International
Workshop
Towards Building a Global Rice Gene Machine
November 11th -12th, 2002
CSIRO Plant Industry,
Plant
Industry Rice Functional Genomics Project Home
PROGRAM & ABSTR
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Inauguration – Jim Peacock |
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Setting the scene – Narayana M. Upadhyaya |
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Session 1 |
Gene knockout systems – T-DNA Chair:
Ko Shimamoto Rapporteur: Chang-deok Han |
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Emmanuel Guiderdoni Abstract A
genome-wide library of insertion lines in rice: distribution of T-DNA inserts
over the rice genome |
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Su-May Yu Abstract T-DNA Insertional
Mutagenesis for Rice Functional Genomics |
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Andrew Eamens Abstract A duel orientation
T-DNA-cum-Ac/Ds gene trap system |
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General
Discussion Report |
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Morning Tea
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Session 2 |
Gene knockout systems – Ac/Ds Chair: Liz Dennis
Rapporteur: Michael Ayliffe |
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Kinya Toriyama Abstract Induction
of independent Ds transposition
using meiosis-associated promoters in transgenic rice |
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Ramachandran Srinivasan Abstract An
Efficient Transposon Tagging in Rice and Large-Scale Analysis of the Ds Flanking Sequences |
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EU consortium on rice
transposon mutagenesis |
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Chang-deok Han
Abstract Transposon Ac/Ds– mediated gene trap systems for rice
functional genomics in |
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Narayana M. Upadhyaya Abstract Can
we produce a substantial “Rice Gene Machine” using the Ac/Ds system? |
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General
Discussion Report |
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Lunch |
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Session
3 |
Retrotransposons,
Naturally occurring alleles & Deletion mutants Chair: Moo
Young Eun
Rapporteur: N.M. Upadhyaya |
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Hirohiko Hirochika Abstract Insertional
mutagenesis in rice using the endogenous retrotransposon |
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Discussion |
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Hei Leung Abstract Deletion
and point mutation stocks for forward and reverse genetics in rice |
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Discussion Report |
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Session
4 |
Gene
Silencing Chair: Eric Huttner Rapporteur: Ming-Bo Wang |
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Peter Waterhouse - RNAi in Plants Abstract |
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Ko Shimamoto Abstract RNAi as a complementing method for functional genomics of
rice |
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Chris Helliwell Abstract High
throughput gene silencing in plants |
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Discussion Report |
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Afternoon Tea |
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Session
5 |
Transgenomics
and TraitMill
Chair: Georges
Freyssinet Rapporteur: Chris Helliwell |
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Andrzej Kilian Abstract TransGenomics: a platform for gene discovery, candidate gene validation and
molecular breeding |
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Discussion |
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TraitMill
- high throughput phenotypic evaluation in rice |
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Discussion Report |
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Session
6 |
Expression
Profiling Chair: Robert
Henry
Rapporteur: Iain Wilson |
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Rudy Dolferus Abstract Molecular
basis of cold-induced male sterility |
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Naoki Kishimoto
Abstract Rice
functional genomics via cDNA microarray |
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Junshi Yazaki Abstract Rice functional genomics
via cDNA microarray: Rice expression database and
the analysis of hormone response genes |
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Nijat Imin Abstract Protein
profiling of male gametophyte development and its response to low temperature
stress in rice |
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Discussion Report |
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Informal
discussions and response to online observers – Industry link room |
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Dinner,
Blue Elephant Indian Restaurant, 29, |
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V.M
Patell Abstract Rice
work at Avesthagen |
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Session 8 |
Bioinformatics
for “Rice Gene Machine” Chair:
Bill
Crosby
Rapporteur: Gavin Kennedy |
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Narayana Upadhyaya &
Leakha Henry - CSIRO Plant
Industry Abstract Towards
developing a web-based "Rice Gene Machine Information Management System
(RGMIMS) |
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Christophe Perin.-
CIRAD Abstract An
overview of Rice Bioinformatics at CIRAD-Biotrop |
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Introduction
to Taiwan Rice Insertional Mutants (TRIM) database |
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Tim
Littlejohn – Biolateral Abstract Bioinformatics
skills for the plant genomicist |
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Bal A. Antonio - RGP Japan Abstract Map-based
rice genomics through INE |
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Andrzej
Kilian- CAMBIA Abstract |
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Bill
Crosby - PBI Open
Source Initiatives for the Integration of Functional Genomics Data |
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Panel discussion
Report |
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Morning Tea |
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Session 9 |
Logistics of “Global Rice
Gene Machine” Panel Discussion Report Chair: V. Sundaresan
Rapporteur: Andrew Eamens
Members: Representatives
from each group and other interested participants |
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Plant Industry facility and
Discovery Centre tour |
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Session 7 |
Case studies of gene identification using FG tools Chair:
Mathew Morell
Rapporteur : Xue-Rong Zhou |
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Qianhao
Zhu Abstract Ds tagged rice mutant defective in anther dehiscence |
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Marcia
Margis Abstract GA
responsive dwarf mutant defective in a early step of GA biosynthesis pathway |
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H. Hirochika |
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CD.
Han |
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Discussion Report |
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Session 10 |
Feed
back and questions from Remote Observer Moderator: N.M. Upadhyaya |
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Session 11 |
Views of outsiders/new comers Report Chair: Bal A. Antonio
Rapporteur: Leakha Henry |
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Joost van Dongen – Max Planck,
Germany Abstract Mutagenesis as a tool for
investigating carbohydrate metabolism in Rice |
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Tayyab Husnain – CEMB, Search of drought tolerant
gene(s) and their over expression in Indica basmati rice |
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Laurie
G. Lewin Abstract The rice gene machine - A
breeder's perspective |
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Peter Langridge The
rice gene machine - A wheat and
barley researcher’s perspective |
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Afternoon Tea |
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Where
to from here? Panel discussion Report Chair: Liz
Dennis/Narayana Upadhyaya
Rapporteur: Qianhao Zhu Members: Representatives
from each group and invited guest members |
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Workshop Conclusion |
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SESSION – 1: GENE KNOCKOUT SYSTEMS – T-DNA
Speaker – 1: Emmanuel Guiderdoni
Title: A genome-wide library of insertion lines in rice: distribution of T-DNA inserts over the rice genome.
Authors: Christophe Sallaud1, Céline Gay2,
Affiliation: 1. Cirad-Amis and 2. Inra-Ensam, Biotrop program, UMR1096, Avenue Agropolis,
F-34398
Montpellier Cedex 5, France
3. Laboratoire Génome
et Développement des Plantes, UMR5096, CNRS/UP, 52, avenue de
Villeneuve, F-66860, Perpignan Cedex
4. Genetrop, Ird, BP5045, F-34032
5. Department of Plant Sciences,
Abstract: In
the framework of the genomics initiative Génoplante,
we have embarked on a project of creation of a genome-wide library of insertion
lines of rice (Oryza sativa L.
japonica cv. Nipponbare). The generation of the
library relies first on a highly efficient Agrobacterium-mediated
transformation procedure for delivery of T-DNA inserts (Sallaud
et al. Theor
Appl Genet, in press). This method allowed the
production of 30,000 primary transformants (T0) with an average
efficiency of 5 independent transformation events per co-cultured callus in an
18 month-time span. The equipment of the pC-4978 and pC-4956:ET15 T-DNAs with a gusA and a gal4:UAS:gfp enhancer trap respectively allows gene
detection through visualisation of GUS activity and
fluorescence in specific cell types and organs. Histochemical
assays conducted on 2,280 T0 plants allowed detection of
GUS-specific activity in leaves, roots and floral organs with respective
frequencies of 26.9%, 6.9% and 3%.
