splicing
and/or RNA
editing of wheat mitochondrial
cox2 transcripts.
Materials

and methods
Winter wheat (Triticum aestivum L. cv. Chihoku-komugi)
seedlings
were grown at 20/1 5°C (day/night)
for 3 weeks. For cold treatment, the seedlings
were exposed to 2/0.5°C (day/night)
for 2 hours, 1 day, 3 days
and 7 days. After cold treatment for 7 days, the seedlings
were returned to 20/15°C(day/night)
and kept for 1
day.
For Northern blot analysis,
wheat total RNA was probed by two kinds ofcox2 DNA probes,
corresponding
to the regions of exon 1 -intron and only intron, respectively.
In order to check the RNA editing status, we used total RNA from control and cold treated plants for 7
days. RT-PCRs were performed with cox2 specific primers, and we got the cDNAs corresponding
to the maturetranscript
and the precursor transcript.
These products were cloned and sequenced.

Results

and discussion
Northern blot analysis with exon 1-intron probe identified
two major transcripts
of 2.7,and 1.5 kb in size. The steady-statelevel of2.7 kb transcript
clearly increased after 7 days ofthe cold treatment. In contrast, the level of 1.5 kb transcript
was not changed (Fig. 1A). In
order to further characterizethese two transcripts,
we performed second Northern blot
experiment using the intron-specificprobe. This probe hybridized
only to the 2.7 kb
transcript
and no 1.5 kb transcript
was detected at all (Fig. IB). These results indicate that a
2.7 kb transcript
corresponds to a precursor transcriptof cox2 with the intron, and a 1.5 kb
transcript
contains the mature message after splicing
out of the intron. These results provide
a clear evidence that the level of cox2 precursor transcript before intron-splicing
is increased
by low temperature.
Seventeen RNA editing sites were reported on the cox2 transcripts
of wheat (Covelloand Gray 1990; Albertazzi
etal. 1998) (Fig. 2A). One of them is located on the IBS1 whichplays an important role for splicing.
In order to investigate
whether low temperature affects
the RNA editing status ofcox2 gene in wheat, and especiallythe editing event on the IBS 1,
wecarried out sequencing analysis of 23 to 29 CDNA clones derived from each transcript.
All
CDNAclones from mature transcripts were almost completely edited regardlessoftemperature condition.
On the other hand, cold temperature suppressed the RNA editing of
precursor transcripts.
Particularly
the editing frequency of the site #6 under the cold
condition is very low (14%), which locates on the IBS1(Fig
2B). It was only 21% of that
1 Correspondence:
S. Kurihara-Y.
Key Words : mitochondrial-encoded, E-mail : shiho@cryo.affrc.go.jp
Fax : (81) 1 1 859 2178
gene expression, cox2, group II intron, RNA editing,intronsplicing

under control condition (Fig. 2C).
These results suggest that there might be some relations between intron splicing
andRNA editing event, especiallyon the IBS 1, of wheat mitochondrial
cox2 under low
temperature.
Our working model includes two possible
variations.
Low temperature affects
the tertiary structure of cox2 intron and the structural change of intron results in the reductionof RNA editing frequency on the IBS 1. Alternatively,
low temperature directly reduces RNA
editing frequency on this site and the exon-Intron binding by unedited IBS I sequence is notefficient
for intron splicing.
This is a first report for that low temperature affects a posttranscriptional
regulationof mitochondrial
gene In higher plant.
References
Albertazzi,

F. J., J. Kudla and R. Bock. 1998 The cox2 locus of the primitive
angiosperm plant Acorus
calamus: molecular structure,transcript
processing
and RNA editing. Mol. Gen. Genet. 259: 591-600
Bonen, L., P. H. Boer and M. W. Gray_. 1984. The wheat cytochrome oxidase subunit II gene has an intron
insert and three radical araino acid changes relative
to maize. EMBO J. 3: 253 1-2536
Covello,P. S. and M. W. Gray. 1990. Differencesin editing
at homologous
sites in messenger RNAs fromangiosperm mitochondria.
Nucleic Acids Res. 1 8: 5 189-5 196

Fig. 1 : Northern blot analysisof wheat mitochondria!
cox2 transcripts
using total RNA isolated
from 21 -daygreened seedlings
as control (lane 0), 2-hour, 1 -day, 3-day and 7-day cold treated seedlings(lane 2h3 Id, 3d,7d), and the seedlings
returned to control condition
after cold treatment for 7 days (lane -Id).
RNA blots were
hybridized
with exon 1-intron probe (A) and intron probe (B).

Fig. 2 A : Seventeen editing sites are reported in wheat mitochondria!
cox2 (Covelloand Gray 1990; Albertazzi
et al. 1998).
B : RNA editing frequency of precursor transcripts at 17 sites under the control and the cold
conditions.
C : The influence of cold treatment upon RNA editing frequency.

159

Mapping QTLs for tissue

culturetraitsin barley

Yoshiro Mano1'2 and Takao Komatsuda1 (National
Tsukuba, Ibaraki 305-8602
Japan. 2Present address:
Nishinasuno,
Tochigi 329-2793
Japan.)

Institute
National

Key words: albinism,shoot differentiationbarley,QTL, recombinant

inbred,of Agrobiological
Resources,
Grassland Research Institute,

Introduction
Identificationof genes controllingtissue culture traits will make possible the transfer of genes
for desirable
tissue culture traits into recalcitrant
cultivars or species. In barley, QTLs forshoot differentiation
ability
have already been mapped on several chromosomes (Mano et al.
1996; Takahashi
et al. 1997; Manninen 2000), however, major QTLs for this trait have notyet been identified.
Here we report on the identificationof loci controllingtissue culture traits
using recombinant inbred lines (RILs)
derived from the cross between the parents showing
extremely wide difference for their tissue culture traits.
Materials and methods
Ninety-nine RILs of 'Azumamugj' (AZ, low differentiation
ability)x 'Kanto Nakate Gold'(KNG, high differentiation
ability)were used. Immature embryo culture was performed in 3
trials, F7 without replicationand ¥\o evaluated by 2 persons (Fi0 repl and Fl0 rep2), and wasdone essentiallyas described by Komatsuda et al. (1989).
Two tissue culture traits of shootdifferentiation
ratio (SD) and percentage of green shoot ratio (GS) were evaluated.
Weconstructed a "base map" with 100 markers selected from 273 AFLP-STS map(Mano et al 1999) using MAPMAKER 3.0 (Lander et al. 1987). QTL mapping analysis fortissue culture traits were performed by single-point
analysis using QGENE (Nelson 1 997).
Results and discussion
Among3 trials, QTLs for SD were detected on chromosomes 1H, 2H, 3H, 5H, and 6H (Fig.
1). A consistent locus with larger effect across 3 trials was found on chromosome 3H [nearest
marker is uzu, KNG alleles increased SD]. This locus was identical with or very close to uzu
locus and the peak LOD ranged from 6.68 to 12.61. The uzu gene has pleiotropic
effects not
only on several agronomic characters but to less auxin production (Kuraishi1974), which may
have effect on tissue culture response. It may be possible that QTL for SD on chromosome
3H results from the pleiotropic
effect of uzu gene, but further study is necessary. The
positionof QTL for SD on chromosome 2H [nearest marker is MWG503, KNG alleles
increased SD] found in F10 rep 1 and rep2 is in good agreement with Shdl previously
reportedby Komatsuda et al. (1995).
Three QTLs for GS were identified
on chromosome 2H, 3H, and 7H (Fig. 1). In
particular,
QTL on chromosome 7H [nearest marker is el5m27-4]
in F10 rep1 shows large
effect (LOD=12.66),
and allele of non-differentiating
cv. AZ increased GS. QTL for GS on
chromosome 7H could be valuable to increase GS in anther culture, which has a serious
problem of frequent albino shoots formation.
Comparison of the map position
between QTLs detected in this study and those inother studies (Mano et al. 1996, Steptoe x Morex cross; Manninen 2000, Rolfi x Botnia cross),the positionsof QTLs on chromosome 2H and 3H were identical or nearly same. Furthermore,QTL for SD on chromosome 2H also mapped
Harrington x TR306 cross (Takahashi
et al. 1997).
can identify the commonfunctional QTL distributedat the same or nearly same positionin
By using commonmolecular markers, wein the 3 mapping populations.