Observations of mature leaf and floral organs of 2,082
pC-4956:ET15 primary transformants allowed fluorescence detection in
more than 15 % of the plants. The T0 plants allowed to set seeds in
the greenhouse have been first selected at the in vitro stage for amplification of a unique product of the genomic
region flanking the T-DNA left border. 15,316 plants have been so far selected
with an overall frequency of 60%, which is remarkably stable over
transformation experiments. To date, 9,700 (90%) of the 10,862 PCR2 products
sequenced with a T-DNA specific primer produced a readable sequence. BlastN search allowed identification of the T-DNA footprint
in most (94.5%) sequences. Survey of the 5,603 (57.8%) genomic sequences larger
than 30bp (average length 250 bp)
against
the rice B
Acknowledgements:
This work is funded by the genomics initiative Génoplante (France) under project "Creation of a
genome wide library of rice insertion lines" and by a bbsrc
(
Speaker – 2: Su-May Yu
Title: T-DNA insertional mutagenesis for rice functional genomics.
Authors: Su-May Yu
Affiliation: Institute of Molecular Biology, Academia Sinica, No. 128, Sect. 2, Yen-Ju-Yen Rd, Nankang, Taipei, 115, Taiwan
Abstract: T-DNA insertional mutagenesis approach has been employed to generate gene knockout rice mutant population for functional genomics. The T-DNA carries a promoter-less reporter gene next to the right border. Insertion of T-DNA downstream of functional genes would lead to identification of promoters that are regulated by physiological, developmental or environmental signals in distinct tissues of transgenic rice plants. The T-DNA tagged rice lines will be used for phenotypic screening under various selective conditions. The rice genomic DNAs flanking T-DNA are also isolated and sequenced. A database for the rice/T-DNA junction DNA is currently being established.
Title: A duel orientation T-DNA-cum-Ac/Ds gene trap system.
Authors: Andrew Eamens1,2,3, Chris Blanchard2,3, Kerrie Ramm1,4
Affiliation: 1. CSIRO Plant Industry, GPO
2.
3. Cooperative Research Centre
for Sustainable Rice Production, C/- Yanco
Agricultural
Institute, Private Mail Bag, Yanco, NSW 2703
4.
NSW Agricultural Genomics Centre, Wagga Wagga, NSW,
Abstract: T-DNA and Ac/Ds have proven to be efficient genomic tools for the production of insertional mutants in rice and other species. Previous studies have raised a number of concerns with the use of either system for gene or enhancer tagging in plants, including low levels of tagging efficiency, localised transposition, complex integration patterns and the incorporation of vector back-bone (VB) sequences. We have developed a T-DNA gene trap-cum-Ds launching pad construct. This construct offers a number of new features that allow it to initially be used as a dual orientation T-DNA gene trap housing two reporter genes (gus and gfp) to increase trapping efficiency. T-DNAs found not be inserted in a coding region of the genome can be used as a subsequent launching pad for Ds transposon-based localised dual orientation gene tagging. A killer-gene (barnase) has also been included in the construct’s VB to remove interfering VB sequences and to reduce the occurrence of complex integration patterns within screening populations. We have evaluated the tagging efficiency of the T-DNA/Ds construct in rice and Arabidopsis and shown an approximately four-fold increase in screening efficiency in both these species.
SESSION – 2: GENE KNOCKOUT SYSTEMS – iAc/Ds
Speaker – 1: Kinya Toriyama
Title: Induction of independent Ds transposition using meiosis-associated promoters in transgenic rice.
Authors: Kinya Toriyama and Ryouhei Morita.
Affiliation: Graduate
Abstract: The maize Ac/Ds
transposon has been expected to be useful for the transposon tagging in rice.
In most case, the transposition of a Ds
element is achieved by crossing plants harbouring the Ac transposase (AcTP), which is usually driven by CaMV
35S promoter. However, employment of 35S promoter sometimes results in high
frequency of somatic excisions, creating undesirable mutations by footprints,
but low frequency of germinal independent transposition of Ds. Individual progeny derived from the same inflorescence has
often been found to carry transposed Ds
elements derived from the same transposition event, thus reducing the
transposon tagging efficiencies. In order to suppress the somatic excision and
increase the germinal independent transposition events in Ac/Ds transposon tagging
system in rice, we are planning to use a meiosis-specific promoter to control
the expression of AcTP.
One of a meiosis-specific promoter was taken from a Lily gene, Lim10 (Lily messages induced at
meiosis). Lim10 has been reported to
encode a protein similar to a small heat shock protein and be expressed in meiocyte starting from zygotic stage and in tetrads. We
first examined the activation pattern of Lim10
promoter in rice by introducing a fusion between Lim10 promoter and GUS gene. Histochemical GUS assay showed the GUS
staining in anthers but not in other floral organs or leaves. GUS expression
was observed in pollen mother cells, tetrads and young
microspores. A fusion between Lim10
promoter and cDNA for AcTP
was constructed and introduced into rice. Then a plant carrying the Lim10::AcTP was crossed with a plant carrying a Ds transposon, Ds-GUS
T-DNA. Excision of a Ds element was
detected by using PCR. The somatic excision was not detected in leaves of F1
plants as expected, although it was detected in F1 plant obtained by
crossing a plant with Ds-GUS T-DNA
and a plant with AcTP
regulated by CaMV35S promoter. F2 plants with germinal excision were
selected based on herbicide resistance, as Ds-GUS
T-DNA has been designed to confer chlorsulfuron resistance
once a Ds element was excised.
Thirty-three plants were identified to be chlorsulfuron
resistance
out of 348 F2 plants tested. Transposed Ds was detected by southern blot analysis. A band at different position indicates that the transposed Ds was derived from independent transposition event. In total 14 plants were identified to contain a unique band of transposed Ds. The frequency of independent transposition was as high as 4%. Lim10 promoter is shown to be powerful to induce independent germinal transposition in rice. We are now making a cross between plants with Ds-GUS T-DNA (35 lines) and plants with Lim10::AcTP (6 lines) and growing F1 plants between them. We are expecting to obtain many numbers (infinite numbers in theory) of tagging lines in rice. Mapping of Ds-GUS T-DNA in rice chromosomes is also in progress.
Speaker – 2: Ramachandran Srinivasan
Title: An efficient transposon tagging system in rice and large-scale analysis of the Ds flanking sequences.