References
Komatsuda, T., S. Enomoto and K. Nakajima. 1989. Genetics of callus proliferationand shootdifferentiation
in barley. J. Hered. 80: 345-350
Komatsuda T., F. Taguchi-Shiobara,
S. Oka, F. Takaiwa, T. Annaka and HJ. Jacobsen. 1995.
Transfer and mapping of the shoot-differentiation
locus Shdl in barley chromosome 2.
Genome 38: 1009-1014
Kuraishi,S. 1974. Biogenesisof auxin in the coleptileof a semi-brachyticbarley, uzu. Plant
Cell Physiol.
15: 295-306
Lander, E.S., P. Green, J. Abrahamson, A. Barlow, M.J. Daly, S.E. Lincoln and L. Newburg
1987. MAPMAKER: An interactive
computer package for constructing
primary genetic
linkage maps of experimental and natural populations.
Genomics 1 : 1 74-1 8 1
Manninen, O.M. 2000. Associations
between anther-culture
response and molecular markers
on chromosomes 2H, 3H and 4H of barley {Hordeum vulgare L.). Theor. AppL Genet.
100:57-62
Mano, Y., S. Kawasaki, F. Takaiwa and T. Komatsuda. 1999. STS-AFLP based map
constructionin the RILs from Japanese barley cross, Azumamugi / Kanto Nakate Gold, (inJapanese). Breed. Res. 1 (Suppl.
2): 149
Mano, Y., H. Takahashi, K. Sato and K. Takeda. 1996. Mapping
genes for callus growth andshoot regeneration in barley {Hordeum vulgare L.). Breed. Sci. 46: 137-142
Nelson, J.C. 1997. QGENE: software for marker-based genomic analysis and breeding. Mol.
Breed. 3: 239-245
Takahashi,
H., Y. Mano, K. Sato and K. Takeda 1997. Mapping genes for callus growth andshoot regeneration in barley {Hordeum vulgare). II (in Japanese).
Breed. Sci. 47 (Suppl.
1):116

Fig. 1 Chromosome locations of QTLs for shoot differentiation(SD) and green shoot ratio (GS) in the RILs
of AZ/KNG. Significant QTLs are indicated to the right of the nearest markers. QTLs for tissue culture traits
in F7, Fj0 repl, and Flo rep2 are shown on the top, middle, and bottom, respectively.
-: Not significant.

161

Mapping
Katsuhiro

of a fertility
restoration
locus (Rfm1) for cytoplasmic
sterility
in barley using RAPD and STS markers

maleMatsui1' 4, Yoshiro Mano2'5, Shin Taketa3, Naoyuki Kawada1, Takao Komatsuda2

1. National Agriculture
Research Center, Kannondai, Tsukuba 305-8666,
Japan
2. National Instituteof Agrobiological
Resources, Kannondai, Tsukuba 305-8602,
Japan
3. Faculty of Agriculture,
Kagawa University,
Miki-cho, Kagawa 76 1 -0795, Japan
4. Kyushu National Agricultural
Experiment Station, Nishigoshi,
Kumamoto 86 1 -1 1 92, Japan
5. National Grassland Research Institute,
Senbonmatsu, Tochigi 329-2793,
Japan

Abstract
The Rfinla gene restores the fertilityof msml cytoplasmic male sterile lines in barley. We
have identified
three RAPD markers linked to the Rfml locus, CMNB-07/800,
OPI-1 8/900,
and OPT-02/700,
using segregating
BC1F1 and F2 populations.
The CMNB-07/800
waslocated beside MWG2218 on the short arm of chromosome 6H using formerly developed
linkage map of the cross Azumamugi and Kanto Nakate Gold. The MWG2218 was also
linked to the Rfml locus. These results showed that the Rfml locus was located on the shortarm of chromosome 6H. The synteny of Triticeae
suggests that the Rfml gene ishomoeologous to the restorer genes identified
in group-6 chromosomes of wheat and rye.
Introduction
Twomale-sterile cytoplasms, designated msml and msm2, were found in 2 strains of the wildbarley, H. vulgare ssp. spontaneum (C. Koch) Thell. (Ahokas 1 979, 1 982). The spontaneum
subspecies strain with the msm1cytoplasm carried a dominant restorer gene designated Rfmla(Ahokas 1979). DNA markers tightly linked to the Rfml locus enable the molecular study of
the CMS system. In the present study, we performed the molecular mapping of the Rfml
locus of barley in order to apply the linked markers to marker-assisted
selection andmap-based cloning of the relevant gene.
Materials and Methods
The CMS line, the restorer, and Adorra were kindly provided by Dr. H. Ahokas, Agricultural
Research Centre, Finland. In this paper, the cytoplasm type is designated by italics and the
genotype of the restorer gene is designated by parenthesized
italics. We used a total of 200
1 2-meric oligonucleotides,and 684 1 0-meric oligonucleotide
primers to detect polymorphism
between msml-(RfmlalRfmla)and msml'-(rfml a'rfml a) in the RAPD profiles. An F2 family
consisting of 233 plants was derived from the msml-(RfmlalRfmla)x N-(rfmlalrfmla)cross.
A BC1F1 family consisting of 242 plants was produced by backcrossing of the Fi plants to
msml-{rfmlalrfmld).
In order to map the RAPD markers on the known linkage group,
recombinant inbred lines (RILs) of the Azumamugi (AZ) x Kanto Nakate Gold (KNG) crossand the molecular maps were used (Mano et al. 1999).
Results and Discussion
The segregations of fertility
in F2 and BC1F1 populations fitted the expected monogenic
segregation ratios of3:1 (x2 = 0.71, 0.25 < P < 0.50) and 1:1 (%2 = 0.0, 0.95 < P), respectively.
Three fragments generated by CMNB-07, OPI-1 8 and OPT-02 were linked to the Rfml locus(Fig. l ). Approximate sizes of the polymorphic fragments of each primer were 800, 900 andFig. 1_ PCR products using the OPT-02 primer. An
arrow indicates polymorphic band.

Fig, 2 Genetic maps of the Rfinl locus on the short armof barley chromosome 6H. The maps were constructedusing (A) RILs from an Azumamugi x KantoNakate
Gold cross, (B) BC1F1, and (C) F2 from a msml(Rfmla/Rfmla)
x N-{rfmlalrfmla)cross. Map distances
are given in cM.

700 bp, and these fragments were designatedas CMNB-07/800,
OPI-1 8/900 and
OPT-02/700,
respectively.
Based on the
populationof242 BCiFi plants, OPI-18/900
was most closely linked to the Rftnl locus(5.2 cM) (Fig. 2B). The location of
OPI-18/900
was similar to that in the F2
population (Fig. 2C). To identify the map
locations of the 3 RAPD markers, we
utilized our previously developed molecular
linkage maps of RILs constructed from theAZ x KNG cross (Mano et al. 1999). RAPD
analysis using the 99 RILs showed that
CMNB-07/800 was linked to MWG2218 on
the short arm of chromosome 6H (Fig. 2A).
In the present study, the restorer
gene Rftnla seems to be located on asatellite,
because the Rftnl locus was distal
to MWG2218, which is itself distal to the
NOR in 6HS. Considering the high degrees
of synteny between barley and wheat, therestorer gene Rftnla of barley might behomoeologous to the wheat restorer genes
Rf4, Rf5 or Rf6 on homoeologous group 6
chromosomes. In wheat-rye addition lines,
the rye restorer gene can restore the malefertilityof wheat that has T. timopheevi
cytoplasm (Curtis and Lukaszewski 1993).
The rye restorer gene (Rfgl) was mappedon the long arm of chromosome 4, which
was homoeologous to the short arm of
homoeologous group 6 chromosome in
Triticeae(Bomer et al. 1998).
These
evidencesindicatethat CMS and therestoration system might be homoeologous
amongwheat, rye and barley.

References
Ahokas, H. 1979. Cytopiasmic
male sterility in barley. Acta Agric Scand 29:21 9-224
Ahokas, H. 1 982. Cytopiasmic
male sterility in barley. XL The tnstnl cytoplasm. Genetics
102:285-295
BorneoA., V. Korain?A. Polley, S. Malyshev? G. Melz. 1998. Genetics and molecularmapping of a male fertility restoration
locus (Rfgl) in rye (Secale cereale L.). Theor. Appl.
Genet. 97:99402
Curtis C.A., A.J. Lukaszewski. 1993. Localizationof genes in rye that restore male fertility tohexaploid
wheat with timopheevi cytoplasm. Plant breed. I l l : 1 06-1 12
Mano9Y, B.E. Sayed-Tabatabaei9
A.Graner, T. Blake9 F. Takaiwa, S. Oka, T. Komatsuda.
1 999. Map construction of sequence-taggedsites (STSs) in barley (Hordeum vulgare L.).
Theor Appl Genet 98:937-946

163

Barleyallele-specificamplicons

useful for wheat-barley

Koji Murai1' *, Shin Taketa2, A. K. M. Rafiqul

introgression

Islam3 and Ken W. Shepherd3

1Department of Bioscience,
Fukui Prefectural
University, Matsuoka-cho, Fukui 910-1195,
Japan, department of Life Sciences, Faculty of Agriculture,
Kagawa University, Ikenobe,
Kagawa 761-0795,
Japan, 3Department of Plant Science, Waite Campus, UniversityofAdelaide,

Glen Osmond, SA 5064, Australia,

Key words: RFLP, STS-PCR, amplicon,*Corresponding

wheat-barleyauthor (murai@fpu.ac.jp)

introgression

Barley (Hordeum vulgare L.) is a potential
new source of genes for wheat (Triticum aestivumL.) improvement.
Recently,
Murai et al. (1997a)
examined heading charactersof wheatbarley chromosome addition lines produced by Islam et al. (1981)
and Koba et al. (1997),
andfound that barley chromosome 5H has genetic effects on accelerating
narrow-sense earliness,decreasing
vernalization
requirement
and/or increasing
photoperiodic
sensitivity.
To
introduce the barley genes for early heading on chromosome 5H into wheat, we are planning
to produce wheat-barley
5H chromosome recombinant lines (Murai et al. 1997b).
A majorproblem for producing
wheat-barley
recombinants
is the low pairing
frequency
betweenwheat and barley chromosomes, resulting in a low frequency of recombinants.
Therefore, itis necessary
to have an effective
selectionsystem to detect
the rare wheat-barley
recombinants.
For the effective screening, it would be suitable to use PCR markers directly
amplifying
barley alleles(allele-specificamplicons)
which can be distinguishedfrom theirwheat counterparts
without restriction
enzyme digestion.
Blake et al. (1996)
reported
135barley allele-specificamplicons amplified
by 115 primer sets developed
from sequences ofpreviously
mapped RFLP clones.
Recently,
we developed
26 barley
allele-specificamplicons (eight for chromosome 5H) detectableby more simplified
detectionsystem (Murai
et al. 2000).
Here, we report ten novel barley allele-specificamplicons useful for identifying
wheat-barley
recombinants involving chromosome 5H.
MATERIALS AND METHODS
Wheat-barley
disomic addition lines possessing
each of 'Betzes' barley chromosomes 2H to
7H in the background of 'Chinese Spring' wheat (Islam et al., 1981) or each of 'New Golden'
barley chromosomes 5H and 6H in the backgroundof 'Shinchunaga'
wheat (Koba et al.
1997)
were used to detect barley allele-specificamplicons.
A total of 23 primer sets
developed from the sequences of previously
mapped barley RFLP clones on chromosome 5H(Kunzel et al. 2000) were evaluated by PCR assay at 55°C as the annealing
temperature.
Plant DNA was extracted by a modified
CTAB method and used as a template
assay.
PCR condition
was same as described in Murai et al. (2000).

for the PCR

RESULTS AND DISCUSSION
Out of 23 primer sets, 13 gave PCR products with the same size in both wheat and barley, andfurther treatments
such as restriction
digestion
would have been necessary to producepolymorphisms.
On the other hand, ten primer sets amplifiedbarley chromosome-specific
PCR products which were confirmed to be located on the same chromosome 5H as the RFLP
markers by using the wheat-barley
chromosome additionlines.
PCR products from theformer primer sets, namely, aABG497, aMWG550, aMWG561, aMWG583, aMWG602,
aMWG835, aMWG877, aMWG891, aMWG2121, acMWG650, can be regarded as barleyallele-specificamplicons.

Based on the RFLP map of Qi et al. (1996),alinkagemap of the barley allele-specificamplicons on
chromosome 5H is shown in Fig. 1. Together with theresults of Murai et al. (2000),
a total of 18 barley allelespecificamplicons can be useful for identifying
wheatbarley
recombinants
involving
chromosome 5H.
However, we did not get amplicons located on distal
positionof short arm. To obtain new amplicons
locatedon this region, alternative
methods such as
developmentof amplicons from barley allele-specific
RAPD markers (Murai 1995) should be introduced.
Fig. 1. Linkage
map of 18 amplicons
on chromosome 5Hdevelopedfrom RFLP markers. Map positionof amplicons wascited from the RFLP map (Qi et al. 1996) with the assumption
that
an RFLP marker and the amplicon
lie on the same locus.
ABA306B is RFLP marker. Centromere position was determinedfrom the barley consensus map, Adelaide.

The authors thank Dr. T. Koba, Chiba University
wheat lines.

for the 'New Golden'

chromosome addition

REFERENCES
Blake,T. K., D. Kadyrzhanova,
K. W. Shepherd,
A. K. M. R. Islam, P. L. Langridge,
C. L. McDonald, J. Erpelding,S.
Larson, N. K. Blake and L. E. Talbert. 1996. STS-PCR markers appropriate
for wheat-barleyintrogression.
Theor.
Appl. Genet. 93:826-832.