Authors: Tatiana
Kolesnik1,2, Ildiko Szeverenyi1,2,
Doris Bachmann1,
Affiliation: 1. Rice Functional
Genomics Group, Temasek Life Sciences Laboratory, 1
Research Link,
NUS,
2. Authors contributed equally
3. Department of Plant
Biology, Life Sciences Addition 1002,
*Author for correspondence
Abstract: We have introduced the modified version of a
two-element Ac/Ds gene trap system developed by Sundaresan et al (1995) into rice
(O. sativa L., sap. japonica, cv. Nipponbare) plants to generate a collection of stable,
unlinked and single-copy Ds transposants. Thirteen different cross combinations of
parental lines were used to generate 4262 F2 families with at least
one putative transposant per family. The average
germinal transposition frequency of Ac/Ds
in rice was estimated as 49% with a range from 16% to 74% depending on cross
combination. Analysis of Ds flanking
sequences of 2057 lines showed that 88% of the insertions were unique rice
sequences and distributed randomly throughout rice genome. The remaining 12% of
insertions were within T-DNA. Although the insertions were randomly
distributed, chromosome 1 had two-fold more and chromosome 9, 11 and 12 had a
few folds less insertions than expected. A putative ‘hot spot’ has been
identified on chromosome 7, where twenty seven Ds flanking sequences (approximately 2% of unique sequences) were
located on a 40 kb region. Over 22% of unique sequences were homologous to
either protein or rice expressed sequence tags (ESTs) suggesting preferential
transposition of Ds into coding
regions. Also 841 genetically mapped sequences were placed on Y
Speaker – 3:
Title: EU consortium on rice transposon mutagenesis.
Authors: A. Pereira1, R. Greco1, L.J.G. van Enckevort1, P.B.F. Ouwerkerk2, C. Sallaud3, A. Kohli4, F. Fornara5, L. Colombo5, E. Pè5, P. Puigdomènech6, E. Guiderdoni3, P. Christou4,7, A.H. Meijer2, J.H.C. Hoge2.
Affiliation: 1. Plant Research International,
2.
3. CIRAD-AMIS, BP 5035, 34032
4. John Innes Centre,
5.
6. CID-CSIC, Jordi Girona 18, 08034 Barcelona, Spain
7. Fraunhofer IME, Auf dem Aberg 1, D-57392 Schmallenberg, Germany
Abstract: An EU consortium on
rice functional genomics was funded in FP5 to develop transposon mutagenesis
systems in rice to address gene functions using forward and reverse genetics
strategies.
To saturate
the genome with inserts in a fewer number of plants, we sought to generate rice
genotypes with multiple transposons. In a construct with the maize Ac element containing a double CaMV 35S enhancer adjacent to the autonomous Ac promoter, all transformants generated
showed very early transposition, and about half displayed Ac amplification (Greco et al., 2001). This allowed the generation
of lines containing multiple transposons (ca. four from a single copy T-DNA
line) that could generate an average of one to two new inserts per progeny
giving a frequency of 15 to 50% of independent transpositions in the next
generation. We observed transposition of Ac
to linked positions, applicable for targeted tagging. The isolation of Ac flanking genomic sequences revealed a
preferential insertion in protein-coding sequences, suggesting that this could
be a valuable asset for generating mutants in rice. Autonomous Ac genotypes are being produced in both
indica and japonica rice. Using 25,000 lines for three to four generations
would generate about 100,000 insertions that are suitable for identifying
knockouts for forward as well as reverse genetic strategies.
However,
the scope of knockout mutations is limited, as the majority of genes display no
obvious phenotype, probably due to functional redundancy. We therefore focused
on developing gene detection strategies using Ac-Ds transposons to
address the function of genes that do not directly reveal a knockout phenotype.
In one strategy we used Enhancer trap constructs that contain a mobile Ds element containing a GUS reporter
gene with a minimal promoter, whose expression depends on transcriptional
regulatory sequences of the adjacent host gene. Another strategy was using
Activation tagging using a Ds element
bearing a strong CaMV 35S enhancer, to generate
gain-of-function phenotypes.
Although
the frequency of Ds transposition in
early generations appears to be high, inhibition of transposition was reported
in later generations (Izawa et al., 1997). Though this might happen in some
lines, active lines have been identified which still show good transposition
activity in the T2/T3 generations, suggesting that Ds inactivation may not be a general
phenomenon. In 2002, about 10,000 Ac-Ds plants are being used to generate Ds flanking DNA to provide a database of
tagged genes.
Speaker – 4: Chang-Deok Han
Title: Transposon, Ac/Ds - mediated gene trap systems for rice
functional genomics in
Authors: Chul Min Kim1, Byoung Il Je1, Hai Long Piao1, Soon Ju Park1, Min Jung Kim1, Sung Han Park1, Jin Young Park1, Su Hyun Park1, Eun Kyeong Lee2, Nam Soo Chon1, Yong Jae Won3, Gi Hwan Lee3, Min Hee Nam3, Doh Won Yun4, Myung Chul Lee4 , Moo Young Eun4, and Chang-deok Han1,2.
Affiliation: 1. Division of Applied Life Science (BK21 program)
2.
3. Division of Rice Breeding and Cultivation Naitonal
Yeongnam Agricultural Experiment
Station (NYAES) Milyang
627-803,
4. Genomics Division National Institute of Agricultural
Biotechnology (NIAB)
441-707,
Abstract: Maize Ac and gene trap Ds has been introduced into rice genome by Agrobacterium-mediated transformation. CaMV 35S promoter was used to express Ac cDNA as a transposase source. For gene trap Ds, a partial intron with three alternative splicing acceptors was fused to GUS. A genetic marker for Ds (BAR) was inserted into trap Ds. To develop a large scale of Ds transposant lines in rice, three strategies have been employed and evaluated. In the first of all, a large scale of genetic crosses have been made between homozygous Ds or Ac starter lines that carry either a single copy of gene trap Ds or a single copy of Ac. The data suggest that the mobility of Ds is highly variable among starter lines. A couple of Ds lines generated high frequency of germinal revertants up to 25%, in the F2 segregation generation. However, the same genetic crosses that were repeated later, the frequency of germinal reversion in the same crosses noticeably decreases down to 10%. This indicates that the activities of Ac or Ds were subjected to modification in rice genomes. In contrast, when plants were re-generated, via tissue culture, from seeds carrying Ac and Ds, over 60% Ds elements were translocated from original sites. The last strategy is to select rice lines carrying high copy of Ds. The possibility is being examined whether a multi-copy Ds can be maintained and normally transmitted into subsequent generations. To maximize the efficiency of characterizing Ds tagged genes, optimal condition of iPCR (inverse PCR) and TAIL-PCR (Thermal Asymmetric Interlaced PCR) have been established to clone Ds flanking DNA in rice genomes. Over 80% Ds adjacent genomic DNA has been successfully amplified by both PCR methods. Over 3,000 Ds insertions have been cloned via iPCR (inverse PCR) and are being characterized. Among these genes, around 500 insertion sites have been analysed in detail. Near 50% showed significant homology with known genes or functional domains. As expected, near half of these tagged genes can express GUS fusion proteins. A national consortium has been organized to develop internationally competitive scale of population mutagenised by Ds, and to construct databases of molecular information on Ds insertion sites. The project includes cloning of 4,500 Ds insertion sites per year and selection of 10,000 Ds lines per year.
Speaker – 5: Narayana Upadhyaya
Title: Can
we produce a substantial "Rice Gene Machine" using the Ac/Ds system?
Affiliation: 1. CSIRO Plant Industry, GPO Box 1600,
Canberra ACT 2601, Australia
2. NSW
Agricultural Genomics Centre, Wagga Wagga, NSW,
Australia
3. Centre for Biotechnology, SPIC
Science Foundation, 111 Mount Road, Guindy, Chennai
600 032, India
SESSION – 3: RETROTRANSPOSONS,
NATURALLY OCCURRING
Speaker – 1: Hirohiko Hirochika
Title: Insertional mutagenesis in rice using the endogenous retrotransposon.
Authors: Hirohiko Hirochika
Affiliation: National Institute of Agrobiological Sciences (NIAS),
Abstract: Insertional mutagenesis is a powerful method for a systematic functional analysis of a large number of genes. Five active retrotransposons have been found in rice and the most active Tos17 was characterized in detail. Tos17 is silent under normal conditions, becoming active under tissue culture conditions. Five to thirty transposed Tos17 copies were found in each plant regenerated from culture. Tos17 becomes inactive in regenerated plants and mutations induced by Tos17 insertion are inherited stably in subsequent generations. Tos17 was shown to transpose preferentially into low copy number, gene-rich regions, indicating that Tos17 can be used as a tool for efficient mutagenesis. A collection of 50 000 regenerated rice lines carrying about 500 000 insertions was generated, and the lines are being used for forward and reverse genetic analyses. We have demonstrated that transposon-tagging is feasible, although the tagging efficiency is relatively low, 5-10%. By using this strategy, causative genes for viviparous, salt-hypersensitive, dwarf, brittle culm, narrow leaf mutations, etc., have been cloned. Several lines of evidence indicated that non-tagged mutations involve deletions and point mutations induced by unknown mechanisms. These mutations may also be utilized by using TILLING and PCR-based screening of deletions. For reverse genetic studies, two strategies are employed. One is PCR-screening of mutants of the gene of interest. DNA pools derived from 40,000 lines have been produced for screening. We screened 31,000 lines and found mutants of 17 genes, including MAPK, MADS-box, and P450 genes, among the 53 genes analyzed. Mutants of five of ten MAPK genes have been identified, but only one mutant of one gene showed clear phenotype. Lack of phenotype can be largely due to gene redundancy. One possible solution is combination of mutations by crossing, as has been successfully demonstrated in Arabidopsis. Existence of frequent allelic mutations indicates hot spots for transposition target sites, which enables us to find and confirm the causality of genes and phenotype, while this may be a limiting factor for saturation mutagenesis. Currently, PCR screening is the most efficient approach for reverse genetics. However, cataloguing of insertion mutants by sequencing the genomic DNA sequence flanking insertions becomes important, considering the need for a systematic approach to find mutants for a large number of genes in the post-sequencing era and the finite nature of DNA pools for PCR screening. To carry out large scale sequencing of the Tos17-flanking sequences, TAIL- and suppression-PCR were adopted. By combining these two PCR methods, about 95% of flanking sequences can be amplified. As of June 2002, 14,300 independent flanking sequences from 3,700 lines have been determined and mutants of different classes of genes have been identified.
Title: Deletion and point mutation stocks for forward and reverse genetics in rice.
Authors: Hei Leung1, Jianli Wu1,
Cailin Lei1, Mayette
Baraoidan1, Alice Bordeos1, Suzette Madamba1,
Bradley Till2, Steve Reynolds3, Luca Comai3,
Steven Henikoff2, Hursong Chang4,
Tong Zhu4, Xun Wang4, Stephen Goff4, Lirong Zeng5, Guo-Liang
Wang5, Changjian Wu1,6, and
Affiliation: 1. International Rice Research Institute,
DAPO
2.
3.
4. Torrey Mesa Research Institute,
San Diego, California, USA
5.
6.
Abstract: IR64 is the most widely grown indica
rice variety in
Morphological variation at vegetative and reproductive stages (between 6-8%), including plant architecture, growth habit, pigmentation, and various physiological characters, are commonly observed in the four mutagenized populations. Subsets of M4 mutant lines are being screened for altered response to multiple stresses (bacterial, fungal and viral diseases, insects, drought, submergence, salinity), phytic acid content, and changes in tillering and root development. To improve the utility of the deletion mutants, we experimented with Syngenta’s Rice GeneChipR (containing oligos for ~21,000 genes) to analyse mutants with known deletions. Results indicated that differential DNA-DNA hybridization signals are sufficient to reveal deleted genes. Provided that allelic mutations with known phenotypes are available, this approach can offer an efficient means for assigning gene function.
For reverse genetics, we apply TILLING (Targeting Induced Local Lesions IN Genome) to detect point mutations in targeted genetic loci. TILLING makes use of enzymatic detection of heteroduplexes formed between wild type and mutant DNA strands and has been successfully used to detect point mutations in Arabidopsis (Colbert et al. 2001 Plant Physiol.126:480). DNA was isolated individually from ~2,000 EMS-induced M2 plants, pooled (8 genotypes per pool), and arrayed in 96-well plates. Primers were designed using CODDLE (http://www.proweb.org/input/) to bracket a 1-kb region most likely containing a deleterious mutation in a target gene. So far, two independent mutations were detected in a gene pp2A4 encoding serine/threonine protein phosphatase catalytic subunit. Sequencing of the mutant loci confirmed two single-base transitions in an intron and an exon of the gene. With available complete rice genome sequence and high-throughput genotyping techniques, efficient strategies for forward and reverse genetics can be applied to the chemical and irradiation induced mutants.
SESSION – 4: GENE SILENCING
Authors: Ming-Bo Wang, Neil Smith, Varsha Wesley, Chris Helliwell, David Abbott, Jean Finnegan, Allan Green, Qing Liu, Surinder Singh, Peter Stoutjesdijk, and Peter Waterhouse.
Affiliation: CSIRO Plant Industry, GPO
Abstract: Many examples of
post-transcriptional gene silencing (PTGS) of endogenous genes, reporter
transgenes or invading viral genes have now been described in plants
transformed with complimentary DNA sequences oriented in either the sense or antisense direction.
However, in most cases, this sense (cosuppression)
or antisense mediated gene silencing occurs only at
low frequency. In relation to PTGS of
invading viral genes, we initially showed in tobacco that transgenic expression
of the Potato Virus Y (PVY) nuclear inclusion protease gene (Pro) in either the sense or antisense orientation conferred immunity to PVY in less
than 10% of transgenic plants.
Subsequent experiments with constructs that contained multiple copies of
the Pro gene in various combinations
of sense and antisense orientation, demonstrated that
increasing the number of either sense or antisense
copies of the gene did not increase the frequency of immunity, however co-expression
of both sense and antisense constructs resulted in a
dramatic increase, with over 50% of such plants being immune to PVY. Experiments in which the sense and antisense Pro
sequences were placed contiguously in a variety of direct or inverted repeat configurations
demonstrated that the immunity to PVY was conferred wherever complimentary
sense and antisense RNA transcripts were encoded,
regardless of the particular structure of the transgenic DNA. Furthermore, when independently introduced
sense and antisense Pro transgenes that individually did not confer PVY immunity were
brought together by crossing, all progeny plants carrying both transgenes were
fully immune.