Islam,A. K. M. R., K. W. Shepherd and D. H. B. Sparrow. 1981. Isolationand characterizationof euplasmic
wheat-barley
chromosome additionlines. Heredity 46:161-174.
Koba, T., S. Takumi and T. Shimada. 1997. Isolation,
identificationand characterizationof disomic and translocatedbarleychromosome additionlines of common wheat. Euphytica
96:289-296.
Kunzel, G., L. Korzun and A. Meister. 2000. Cytologicallyintegrated
physicalrestriction
fragment
length polymorphism
maps for the barley genome based on translocation
breakpoints.
Genetics 154:397-412.
Murai,
Murai,
Murai,

Murai,

K. 1995. Developmentof barley chromosome-specific
RAPD markers for identification
added to the wheat genome. Bull. Res. Inst. Agr. Resources, Ishikawa Agr. Coll. 4:31-37.
K., T. Koba and T. Shimada.
1997a.
Effectsof barley chromosome on headingof barleycharacters

chromosomes
in wheat-barley

chromosome additionlines. Euphytica
96:281-287.
K., S. Taketa, A. K. M. R. Islam and K. W. Shepherd. 1997b. A simple procedure for the productionof wheat-barley
5H chromosome recombinantlines utilizing
5B nullisomy
and 5H-specificmolecular
markers. Wheat Inf. Serv.
84:53-55.
K., S. Taketa, A. K. M. R. Islam and K. W. Shepherd. 2000. Barley allele-specificamplicons
useful for identifying

wheat-barley
recombinant
chromosomes. Genes Genet. Syst. 75:131-139.
Qi, X., P. Stem and P. Lindhout.
1996. Comparison and integrationof four barleygenetic

maps. Genome 39:379-394.

165
Geographicalvariation of the gene, lox-3(t), causing
deficiency in Asian rice varieties

Tsukasa Nagamine1*, Yasuhiro Suzuki2) and Kazuo Ise3)
1) National
Instituteof Agrobiological
Resources, Tsukuba
2) National
Agriculture
Research Center, Tsukuba Ibaraki
3) Japan International
Research Center for Agricultural
305-8686
Japan

lipoxygenase-3

Ibaraki
305-8602Japan
305-8666
Sciences,
Tsukuba Ibaraki

Introduction
Deteriorationof rice grains and development of stale flavor during the storage areserious problems,
which reduce the qualityof stored grains. Lipoxygenase(LOX)
catalyzes
the peroxidationof linoleic
and linolenic
acids to form hydroperoxides.
The absence of the LOX enzymes in rice grains may thus alleviate
the oxidative
deterioration.
Three isozymes, LOX-1, LOX-2 and LOX-3 were found in riceembryos and LOX-3 is the major component of the isozymes accounting 80-90% of thetotal LOX activities(Ida et al 1983).
We detected one LOX-3 deficient variety, Daw
Dam originatedfrom Thailand
after a preliminary
survey (Suzuki et al. 1993) and wetentatively
designatedthe Lox-3 null characteras lox-3(t) (Suzuki
et al. 1996).
However, the geographicalvariationof the lox-3(t) gene in cultivatedrice is notknown well, we have screened the gene in Asian-wide varieties.
Materials and Methods
To analyze the geographicalvariationin the occurrence of the lox-3(t) gene, 471
Asian varieties
conserved in the Genebank of NIAR, were assessed using monoclonal
antibodies(Suzuki et al. 1992).
The LOX-3 content was graded by visual inspection
according to the descriptor
below; 1 (deficient),
2 (less than content of Koshihikari),3(almost same as of Koshihikari)and 4 (more than that of Koshihikari).
Analysis of
the LOX-3 was conducted using at least two grains per one accession.
Results and Discussion
A total of21 varieties
were found to have lox-3(t) gene and among them seven were
heterogeneousat the Lox-3 locus (Table1). The LOX-3 deficient varieties
werefound in China, Vietnam, Laos, Thailand
and Myanmar. It is recognizedthat thedeficiency
occurred in the limitedarea of the Asian countries
and the area waspointed out to be the center of genetic diversityof rice (Nakaghara
et al. 1977).
Based on the change of hull color in phenol solution of 1.5%, Asian rice varieties
areroughlyclassified
into two varietalgroups; Japonica
and Indica.
For the 441
varieties
except some varieties
from China, the phenol color reaction of hull were
examined (Table 2). As a result, LOX-3 deficientvarieties
were found in the both
Japonica
and Indica
groups and it is assumed
that the lox-3(t)gene were
spontaneously
occurred in the both varietalgroups.
As the hydroxides
are transformed
into volatilecompounds causing flavors, LOX
is related
to the formation of the volatilecompounds (Suzuki et al. 1999), these LOX3 deficientvarieties
will be utilized
as novel genetic
resources
for increasingstorabilityof grains.
References
Ida, S., Y. Masaki

and Y Morita

1983. The isolationof multiple

forms and productspecificity
of rice lipoxygenase.
Agric. Biol. Chem.. 47. 637-641.
Nakagahra,
M. 1978. The differentiationand center of genetic
diversityof cultivatedrice by isozyme analysis.
Trop. Agr. Res. Ser. ll: 77-82.
Suzuki, Y., T. Nagamine, A. Kobayashi
and K. Otsubo 1993. Detection
ofa new rice
varieties
lacking
lipoxygenase-3
by monoclonal antibodies.
Jpn. J. Breed. 4,405-409
Suzuki, Y., T. Nagamine, and K. Gkuno 1996. Genetic analysis
ofa null-alleleforlipoxygenase-3
in rice seeds.
Euphytica
91: 99-101.
Suzuki, Y, K. Ise,C. Li, I. Honda, Y. Iwai and U. Matsukura
1999. Volatilecomponents in stored rice (Oryza sative h.) of varieties
with and without
lipoxygenase-3
in seeds. J. Agric. Food Chem. 47 1119-1124.

Table

1. Geographical

Note:

Table

2

variationof LOX-3 deficiency

in Asian rice varieties

Number in the parenthesisshows the number of varieties
heterogeneousfor Lox-3 locus.
LOX-3 deficient varieties
found in the two varietal
based on the phenol color reaction of hullgroupsof

167

A Molecular Phylogenyof genus Lilium in matK gene Sequences,

Tomotaro

Nishikawa1,

Okazaki2,

Agrobiol.

Resour., 2. Fac. Agr. Niigata

Keiichi

KatsuroArakawa3,

U., 3. Yurigahara

TsukusaNagamine1(1. Natl.

Inst.

Park)