These results
suggested that the formation of double-stranded RNA (dsRNA)
duplexes may be the critical factor in triggering PTGS in plants, a feature
which would have striking similarities with RNA interference (RNAi) described in nematodes and Drosophila, and quelling observed in fungi. Such a phenomenon could represent a
ubiquitous system in plants for natural defence
against dsRNA associated with viral infection. The molecular mechanism currently postulated
by a number of research groups involves the non-specific recognition of dsRNA by surveillance nucleases which subsequently degrade
the RNA in a manner that initiates a sequence-specific degradation of
complimentary RNA. This hypothesis is
supported by the regular observation of 23nt RNA degradation fragments in
association with the PTGS phenomenon in several different plant/transgene
systems. Once initiated, the
sequence-specific dsRNA degradation can occur
throughout the plant and is graft-transmissible, indicating that a critical
component of the mechanism is mobile in the plant.
Based on these
mechanistic insights, we have now developed a PTGS system that can specifically
silence genes with much greater efficiency than either antisense
or cosuppression.
We have utilised inverted-repeat DNA
constructs that have inbuilt self-complimentarity and
are hence capable of regularly producing dsRNA
through the formation of transcripts having hairpin structures (hpRNA). Such constructs regularly give high
frequencies of PTGS that is specific for sequences having complimentarity
to the sense and antisense arms of the construct,
which may be relatively short regions (~ 120nt) of either the translated or untranslated regions of the target gene. The particular sequence of any intervening
DNA between the arms does not affect the specificity of silencing and is
required only to facilitate the manufacture of the inverted-repeat constructs
in bacteria. In fact, the use of a splicable intron as the intervening sequence further
enhances the frequency of PTGS to near 100% efficiency, without altering its
sequence specificity. We have now
designed and evaluated in Arabidopsis
several hpRNA constructs targeted against either viral genes (e.g. PVY),
reporter transgenes (e.g. GUS), and endogenous genes (e.g. fatty acid desaturases, pigment biosynthesis). Additionally, we have already utilised the technique in cotton (Gossypium hirsutum) to achieve high level
seed-specific silencing of both stearoyl-
Besides being a
fascinating attribute of cells, that was completely unsuspected 15 years ago, dsRNA-mediated PTGS promises to be a powerful tool for
plant biotechnology and gene discovery.
The value of the method is considerably enhanced by the high frequency
and specificity of silencing that can be achieved through the use constructs
encoding intron-spliced hpRNA.
Speaker – 2: Ko Shimomoto
Title: RNAi as a complementing method for functional genomics of rice.
Authors: Ko Shimamoto, Satoru Moritoh, and Daisuke Miki.
Affiliation: Nara Institute of science and technology, 8916-5, Takayama,
Ikoma,
Abstract: To suppress functions of genes whose DNA sequences are available RNAi is an effective method in various organisms including rice. When we want to suppress functions of genes of interest we often find they are members of a gene family. This is particularly true in rice compared with Arabidopsis. To individually suppress functions of each member of a gene family one may have to use regions of genes which are not well conserved to transcribe trigger dsRNA. However, it is known in plants and C. elegans that siRNA is produced from the region outside of the genes dsRNA is originally targeted by RNA dependent RNA polymerase resulting in spreading of RNAi to non-conserved regions. If this “spreading of RNAi” occurs it would be difficult to specifically suppress function of a member of a gene family by RNAi.
We addressed this question by applying RNAi method to the OsRac gene family. The OsRac genes encode small GTPases involved in various cellular functions including defence signalling. We have so far identified 10 rice genes in this family consisting of 7 groups. Most of their coding regions are highly conserved, however their 3’ UTRs are less conserved. Therefore, we designed RNAi constructs from which dsRNA of ca. 300 bp UTR regions are produced from 7 different genes and transformed them into rice. Results of the analysis of transgenic rice plants indicated that each of 7 group members of OsRac gene family was specifically suppressed by the respective RNAi construct with high frequency. Furthermore, analysis of RNA showed that siRNA was only detected by 3’ UTR regions and not by the highly conserved coding region.
These results suggested that RNAi by dsRNA for 3’UTR is a useful method to identify gene functions in rice for those genes for which tagged lines are not available.
Title: High throughput gene silencing in plants.
Authors: Chris Helliwell, Varsha Wesley, Anna Wielopolska, Rong-Mei Wu, David Bagnall and Peter Waterhouse.
Affiliation: CSIRO Plant Industry, GPO
Abstracts: A major challenge in the post-genome era of plant biology is to determine the functions of all the genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. The efficacy of gene silencing in plants using inverted repeat transgene constructs that encode a hairpin RNA (hpRNA) has been widely demonstrated and offers a direct method of determining gene function. To allow gene silencing to be used in functional genomics applications in a straightforward manner we have developed a series of vectors using the Gateway recombination system to facilitate the easy and rapid production of hp RNA constructs. The latest advances in design of these vectors will be described. The sequence of the Arabidopsis genome has shown that large portions of the genome are duplicated and therefore likely to be functionally redundant. This means that conventional single gene knockout approaches are unlikely to reveal the function of these genes. As gene silencing is sequence dependent it can potentially be used to silence multiple members of a gene family. We will present the results of experiments to define rules for construct design to silence multiple members of a gene family.
SESSION – 5:
Authors: Andrzej Kilian
Affiliation: CAMBIA, GPO
Abstract: TransGenomics builds on the
recent progress in the understanding of the important role gene regulation
plays in plant evolution and adaptation. TransGenomics
attempts to create changes in gene regulatory networks using synthetic transactivators. The controlled manipulation of expression
of practically any gene in rice, offers an opportunity to develop and test
specific hypotheses of linkages between gene activity and the resulting
phenotype. It is expected that rice
lines with improved characteristics could be produced as a result of the TransGenomics project. These lines could be isolated
through appropriate screens of TransGenomics
materials and then used as improved germplasm in breeding programs.
TransGenomics is a three-step process.
First, we capture a large number of rice genomic regions using an Enhancer Trap
system, which employs a transcriptional activator to generate TransActivator Facilitated Enhancer Trap (TAFET) lines. Second, we insert into the rice genome, a number of
copies of an Upstream Activating Sequence (UAS) that are
the targets for binding by the Transactivator.
Transgenic lines containing UAS element(s) are called random TARGET lines. Third, crosses between the TAFET lines and the
random TARGET lines are performed. The transactivator
is then able to create changes in the expression patterns of the chromatin
regions containing UAS insertions. Such transactivation
of a UAS-tagged gene-containing region is expected to result in “Gain of
Function” mutations. Using this strategy we will be able to scan the rice
genome for novel genes which may be missed by other approaches, especially
those focusing on “Loss of Function” mutations.
TAFET lines can be
also used for candidate gene validation. In this case, the UAS promoter is
fused with a candidate gene “in vitro”
and then introduced into rice genome via
transformation, creating a specific
TARGET line. Genetic crosses between selected TAFET line(s) and a specific
TARGET line allows rapid testing of candidate gene with various expression
patterns imposed by the transactivator.
The current stage
of the rice TransGenomics project, including
informatics component, will be reported, with emphasis on the development and
analysis of a comprehensive population of TAFET and UAS lines.