Introduction
The genus Lilium includes
approximately
100 species, and it is native to the Northern
Hemisphere, in Asia, Europe, and North America. In Japan, 14 species are distributed,
andmost of them are important genetic resources of Oriental or Asiatic hybrids,
which are main
divisionsof cultivars. Recently, crossing between distantly related species have been tried for
introductionof new characteristics
to existing cultivars,
but detailed
relationships
betweensections are not clear in spite of phylogenetic
estimation of Comber (1949) and Lighty (1968)
based on morphologicalcharacters and germination form. The objectivesof this study are toclarify relationships
among sections in genus Lilium based on the sequence data of the matK
gene.
Materials and methods
Twenty-six taxa of Lilium were sampled and analyzed in the present study. Fritillaria
verticillata
was used as an outgroup. DNA sequences were determinedusing cycle
sequencing,and multiplyaligned using Clustal
X version 1.81 (Tompson et al. 1997).
Phylogenetic
analysis was performed as a maximum parsimony phylogeny
estimation using
the PAUP* version 4.0b4a (Swofford 1998) using 10,000 bootstrap replications.
Results and discussions
The alignment of 27 entire matK sequences provided a matrix of 1,545 base pairs. The
parsimonious phylogenetic
tree was divided into three major clades (Fig. 1), designatedasfollows:
Group I comprised section Mqrtagon, Sinomartagon, Daurolirion,
subsection
6b(Leucolirion)and section Liriotypus;Group II comprised only section Pseudolirium;Group
III comprised section Archelirion
and subsection 6a {Leucolirion).
The orders of divergencesamong these three groups were not clear, but they were clearly monophyletic,
respectively.
The distributionof Group I taxa extend from East Asia to Europe. Section Liriotypus,
which is distributedin Europe and West Asia, was divergent from them first. But in the rest of
taxa belonged this clade, the order of divergenceswere ambiguous among three sections,Martagon, Sinomartagon and Daurolirion.
In the phylogenetic
tree constructedfrom theinternal transcribedspacer (ITS) region of ribosomal DNA (Nishikawa et al. 1999), Martagon
was clearly monophyletic,
and had sister relationshipto the clade included
subsection 6b,
Sinomartagon and Daurolirion.
But in the tree constructedfrom matK gene, Martagon,
Sinomartagon and Daurolirion
were monophyleticand had sister relationshipto subsection

6b. Therefore,

it was difficult

to decidewhich was divergent

earlier,

Martagon or subsection

6b.

Fig. 1Phylogenetic
tree constructed from matK gene sequences of 26 Lilium taxa by maximum
parsimony method. The numbers beside the nodes are the bootstrap value (%).

In Group
constructedIII

includedrelationshipII andIII,

from only
taxa

each
sectiondistributed

section(or

subsection)

Pseudolirium,

which

in Eastand Archelirion

as well as ITS (Nishikawa

Asia,

was divergent
is distributed

clearly.

Group

II wasover North

America.

Group

and

subsection

6a had sister

et al. 1999).

Acknowledgement
Wethank Ms. N. Nakamaru and Ms. Y. Obata for their assistance in DNA sequencing. Wealso acknowledge
Mr.T. Nobori, Mr. T. Komatsuzaki, Mr. M.Akiba, Mr. T. Ohmizu and Mr. T. Taguchi, who are the staff of theFarm Management Division of the National Instituteof Agrobiological
Resources for their management ofmaterials.
Reference
Comber, H.F. 1949. A new classificationof the genus Lilium. Lily Year Book of RHS 13: 86-105.
Lighty, R.W. 1968. Evolutionary
trends in lilies. Lily Yeai Book of RHS 31: 40-44.
Nishikawa,T., K. Okazaki, T. Uchino, K. Arakawa and T. Nagamine. 1999. A Molecular PhylogenyofLilium in
the Internal Transcribed Spacer Region of Nuclear Ribosomal DNA. J. Mol. Evol. 49(2): 238-249.
Swofford, D. L. 1998. PAUP*. Phylogenetic
AnalysisUsing Parsimony (*and Other Methods).
Version 4.
Sinauer Associates, Sunderland,
Massachusetts.
Tompson, J.D., T.J. Gibson, F. Plewniak,
F. Jeanmougin and D.G. Higgins.
1997. The CLUSTAL_X
windows interfaceflexible
strategies
for multiple
sequence alignment
aided
by quality
analysistools. Nucleic Acids Res. 25(24):4876-4882.

Diversity

in Pakistan

Rice Genetic

Resources

for Endosperm

Storage

Sadar Uddin SIDDIQUI,
Toshihiro KUMAMARU and Hikaru SATOH
Lab. of Plant Genetic Resources, Kyushu University,
Japan
Pakistan rice germplasm is very diverse in terms of qualitativeand quantitative
traits.
For the improvement of rice grain quality, local genetic resources is needed to be investigated.
Research in Pakistan is mainly concentrated for increasing the rice yield; lacking studies about
the grain protein and starch quality. In order to ascertain the diversity
for its possible
utilizationin rice improvement, in this report we describe the diversity
observed in Pakistan
local rice genetic resources for storage proteins and starch characteristics
for its utilizationinthe enhancement of the grain quality In terms of storage proteins and cooking quality In viewof the starch characteristics.
475 accessions of Pakistan local rice genetic resources obtained from MAFF genebank,
Japan were used as materials. The endosperm storage proteins and waxy proteins of theaccessions were analyzed by SDS-PAGE and glutelin by IEF. Starch characteristics
pertaining
to cooking quality are related to gelatenization
temperature, which is related to alkali
digestibility,
hence it was measured using 1.7% KOH solution.
Rice storage proteins are composed of glutelin and prolamin. Major component of
prolamin is 13/15 kD polypeptide.
Rice glutelin is consist ofa and p subunits which werecomposed of4 and 3 bands, respectively,(Uemura et al 1996).
A wide variation was found in Pakistan.rice cultivars for the two major storage proteins,
i.e. prolamin and glutelin. Prolamin variation was apparently
grouped Into 4 types; 16% of thetotal accessions showed low levels of 13 kD, while presence of the 14 kD (not found In IR36),
was observed in 1% accessions, 3% of the accessions had low level of both 13 and 15 kD
polypeptide
while, most of the cultivars (80%) were having both bands of 13 and 15 kD ofequal high intensity.

Fig. 1 Showing

variationin glutelina-3 band (A), a-4 band (B) and 57 kD band (C).

Pakistan varieties revealed 6 types of variation for giutelin subunit, Fig. 1A shows thevariation for a-3 band, i.e. fast migration a-3 band type (lane 1), slow migration a-3 band
type (lane 2), a-3 band having both fast and slow migration bands type (lane 3) and a-3 band
deletion type (lane 4). Their respective
frequencies
were 70%, 1 1%, 2% and 0.4%

170respectively.
Fig. IB shows the variation for a-4 band, where fast (lane 1) and slow (lane 2)
migration types were recorded, wild type is in lane 5. Their respective frequencies were 2%
and 80%, respectively,
for the total germplasm. For the variation of 57 kD glutelin precursor,
enriched glutelin precursor (57H) cultivars were observed (Fig. 1C). The frequency of 57H
was 18% of the total germpiasm. Satoh et al (1995)
reported 1.4% of accessions from North
Asian countries showing 57H variation. 98% of varieties bearing 57H variation were
distributed
in low altitude range areas, whereas the highest frequency.of occurrence was in
Punjab region i.e. 90.5%. The distribution
pattern of 57H variation show a high degree ofcorrelationin terms of altitudinaland geographicaloccurrence.
Genetic analysis of the Pakistan 57H variation showed that, the 57H variation iscontrolled
by a single recessive gene, allelic to glup3 gene. The glup3 gene was located within
5.3 cM distance between RFLP markers C1238 and R2370 on chromosome 4.
The classified
variation by SDS-PAGE was analyzed by IEF In detail for glutelin.
Glutelin was separated Into a and p subunits (Uemura etal. 1996), which consisted of 12 and10 bands, respectively.
Where japonica cultivar, KInmaze and indica cultivar, IR36 has
specific bands. Pakistan rice cultivars showed another type besides the T65 and IR36 types,
which consisted of bands 6 (IR36 specific),
and A and B (T65 specific),
data not shown.

51 cultivars' (1 1%) had 60 kD waxy protein band of Wxhtype i.e. low intensity
band in
SDS-PAGE. Majorityof these (80%) were distributedin high altitude region. A wide variation
was found in Pakistan cultivars for alkali digestibility(Fig. above) covering all the range fromlevel 1 to 10, however, majority ofcultivars(74.9%)
had low, 14.95% showed medium and10. 1% were with high alkali digestibility,
further investigation
on starch content are underway.
Our findings confirm the existence of great diversity
in seed storage protein and starchcharacteristics
especiallypertaining to variation in alkali digestibility,that could be utilized forprotein quality and cooking quality improvement and other genetic studies.
References:
SatohH, Kumamara
T, OgawaM, Siraishi M, Gi Im B, Son MY, Takemoto Y. 1995.
Spontaneous mutants in rice. Rice Genetics Newsletter
12: 194- 196.
Uemura YJ3 Sotah H, Ogawa M, Suehisa H, Katayama TC, Yoshimura A. 1996.
Chromosomal location of genes encoding glutelin polypeptides
in rice. In: Khush GS, editor.
Rice Genetics III Proceedings of the 3rd I R G Symp.;16-20
Oct. 1995; Los Banos,
Philippines,
p 471-476.

171

Isolationof microsatellites

Keita
Suwabe1,
O Hiroyuki
Kowyama2 and Masashi Hirai1

in Brassica

rapa.

Iketani1,

Takahiro

Kage1,

1)

National
Research
Instituteof Vegetables,
360 Kusawa, Ano, Mie, 512-2392
JAPAN.

Ornamentals

2)

Faculty1515

of Agriculture,

Mie

University,

Tohru

Tsuchiya2,

Yasuo

and Tea (NIVOT),MAFF,

Kamihama,

Tsu,

Mie,

514-8507

JAPAN.
Introduction
Microsatellites
are now recognizedas powerfulmolecular
markers thanks
totheir hypervariability
and omnipresencein the genome. However laborious
proceduresindispensablefor their development
prevents
the rapid spread into the plant genome
research.
In the Brassica
species,severalresearchgroups have alreadyisolatedanumber of microsatellites
mainly from B. napus, of which only a few are disclosed.
We therefore
startedto develop
enough number of microsatellite
markers
covering the whole genome of Brassica rapa.

for

Materials

and methods.
A doubled haploidline, A9709(B. rapa)
developed
in NIVOT was used for
DNA material.
Genomic DNA digested
with Sau3A I was ligatedinto a X vector. The
plaques
were screened using digoxigenin-labeled(GA)isand (GT)isoligonucleotide
probes.
Positivecloneswere further
screenedand converted
into plasmids,
thensequenced.
PCR primers
for amplifying
the microsatellite
were designed
from theflanking
region sequences.
Polymorphism
among 19 cultivars
belongingto different
cultivargroups of B.
rapa were examined using the obtained
microsatellite
primers.
Results
obtainedand discussion.
A total of 58 clones
containing
microsatellite
from 1.2 x 105 clones.
GA/CT repeats

repeats(Table(Szewc-McFadden

1).

Primerpolymorphism

pairs
using

motifs (4 to 53 repeats)
were more abundantthan

The results
were consistentwithet al. 1996, Uzunova and Ecke 1999).
were designed
electrophoresis

thefor 38 clones.
One
on 2.5 % agarose gelreports
third
(Fig.

on
of
1).

were
GT/CA

B. napusthem showed
For the rests,

polymorphism
was detected
on denatured
5 % polyacrylamide
sequencing
gel (Fig. 2).
Wewill add more markers, and apply them to the genome analysis
ofB. rapa,
including

QTL analysisof the resistantgenes for clubrootdisease.

References
Szec-MaFadden,
1996.

Identification

A. K., S. Kresovich,S. M. Bliek,of polymorphic,

conserved

S. E. Mitchell
simplesequence

cultivatedBrassica
species.
Theor. Appl. Genet. 93: 534-538.
Uzunova, M. I. And W. Ecke 1999. Abundance,
polymorphismof microsatellites
in oilseed
rape {Brassica
napus L.). Plant
Table

1. Microsatellites

isolatedin thisand J. R.McFerson
repeats

and genetic
Breed. 118:

(SSRs)

inmapping323-326.

study.

Figure1. Polymorphismof microsatellites
among 19 cultivarsof B. rapa using
BRMS-006 primers on 2.5 % agarose gel M: molecular marker (k/Hind
III, Hinc II).

Figure2. Polymorphismof microsatellites
BRMS-027
primers
on 5 % denatured(GENESCAN
500). A: A9709.

amongacrylamide

19

cultivarsgel.
M:of B. rapa using
molecular
marker

173

Origins
Linkedof Six-rowed CultivatedBarley deduced
to the vrsl Locus (Row Type Gene).

Ken-ichiTanno1'2, Shin Taketa3, Kazuyoshi

from a DNA Marker Tightly

Takeda4, Takao Komatsuda1

Rowtype of barley is controlled
by gene on the vrsl locus in chromosome 2H. Six-rowedbarley is believed
to have differentiatedfrom two-rowed barley by the mutation at this locus(see Harlan 1995). Given that the six-rowed barley arose from mutational events, its origin
was a probably limited in number. There is, however, a little evidence for the origin of the sixrowed cultivated barley at a DNA level.
A DNA marker, cMWG699, is the most closely linked to the vrsl locus (0.1cM)(Komatsuda et al. 1999). This DNA marker did not show recombination with the vrsl locus in
Japanese breeding cultivars
and had polymorphism within six-rowed cultivars (Tanno et al.
1999). By using this marker, we investigatedthe origin of the six-rowed cultivatedbarley andfound that variationof the cMWG699 was considerably
reduced in six-rowed cultivatedbarley.
Seventy-six
two-rowed cultivated,
204 six-rowed cultivatedand 183 wild (ssp.
spontaneum, two-rowed) accessions
were analysedwith PCR-RFLP method. PCR
amplificationof the cMWG699 was carried out using the internal primers cMWG699 T7-3
and T3-3 (Tanno et al. 1999) and restriction
analysis with Taql were performed following themethods described by Komatsuda et al. (1998).
The Taql digestionof the PCR products showed three types A, K and D as reported
in our previous study (Tanno et al. 1999), and no other type was detected. Types A and D
were found in two-rowed cultivated,
six-rowed cultivatedand wild barley while type K was
absent in six-rowed cultivatedbarley (data not shown). Most of the accessions analysed wereof type A (87.9%).
Types K and D appeared with low frequencies (8.6% and 3.4%,
respectively)
and showed clear geographicaldistributions:type K was prevalent in Europe
and the Middle East and type D distributed
to the Mediterranean region.
To discriminate
further variations,
nucleotide sequence analysis was carried out using
the outer primers cMWG699 T7-2 and T3-2 (Komatsuda et al. 1998). Sequence analysis oftype A accessions revealed that all the 10 six-rowed cultivatedbarleys analysed had an
identical
sequence (Table 1). The sequence, named sequence-type I, coincided with that of assp. spontaneum accession OUH730 from Turkmenistan.
Accessions of type D, which included both two- and six-rowed barley, had the same
sequence, named sequence-type II. The sequence-type II distributed
around the Mediterraneansea region, suggesting that the six-rowed cultivatedbarley of sequence-type II probably
1.Department
8602,

Japan,

3.Faculty

2.1nstituteof Agriculture,

Bioresources,
Instituteof Genetic Resources II, National

and Forestry,

Kagawa University,

Okayama University,

for Bioresources,

Key Words.

of Agriculture

InstituteChuo, Kurashiki

Okayama University,

Six-rowed barley,

Origin,Miki,of Agrobiological
University

Resources,of Tsukuba,

Kagawa 761-0795,
710-0046,

Kannondai, Tsukuba 305-

Tsukuba,

Japan,

Japan.