Speaker – 2:
Title: TraitMill – high throughput phenotypic evaluation in rice.
Authors:
Affiliation: CropDesign General, Technologiepark 3, B-9052
Abstract: CropDesign,
based in
One of CropDesign's main technology platforms is TraitMill. TraitMill closes the application gap between classical genomics and the development of improved or novel crop traits. The TraitMill is designed to determine the effects on plants of changes from only single genes or from specific gene combinations. The TraitMill is a set of high throughput and high resolution technologies and processes for identifying, cloning and inserting genes in plants. It can rapidly validate the effect of a gene at various expression levels and in various tissues in a cereal crop. Plant phenotypes are evaluated using automated plant and seed handling systems, and with digital imaging and analysis. Trait relevant data in rice is available within 15 months. CropDesign also applies extensive gene sequence databases and gene function information to crop trait development. CropDesign's bioinformatics platform "GeneCircle" is an integrated system for the storage, visualisation and editing of annotated gene sequences - facilitating data access, automated annotation and advanced data queries.
SESSION – 6: EXPRESSION PROFILING
Title: Molecular basis of cold-induced pollen sterility in rice.
Authors: Rudy Dolferus1,3, Xiaochun Zhao2,3,
Sandra Oliver1,4, Jane Edlington1,
Affiliation: 1. CSIRO
Plant Industry, GPO
2.
3. CRC for Sustainable Rice
Production, C/- Yanco Agricultural Institute, Private
Mail Bag,
Yanco, NSW
2703, Australia
4.
Abstract: Cold-induced pollen sterility is a problem that affects rice yields in most temperate rice growing areas of the world. Breeding efforts for cold-tolerant varieties have been relatively unsuccessful, due to the lack of suitable germplasm, reliable screening methods, and knowledge about the molecular basis of the problem. We are using a sensitive microarray approach to study cold-induced changes in anther gene expression in order to identify genes that are critically affected by cold. This gene expression profiling approach may lead to the identification of the biochemical and physiological changes that cause pollen sterility. In addition, by comparing the cold response of cold-tolerant and cold-sensitive rice varieties we will be able to identify genes that could be used as molecular markers in breeding programs for cold tolerance in rice.
Speaker – 2: Naoki Kishimoto
Title: Rice functional genomics via cDNA microarray: Expression profiles of stress-responsible genes and Rice full-length cDNA clones for global expression analysis
Authors: Naoki Kishimoto1, Junshi Yazaki1, Masahiro Ishikawa1, Kohji Sato1, Toshifumi Nagata1, Nobuyuki Kawagashira1, Kouji Doi1, Keiichi Kojima2, Takahiro Namiki2, Kanako Shimbo2, Fumiko Fujii1, Tomoya Ohata1, Zenpei Shimatani3, Akiko Hashimoto3, Yuko Nagata3, Sachiko Honda3, Kazuko Toyoshima1, Katsumi Sakata1, Kimiko Yamamoto3, Takuji Sasaki1, Yasuhiro Otomo4, Kazuo Murakami4, Kennichi Matsubara4, Jun Kawai5, Piero Carninci5, Yoshihide Hayashizaki5 and Shoshi Kikuchi1.
Affiliation: 1. NIAS; 2. Hitach
Soft Engineering; 3. STAFF; 4. FAIS; 5. RIKEN,
Abstract: The rice full-length cDNA project has been initiated since 2001. Up to now 28K independent groups are collected from randomly picked-up full-length cDNA clones (170K) , which are from the libraries of seedlings, calli, and both were treated with several environmental stresses, panicles of rice. About 16K clones are completely sequenced and functional annotation of the clones are made with the homology search (BLAST N and X) against NCBI GenBank database. About 7,000 clones have hit to the clones or sequences registered in DB (E<10-100). At this moment about one fourth of the total clones hit the sequences from Arabidopsis genome (BLAST X). About 4,000 clones, which have hit to the gene products (mRNA, Protein) are divided into the functional categories according to the KEGG site (http://www.genome.ad.jp/kegg/). Summarizing the expression data from the Microarray analyses, the full-length sequence data of cDNA clones, gene annotation data from the homology search (especially from the mapping data on the chromosomes) and the genomic sequence data from the Rice Genome Sequence Project, it is possible to assign the promoter sequences of the genes which show common profile in stress responsible expression.
Also, we have embarked on a large-scale functional genomics by Microarray system (using RGP/EST clones as probe; about 11,000 non-redundant set) in order to obtain a global expression profile of rice genes, supported by the Rice Microarray Project by MAFF. In the research project, the system was used to hybridize target RNAs prepared from normally grown rice tissues (as control) and some stress treated tissues, such as some low temperature conditions, a high salt condition, some nutrient deficient conditions, oxidative stress treatment such as UV-B and Gamma irradiation and the treatment with Hydrogen peroxide, the additions of excess hormones and the treatment with hormone biosynthesis inhibitors and so on (http://microarray.rice. dna.affrc.go.jp/).
We analysed gene expression responses of rice plant after UV-B and Gamma rays irradiation. Dosage-dependent expression and time course after irradiation were monitored. In both cases, there are at least two gene sets, which are early (within 8hrs) induced genes and late (about 24hrs) induced gene set. Both irradiations should give DNA damage and oxidative stress to the plant, however, the genes which change their expression are different each other. Similarity of the gene expression profiles of these two stimuli to expression profiles from other stresses will be discussed.
Speaker – 3: Junshi Yazaki
Title: Rice functional genomics via cDNA microarray: The expression profiles of plant hormone responsible genes.
Authors: Junshi Yazaki1, Naoki Kishimoto1, Masahiro Ishikawa1, Keiichi Kojima2, Kanako Shimbo3, Fumiko Fujii1, Tomoya Ohta1, Zenpei Shimatani3, Akiko Hashimoto3, Yuko Nagata3, Katsumi Sakata1, Kimiko Yamamoto3, Takuji Sasaki1, and Shoshi Kikuchi1.
Affiliation: 1. NIAS; 2. Hitach
Soft; 3. STAFF,
Abstract: We have embarked on a large-scale functional genomics using the microarray system in order to obtain a global expression profile of rice genes. More than 11,000 partial cDNA sequences corresponding to unique genes have been identified at the Rice Genome Research Program (RGP). From these sequences, microarrays were constructed using 8987 cDNA clones. These were used as probes to hybridize target RNAs prepared from normally grown callus and plant hormonal treated callus. Normalized data among these several experiments are summarized in our database (RED; Rice Expression Database) and the expression profiles of the genes are compared with over 600 expression profiles. We hope to introduce results of interesting clusters of plant hormonal responsible genes and our in silico functional genomics tool in this workshop.
Title: Protein profiling of male gametophyte development and its response to low temperature stress in rice.
Authors: Imin N., Kerim T., Weinman J.J. and Rolfe B.G.
Affiliation:
Abstract: We used proteomic analysis to investigate the changing patterns of protein synthesis during pollen development in rice anthers and their responses to low temperature treatment that cause male sterility in rice. An anther proteome database of the proteins separated by two-dimensional gel electrophoresis (2-DE) was established and publicly accessible at http://semele.anu.edu.au/2d/2d.html. Over 3,000 protein spots were detected over the pH range of 4-11. Of these, over 350 protein spots were analysed by MALDI-TOF MS (matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry), tandem MS and N-terminal sequencing and putative identities were assigned to more than 100 protein spots. Then, we compared the anther proteome maps of different developmental stages and we detected approximately 150 protein spots changed consistently during development. Of these, 44 proteins were identified including proteins are closely associated with sugar metabolism, cell elongation and cell expansion. Furthermore, we detected 36 protein spots as differentially after one, two and/or four days of cold temperature treatment in the cold sensitive cultivar Doongara. Among them, 32 protein spots were up-regulated and four protein spots were down-regulated. Of these, 12 proteins were identified including proteins potentially associated with exine formation.
SESSION – 7: CASE STUDIES OF GENE IDENTIFICATION
USING FUNCTIONAL GENOMIC TOOLS
Title: A Ds tagged mutant defective in anther dehiscence.
Authors: Qian-Hao Zhu1,2, Kerrie Ramm1,2, Ramani Shivakkumar1, Elizabeth S. Dennis1,2 and Narayana M. Upadhyaya1,2.
Affiliation: 1. CSIRO Plant Industry, GPO
2. NSW Agricultural
Genomics Centre, Wagga Wagga, NSW,
Abstract: Using a
two element Ac/Ds transposon tagging
system we have isolated a rice (cv. Nipponbare)
mutant – anther indehiscence 1 (aid1), showing undehisced
and delayed dehisced anthers resulting in partial to complete spikelet
sterility. Spikelets of aid1 could be
classified into 4 types based on degree of deformities in pollen grain
development, anther dehiscence and its synchronization with spikelet opening.
The aid1 mutant has fewer
tillers and takes 10-15 more days for panicle heading compared to the
wild-type. The phenotype of the aid1
mutant could not be rescued by application of jasmonic acid (JA) at panicle
booting and anthesis stage, and was not affected by light and humidity,
indicating that AID1 is not involved
in the biosynthesis of JA and that light and humidity do not contribute to the
variable phenotypes of aid1 mutant.
The Ds insertion responsible for this
mutation is located on B
Title: A GA responsive dwarf mutant defective in an early step of GA biosynthesis pathway.
Authors: Marcia Margis-Pinheiro1,3, Xue-Rong Zhou1, Qian-Hao Zhu2, Shamsul Hoque2, Ramannee Shivakkumar1, Kerrie Ramm1,2, Elizabeth S. Dennis1,2, Narayana M. Upadhyaya1,2
Affiliation: 1. CSIRO Plant Industry, GPO
2. NSW Agricultural Genomics Centre, Wagga, Wagga, NSW, Australia
3.
Laboratório de Genética Molecular Vegetal
(LGMV)-Depto de Genética, Universidade
Federal do
Rio de Janeiro, Rio de Janeiro, Brasil
Abstract: The gibberellin (GA) sensitive dwarf mutants form a large and very well known group of plant hormone deficient mutants. The characterization of these mutants has led to the elucidation of GA biosynthetic pathway allowing the cloning of genes encoding the enzymes involved in the different steps of GA production. Enzymes of the GA metabolic pathway have been mainly characterized in A. thaliana and no data has been collected up to now concerning the regulation of the early steps of GA biosynthesis in rice. Here we report the identification and characterization of a GA sensitive dwarf mutant in rice, produced by transposon tag. The rice transgenic line TT14-1-3-3-1 was obtained by super-transformation of transgenic calli carrying a Ds transposon. This plant produces in its progeny short and sterile plants in the proportion of 15:1 normal/dwarf. The dwarf plants show normal viability and germination, but can be differentiated early in development by extreme short sized seedling with dark green leaves. In addition, these plants did not flower. However, in spite of its acute dwarfism, the mutant shows normal root development. Southern experiments using probes derived from both transposon and T-DNA, showed that the dwarf plants contain two different insertions but only one of them, a HindIII 8 kb band, is not present in the homozygous normal plants suggesting that this DNA fragment contains the gene that has been knocked out. TAIL-PCR was used to rescue the flanking region of the Ds transposon with genomic DNA of dwarf plants as template. The sequence obtained encodes an ent-kaurene synthase (KS) protein, the enzyme catalyzing the second step of the GA biosynthesis pathway. Consistent with this result, the osks1 mutant seedlings are able to respond positively to exogenous gibberellin (GA3), by increasing their size to similar levels as wild-type plants. The osks1 transcripts of about 2.3 kb were detected in leaves, stem and panicles, but not in roots. This result suggests that other genes encoding KS could be responsible for the production of ent-kaurene in rice roots. osks1 is not implicated in seed germination since RNA isolated from imbibed seeds of 2, 4 and 6 days did not hybridize to the osks1 probe. Searches on four different databases revealed the presence of at least 4 KS genes in addition to osks1. The phylogenetic analysis of the deduced amino acid sequences of the 5 putative ent-kaurene synthase genes revealed that they all cluster with ent-kaurene synthase. Our results suggest that the osks1 gene may be responsible for the production of ent-kaurene through the most of the rice life cycle, such as stem elongation, leaf expansion and flowering. However, this gene apparently is not implicated in root development or seed germination.
SESSION – 8: BIOINFORMATICS FOR THE
“GLOBAL RICE GENE M
Speaker – 1: Narayana Upadhyaya and Leaka Henry
Title: Bioinformatics for the Global Rice Gene Machine - Towards developing a web-based Rice Gene Machine Information Management System (RGMIMS).
Authors: Leaka Henry2,3, Narayana M. Upadhyaya1,2
Affiliation: 1. CSIRO Plant Industry, GPO
2. CSIRO Mathematical and Information
Sciences, GPO
3. NSW Agricultural Genomics Centre, Wagga Wagga, NSW,
Abstract: A labnote-cum-relational database was originally created using the Microsoft (MS) Access program to catalogue Ac and Ds lines, Ds trap lines, flanking rice genomic sequences, mutant phenotypes, expression patterns, sequence information and sequence homologies. Recently the database has been upsized to MS Access Project with the MS SQL server/client setup - a more robust relational database management system. A tagged sequence database has also been set up in the Unix system using Wisconsin GCG programs and is being made available to registered users for “BLAST” searches using GCG software. Simultaneously, searches for sequences homologous to our “tagged sequences” are being made in publicly available databases. A web site has been setup (http://www.pi.csiro.au/fgrttpub/ ) for the release of information on lines with tags in known gene sequences.
This group’s bioinformatics capabilities are being enhanced with the involvement of CMIS through the new “Centre for Agricultural Genomics”. In the pipeline, is the development of a web-based Rice Gene Machine Information Management System (RGMIMS) to manage all information regarding the “Rice Gene Machine” being developed here at CSIRO Plant Industry. The existing MS Access Project is being streamlined for a robust RGMIMS. The system will interface with a barcode reader/writer to track transgenic plant lines, DNA samples and seeds. Possible migration to other platforms are being taken into consideration while revising the MS Access Project structure in view of future intra- and inter-system unification and/or information filtering “Towards Building a Global Rice Gene Machine”.