Present

address:

Chuo 2-20-1 , Kurashiki

7 1 0-0046,

Japan

the vrsl locus, Nucleotide

sequence

305-8572,

4.ResearchInstituteJapan,
forK. Tanno, Researchdifferentiatedfrom the two-rowed barley of sequence-type II in this region.
As table 1 shows, the ssp. spontaneum accessions
were more variable than thecultivatedaccessions,where 9 sequence-types
were found among 10 accessions studied. The
two-rowed cultivatedbarleywas less variable,
including
5 sequence-types
among 8accessions. In contrast, the six-rowed cultivatedbarleys had only 2 sequence-types
among 1 3
accessions.
The reason for the reduction of variation in six-rowed cultivatedbarley is probablythat the origin of the six-rowed barley was a few and that there is a tight linkage between thevrsl and cMWG699. If some favorable genes are able to transfer from two-rowed barley (e.g.
a gene for lateral awn development is located at/near the vrsl locus), it may contribute an
improvement of the six-rowed cultivatedbarley.
Acknowlegements.
The authors would like to thank Dr. H. Takahashi
Mano for DNA isolation of ssp. spontaneum accessions.

for raisingand Dr. Y.

References.
Harlan JR (1995).
Barley. In: Smartt J and Simmonds NW (eds) Evolution
crop plants, 2nd ed. Longman, UK, pp140-147
Komatsuda T, I. Nakamura, F. Takaiwa, S. Oka (1998).
Genome 41 : 680-685
Komatsuda T, W. Li, F. Takaiwa, S. Oka (1999).
Genome 42: 248-253
Tanno K, F. Takaiwa, S. Oka, T. Komatsuda (1999).
Hereditas
130: 77-82.
Table

1 Nucleotide

polymorphisms

at cMWG699 in cultivatedand wild barley

of

175

Constructionof microsatellite-enrichedlibrary

in azuki bean

Xin-Wang Wang, Akito Kaga, Norihiko Tomooka and Duncan Vaughan
NIAR, Tsukuba, JapanThe azuki bean [Vlgna angularis (Wild) Ohwi and Ohashi] is an important cropin East Asia and is closely related to several other Asian cultivatedlegumes such asmung bean (V radiata)
and black gram (V mungo). To date the information at the
genome level for azuki bean is limited and the most recent genome map resolved 14
linkage groups for the 12 chromosomes of azuki bean using RFLP and RAPD markers(Kaga et al, 2000). The objectivesof our research are to seek an efficient method to
identify
microsatellite
markers that may be used to improve our understandingof theazuki bean genome and to provide codominant markers that may be of use inunderstanding
the population dynamics of Vigna species complexes such as wild, weedy
and cultivated forms of azuki bean.
Materials

and Methods
Vigna angularis cv. Erimo shouzu was used for the construction
library.
The main procedures are summarized in Fig. 1. The method
modifiedversion of the method employed for finding microsatellites(Takahashi et al. 1996).

of genomic
used was ain chicken

Results

and discussion
A random genomic library,containing
1/10 of an azuki bean genome
equivalent,
was used for single strand plasmid DNA preparation. After extension ofcomplementary strands using a (CA)i2 primer, approximately
3000 transformed cells
were identified and are referred to as the initial SSR library. By colony hybridization,
600 positive clones were identified as containing
poly(CA)
motifs and constitutethefinal SSR library. The initial SSR library represents
a 20x improvement over direct
selectionfrom the random genomic library.
96 of the 600 positive
clones weresequenced and 90 sequences were found to be non-duplicates,of which 60 sequences
can be used to design PCR primers flanking the microsatellite
motifs (Table 1). This is
the first report of developing a microsatellite
enriched library for azuki bean.
This research continues with the objectivesof (a) improving the efficiency ofenrichment of positive clones from genomic library; (b) evaluating
the extent to which
identified
microsatellites
are polymorphic
among and within Vigna species;(c)applicationof useful markers identified to populationand genome studies.
Refe rences
Kaga A., T. Ishii, K. Tsukimoto, E. Tokoro and O. Kamijima 2000. Comparative
molecular mapping in Ceratotropis species using an interpecific cross between
azuki bean {Vigna angularis)and rice bean (V umbellata).
Theor. Appl. Genet.
100:207-213
Takahashi H., K. Nirasawa and T. Furukawa 1996. An efficient method to chicken
microsatellite
repeats sequences. Jpn. Poult. Sci. 33:292-299.

Fig. 1. The flow diagram of the construction

Table 1. The (CA)nrepeatsin the obtainedof microsatellite-enrichedlibrary

sequences in the genome of azuki bean.

177

Diversityof chloroplast
DNA SSRs in wild and cultivatedsoybeans: anevidence for the multiple origin of cultivatedsoybeans with different organelle
genomes fromwild soybeans
Donghe Xu, Jun Abe and Yoshiya Shimamoto
Laboratory of Plant Genetics and Evolution,
University, Sapporo, 060-8589, Japan
Key words, soybean, chloroplast

Graduate

Schoolof Agriculture,

Hokkaido

DNA, simple sequence repeat, diversity

The soybean (Glycine max) is one of major crops in the world and had been establishedinEast Asia. Its wild relative, G. soja, is distributed
throughout China, Korea, Japan, Taiwan andareas of Russia adjacent to China. A series of studies on the genetic diversity of chloroplastand mitochondrial
genomes of soybeans has revealed a rich variabilityof organelle genomes
in both wild and cultivated soybeans (Tozuka et al. 1997; Abe et al. 1999; Shimamoto et al.
2000).
We have developed
chloroplast
DNA SSR (CPSSR)
markers to gain a better
understanding of the genetic diversityof chloroplast
genomes and the evolutionary process ofdomesticationand genetic differentiation
in soybeans.
The genetic variation of six CPSSR markers was evaluated for a total of 301 wild(144) and cultivatedsoybean (157) accessions that were introduced and/or collectedfromvarious regions of Asia by using sequencer. A total of 23 variants were found in the six
markers (Table 1). All of the variants were found in the wild soybean, while only thirteen
variants was observed in the cultigen. Based on the combination of variants at the six markers,
chloroplast
genomes of the accessions tested were classified
into 52 haplotypes. All of the
haplotypes were found in the wild soybean, and only eight were detected in the cultigen,suggesting a high diversity of chloroplast
genomes in the former. The haplotypes found in thewild soybean were mostly region-specific.
The limited distributionof haplotypes in the wild
soybean may be due to the limited dispersionof seeds because it has no device for longdistance seed dispersal.
Of the eight haplotypes,
haplotype no.49 was predominant in the cultivated soybean
and was commonly observed in any regions of Asia (Table 2). On the other hand, theremaining seven haplotypes
each was distributed
in different
regions. These region-specifichaplotypes
were also found in the wild soybean populations
that were sympatric to the
distributionof the cultivatedsoybeans with respective
haplotypes.
This result strongly
suggests that cultivatedsoybeans with different CPDNA haplotypes had been establishedindifferent regions from wild soybean plants of respective haplotypes
as a result of multiple
domesticationor hybridization
between the two species.
Haplotype no.49 was detected only in seven wild soybean accessions from southern
Japan and southern China. When combined with the classificationof chloroplast
genome with
RFLPs (types I, II and III; Shimamoto et al. 1992), this haplotype
was further divided into
two groups, the one for type I and the other for type II, the former of which was the most
predominant in the cultivatedsoybean. This chloroplast
haplotype was observed only in one
wild accession collectedin southern Japan. The predominant haplotype
in the cultivatedsoybean may therefore have derived from a rare type of the wild soybean. The chloroplast
DNA SSR markers are useful for understanding
the evolutionary process from the wild tocultivated soybeans.
Ackno wledge ments
This research was partly(JSPS)(Grant
No. PI98120

supported by the Japanese Society
and Grant No. 12460142).