Acknowledgements: PI - Qianhao Zhu, Kerrie Ramm, Ramani Shivakkumar, Shamsul Hoque, Marcia Margis, Liz Dennis
CMIS – Ian Saunders, Bella Robinson, Mark Cameron, Gavin Kennedy
Speaker – 2: Christophe Perin
Title: An overview of rice bioinformatics at CIRAD.
Authors: Biotrop, P., Larmande,
M., Ruiz, Perin, C., and
Courtois, B.
Affiliation: CIRAD, TA 40/03 Avenue Agropolis, Montpellier,
Hérault, 34398,
France
Abstract: Three bioinformatics projects dealing with rice are
underway at CIRAD-Biotrop. Two were developed in
order to analyse and store molecular and phenotypic data generated in our
institute. The third is a prospective project to connect several sets of
heterogenous data concerning functional genomics. A Perl
pipeline previously developed for automatic annotation of FSTs (Flanking
Sequence Tags) in Arabidopsis was
adapted and extended for FST annotation of our rice T-DNA insertion library.
This pipeline work is carried out in three steps: vector sequences are removed,
all of the good quality FSTs are blasted against the B
Speaker – 3:
Title: Introduction to
Authors: Yao-cheng Lin1, Chun-I Chung1, Su-may Yu2, Teh yuan Chow1, Chih-kuan Chern3, Ming-sher Fan3 and Yue-ie C. Hsing1
Affiliation: 1.
2.
Abstract: As part
of the effort for rice functional genomics research, a team was funded to
address gene functions in
Title: Bioinformatics skills for the plant genomicist.
Authors: Tim Littlejohn
Affiliation: BioLateral,
Abstract: Molecular life sciences are information driven: as a consequence investigators need to be part of expert teams that can both create high value datasets and have the skills and technologies to mine them for IP. Bioinformatics skills are essential for this discovery process, and they include:
*A deep understanding of information biology;
*A broad knowledge of available bioinformatics resources (databanks, software, computing), and;
*An ability to engineer new technologies when existing ones are lacking or rate limiting
This presentation will focus on the engineering skills needed by the modern plant genomicist.
Speaker – 5: Bal Antanio
Title: Map-based rice genomics through INE.
Authors: B.A.
Antonio, Y. Mukai, N. Namiki,
T. Matsumoto and
Affiliation: Rice Genome Research Program, National Institute of Agrobiological
Sciences / Institute of the Society for Techno-innovation of Agriculture,
Forestry and Fisheries, Tsukuba,
Abstract: The rice genome database INE (Integrated Rice Genome Explorer) integrates all the genomic information that has been accumulated from large-scale analysis of rice including cDNA analysis, genetic mapping, physical mapping, EST mapping and genome sequencing. So far, the database contains more than 422 Mb of sequence data resulting from the international genome sequencing effort. As the high-quality draft sequence of the entire genome will be completed within this year, INE will facilitate a more detailed and accurate correlation of mapping information with the genome sequence. This will be very useful for map-based genomics including comparative analysis of plant genomes, map-based cloning of biologically important genes and functional characterization of genes.
Title: Open Source Initiatives for the Integration of Functional Genomics Data.
Authors:
Affiliation: Plant Genomics Program, NRC Canada,
Plant Biotechnology Institute,
Abstract: The increasing complexity and volume of functional genomics data presents new challenges for deriving data for the rapidly-growing global datasets. Recent developments in the 'open source' community offer guiding principles and effective tools to assist life scientists in capturing full value from the output of functional genomics program. Specific examples will be discussed, with special reference to the needs of the plant and agricultural genomics community.
SESSION – 11: VIEWS OF OUTSIDERS/NEW COMERS
Speaker – 1: Joost T. van Dongen
Title: Mutagenesis as a tool for investigating carbohydrate
metabolism in Rice.
Authors: Joost
T. van Dongen, Peter Geigenberger
Affiliation: Max Planck Institute of Molecular Plant Physiology, Am
Muehlenberg 1, 14476
Abstract: Many questions exist on how sucrose is converted into starch within the
seeds of monocotyledonous plants like rice. What is clear yet,
is that it is significantly different from starch synthesis in e.g. potato
tubers, which is already being investigated extensively in our group.
For example, the transport pathway of carbon through the rice kernel is rather complex, with many different cell types being involved. Besides, starch synthesis in rice seems to be under strict developmental control and the time frame, in which it takes place, is very short: about three weeks only.
The advantages of the sequenced rice genome, the possibility to transform rice, and the increasing availability of different types of mutants will be discussed as tools for investigating the regulation of sucrose to starch conversion in developing rice seeds. Special attention will be paid on the TILLING technique.
Speaker – 2: Tayyab Husnain
Title: Search
of drought tolerant gene(s) and their over expression in Indica
basmati rice (Oryza sativa L).
Authors: Husnain. T.1, Fatima. T.1, Naseem. M.1, Shinwari. Z. K.1, Hussain. S. S.1, Ahmed. Z.1, Riazuddin, S.1, Ito, Y.2, Katsura. K.2 and Shinozaki. K. Y2
Affiliation: 1. National
Centre of Excellence in
2.
Biological resources division, JIRCAS 1-1, Ohwashi, Tsukaba,
Abstract:
Drought, excessive salts and
freezing are stresses that cause adverse effects on the growth and productivity
of plants. Drought and salinity are significant environmental hazards in
Late embryogenesis abundant proteins (e.g HVA1) have been reported to correlate with seed desiccation. We are searching for proteins (genes) involved in somatic embryogenesis in cotton varieties growing in semiarid climate. Histological studies have revealed that the expression of certain proteins is correlated with essential histomorphological changes. During these attempts, proteins have been extracted from local cotton varieties (Gossypium hirsutum L) as well as non-adapted variety G. hirsutum Coker. Hypocotyl segments, regenerating calli at different stages and cell in suspension were used as a source. At present, difference in protein patterns after 2-D gel electrophoresis revealed eight different proteins. Our future goal is to narrow down these proteins and then carry out the molecular characterization of the candidate proteins.
The transformation of plant using regulatory genes is an attractive approach for producing dehydration stress tolerant plants. Stress resistant genes (DREB1A and DREB1B) reported to have significant role in regulating gene expression in response to drought, salt and cold stress. The constructs containing DREB1A and DREB1B under ubiquitin and lip9 promoters are being used to transform Basmati rice. The scutellum-derived calli from mature embryos of Basmati rice were used in biolistic gun transformation. These experiments resulted in seventy putative transformants that were selected on a medium containing hygromycin.
Title: The Rice Gene Machine - A Breeder's Perspective
Affiliation: Cooperative Research Centre for
Sustainable Rice Production and NSW
Agriculture,
PMB Yanco NSW 2703,
Abstract: Rice breeding will benefit from a better understanding of genetic control of the many characters that must be manipulated in a breeding program. This is illustrated by any recent examples including control of amylose content in rice.
The Rice Gene Machine has potential to build on the developments in rice genomics by linking function to structure. The molecular techniques are elegant and have great potential to revolutionise breeding. Describing the link between a gene and its function (phenotyping) is not a trivial exercise and similar elegance will be required to achieve the desired outcome.
Once function has been described, it is variation within a gene that may be important. This variation may already be available within the rice gene pool or may have been created by the Rice Gene machine itself.