for the Promotionof ScienceReferences
Abe, J., A. Hasegawa., H. Fukushi,
T. Mikami, M. Ohara and Y. Shimamoto (1999)
Introgression
between wild and cultivatedsoybeans of Japan revealed by RFLP
analysis for chloroplast
DNAs. Econ. Bot. 53: 285-291
Powell, W., M. Morgante, J. J. Doyle, J. McNicol, S. V. Tingey and A. J. Rafalski (1996)
Gene pool variation in genus Glycine subgenus Soja revealed by polymorphic
nuclearand chloroplast
microsatellites.
Genet. 1 44: 793-803
Shimamotc, Y., A. Hasegawa, J. Abe, M. Ohara and T. Mikami (1992)
Glycinesoja
germplasm in Japan: isozymes and chloroplast
DNA variation. Soybean Genet. Newsl.
19:73-77
Shimamoto, Y., J. Abe, Z. Gao, J. Gai arid F. Thseng (2000)
Characterizing
the cytoplasmic
diversityand phyletic
relationshipof Chinese landraces of soybean, Glycine max,
based on RFLPs of chloroplastand mitochondrialDNA. Genet. Resour. Crop Evol. (inpress)
Tozuka, A., H. Fukushi, T. Hirata, M. Ohara, A. Kanazawa, T. Mikami, J. Abe and Y.
Shimamoto ( 1 997) Composite
and clinal distributionof Glycine soja in Japan revealedby RFLP analysis ofmitochondrialDNA. Theor. Appl. Genet. 96: 170-176
Table 1. Variants found in six CPDNASSR markers of soybean

Table 2. Distributionof cultivatedsoybeans with different

Figures in table show the number of accessions

chloroplast

SSR haplotypes

which were grouped into respectivein Asia

haplotypes

179

A syntenic approach for the functional genes conveying
salinity tolerance in the family Gramineae
Kouki Yoshida1*, Nobuyuki Imaizumi2^, Miho Akiyoshi1^ and Noboru Endo1^
1 ) Biotechnology
Research Center, Taisei corp., Yokohama, 245-005 1 Japan
2) Japan Turfgrass Inc., Tokyo, 103-0004 Japan
Seawater tolerance is the term defined for the plants which are capable of surviving under
the salinity
of seawater, 400-500 mM NaCl, whilst salt tolerance is generally
used more
widely for the salinity
higher than 50 mM NaCl (Akiyoshi
et al 1998). In the family
Gramineae, five sub-families
are known. Among them, it was found that plants tolerant toseawater were rich in the Eragrostoideae,
followed by the Panicoideae(Endo et al 2000).
Based on this observation,
we selected Paspalum in Panicoideae
as a representativeofseawater tolerance, and Oryza sativa (rice) in Pharoideae
as a susceptible
check for theseawater tolerance. One of the rice variety, Pokkali, however survives for its vegetativetomaturity stage at 50 mM NaCl.
In the seawater tolerant Paspalum, we isolated 6 CDNA clones differentiallyexpressed
under salinity of 400 mMNaCl. We also selected 9 of 75 enzymes whose activities
increasedfrom 1.5 to 30 fold in response to the same salt stress. Anatomical observation of the tissue
sections showed that the degree of waxy layer development was more intensive in Paspalum
than other grasses (Endo et al 1999). In conjugatingthese available data, there was a possible
metabolic link between the development of a waxy layer and the enzyme activity (Fig. 1). Of
the 9 enzymes selectedj3 -(1,3;l,4)-glucanase
was rather peculiar in terms of the 20
fold-increase
in its activity(Fig.2).
This gene may supply sugars, followedby theenhancement of the pentose phosphatepathway, being supported by the activationofglucose-6-P dehydrogenase. This pathway produces NADPH and supply H2O2 into the cellwall by the catalysis ofNADPH oxidase. The role of H2O2 in the cell wall seemed to link to
the waxy layer development, hence a closed metabolic pathway was suggested (Fig.l).
Differentiallyselected cDNAs were also incorporated
into this metabolic pathway. It was,then, suggested that UDP-galactose-epimerase
homologue (Cyb) can occupy a suitable
position between cell wall metabolism and pentose phosphate pathway (Fig.3). Second CDNA,
Cyf, is a type I-metallothionein(Fig.4).
Northern analysis successfully
showed that Cyf wasexpressed specific to Paspalum but not in Oryza, Although its function is not fully understood,
metallothioneinof plants may work as a defender against the oxidative stress like reported forthe animal cell. Now we are in the step of final proof via transgenic rice. Meanwhile, acomparative DNA microarray assay is applying for rice to know the metabolic basis available
under salinity. A preliminary result suggested that many kinds of CDNAreduced their activity
much under saline condition, however the reduction rate was less in the tolerant Pokkali thansusceptible
Nipponbare (Fig.5).
It seems that the maintenance of basic metabolism is moresignificant
than a specific genes inducible. Based on this syntenic approach, the genes for salt
tolerance will be discussed systematicallyfor the family Gramineae.
Acknowl edgment
The authors will be thankful to Dr. S. Kikuchi, Dr. N. Kishimoto and Dr. J. Yazaki for theirorganized supervisionand collaborationin the DNA microarray assay granted by Rice
GenomeProject (MA-2 103). The study for seawater tolerance was partly supported by a grantfrom the Petroleum Energy Center subsidized
by the Ministry of International
Trade andIndustry.

References
Akiyoshi,
M.?M. Yaneshita, R. Nagasawa and N. Endo. 1998. Sea water tolerance
Zoysiagrasses
In relation to morphologicaland genetic classification.
Grassland
44:743.
Endo, N., K. Yoshida and M. Akiyoshi.
greening. Green Age 20: 5-8.

2000.

Genetics

for seawater

toleranceof
Science

toward dryland

Fig. 2 Enzyme activlty(above)and northern
analysis (below) of glucanase paralogs

Fig. 1 Possible

metaboliclink for seawater tolerance

Fig. 4 Northern analysis

Fig. 3 Estimatedmetaboliclink for Cyb (CDNA clone )of cyf (CDNA clone)

Fig. 5 DNA microarray pattern of rice
with/without
salt stress

181

MAFF International
1.

Workshop on Genetic

Root andTuber

Resources

SeriesCrops

2. Soil Microorganisms
3.

Animal Genetic Resources:

4. Plant
5.

Genetic

Diversity

6. Genetic

Resources:

EfficientCharacterizationand Use of Agricultural
Diversity

Conservation

and Conservationand Effective

Useand Evaluation

Microorganismsof Animal Genetic Resources

7. Part 1. Wild Legumes*
Part 2. In-situ
8.

Integration*Published

Conservationof Biodiversity

separately

Research*
and Genome Technology

for Crop Improvement

Editors
K. OonoT. Komatsuda
K. Kadowaki
D. Vaughan

Published

November 2000

National

InstituteISBN 4-93151

of Agrobiological1-04-X

Resources,

Tsukuba 305 8602, Japan