Cassava Japanese Initiativegcp21.org/Bellagio/13-MotoakiSeki.pdf · 2000 - 2005 Millennium project...

Cassava Japanese Initiative

Motoaki Seki1. Plant Genomic Network Research Team,

RIKEN Plant Science Center;2. Kihara Institute for Biological Research,

Yokohama City University

Cassava RIKEN Initiative




1. Outline of RIKEN and Plant Science Center (PSC)




2. Asia-Africa Science and Technology Strategic Cooperation Promotion Program (2009-2011) (Supported by the Special Coordination Funds for Promoting Science & Technology) “Towards food security in Asia and Africa by development and application of advanced molecular breeding technologies for the tropical crop, cassava”

3. Biomass Engineering Program (BMEP, 2010-2019)

TAKAMINE Jokichi

SUZUKI Umetaro

TOMONAGA Shinichiro

OKOCHI Masatoshi

NOYORI Ryoji

SHIBUSAWA Eiichi

History of RIKEN

2003-2007 1st 2008-2012 2nd RIKEN Independent Administrative Institution

1958– RIKEN public

corporation

1948– Scientific Research

Institute Ltd. (KAKEN)

1917– RIKEN private

foundation

RIKEN Konzern (Industrial Group) In 1939, 63 companies

and 121 plants

NISHINA Yoshio

RIKEN started in 1917 as the Institute of Physical and Chemical Research, but now biological and medical sciences are major parts of the research and development.

RIKEN: 理化学研究所（理研）

2017

100 years anniversary

4

(Nagoya)

▲Wako Institute (Main Campus)Advanced Science Institute （基幹研究所）

▲Harima Institute (SPring8)

▲Kobe Institute (CDB)▲Bio-Mimetic Control Center

▲Yokohama Institute

▲Tsukuba Institute (BRC)

▲ Sendai Facility

RIKEN has 7 campuses. Main campus is located at Wako. Advanced Science Institute, Nishina Center, and Brain Science Institute in Wako. Bioresource Center in Tsukuba, SPring -8 in Harima, Center for Developmental Biology in Kobe, and Plant Science Center in Yokohama.

RIKEN's Research Network in 2009

RIKEN

135 134 134 132 133 131 130 131 131 131 133 147 161 175 204 230 243 269 266 253 239 2290 16 32 42 94 108 124 136 139 172 189 192477 478 478 479 479 482 483 482 483 483 481 472 463 456 441 420 413 403 397 408 396 381533

725 8691,485

2,606 2,392

0 0 0 0 0 0 0 0 0 0

29 29 41 66 106 135 143 159 176 245 340

1,8041,962 2,230

2,3502,507

0

500

1000

1500

2000

2500

3000

3500

4000

FY1986

FY1987

FY1988

FY1989

FY1990

FY1991

FY1992

FY1993

FY1994

FY1995

FY1996

FY1997

FY1998

FY1999

FY2000

FY2001

FY2002

FY2003

FY2004

FY2005

FY2006

FY2007

Num

ber o

f Sta

ff

Fixed-term

Researcher

Perm anent

Researcher

Fixed-term

Adm inistrative

Staff

Perm anent

Adm inistrative

Staff

Data as of 31 March 2008

Number of employees

Rapid increase since 1997

RIKEN became IAI

6

ASI, 597

RN C , 147

B SI, 482

RSC , 94

C D B , 274

C IPS, 91

O ther, 683

RC AI, 177

C LSP*, 214

C RN ID , 10

C G M , 90

PSC , 100

B RC , 116

Data as of 1 April 2008

*CLSP: RIKEN Cluster of Life Science Platform (OSC, SSBC, BASE)

Recent Expansion

ASI: RIKEN Advanced Science Institute RNC: RIKEN Nishina Center for Accelerator-Based Science BSI: RIKEN Brain Science Institute BRC: RIKEN BioResource Center RSC: RIKEN SPring-8 Center PSC: RIKEN Plant Science Center (PSC) CGM: RIKEN Center for Genomic Medicine RCAI: RIKEN Research Center for Allergy and Immunology CRNID: RIKEN Center of Research Network Infectious Diseases CDB: RIKEN Center for Developmental Biology CIPS: Center for Intellectual Property Strategies OSC: RIKEN Omics Science Center SSBC: RIKEN Systems and Structural Biology Center BASE: RIKEN Bioinformatics And Systems Engineering division CMI: Center for Molecular Imaging

PSC 100

Total No.:~3,000 (in2009)

Personnel & Administration 10,480

Technology Transfer 1,700

President’s discretionary fund and development of young scientists 6,872

Advanced Science 6,101

Accelerator-Based Science

3,851 Synchrotron Radiation Science　　　2,098

Plant Science　1,519 Genomic Medicine　1,600

Developmental Biology 4,467

Grants 6,482

Life Science Platform 　1,000

Bioresources　　　3,181

Brain Science　9,321

98.00billion

JP Yen ($980M.)

Allergy & Immunology　3,261 Yokohama Institute shared research funds　 3,950

Operation of large-scale facilities （SPring-8, XFEL, Supercomputer） 23,557

Construction　 7,500

(unit: million JP Yen)

Molecular Imaging 1,065

7

RIKEN Budget for 2008 Fiscal Year

1. Agricultural science 2. Biology & biochemistry 3. Chemistry 4. Clinical medicine 5. Computer science 6. Economics & business 7. Engineering 8. Environmental/ecology 9. Geosciences 10. Immunology 11. Materials science

RIKEN ranks within the top 1% in the world in 12 fields. Plant science is one of the top research fields in RIKEN. PSC is one of the top research centers in plant science among all the institutes and universities based on citation data by Thomson ISI.

12. Mathematics 13. Microbiology 14. Molecular biology & genetics 15. Multidisciplinary 16. Neuroscience & behavior 17. Pharmacology & toxicology 18. Physics 19. Plants & animal science 20. Psychiatry/psychology 21. Social sciences, general 22. Space science

Thomson Essential Science Indicators, 22 fields. Updated May 1, 2007, for period January 1, 1997－February 28, 2007.

22 Fields of Essential Science Indicators (ESI)

RIKEN RIKEN’s fields of specialty

8

Crops for food, plants for health, crops and trees for biomaterials and energy supply, trees for environmental sustainability

RIKEN Plant Science (PSC1) Genetics, Biochemistry, Molecular biology, Resources in functional genomics 2000 - 2005 Millennium project Director: Tatsuo Sugiyama

Final Goal after 15 years of PSC2Contribution for the improvement of plant productivity

for more and better food and materials

Research Goal of RIKEN PSC2

Research goal in 5 years

・Construction of Metabolomics platform for detecting > 1000 metabolites ・Discovery of important metabolic networks（primary metabolites, secondary metabolites and vitamins）

・Bioinformatics for data mining and simulation (Systems Biology) ・Discovery of new genes and compounds in metabolism (Gene discovery) ・Gene discovery in plant environmental responses ・Gene networks for plant immunity ・Comparative Genomics for crops and trees

Plant Science Center (PSC2) 2005 - 2020 Director: Kazuo Shinozaki

10

Crops for food, plants for health, crops and trees for biomaterials and energy supply, trees for environmental sustainability

RIKEN Plant Science (PSC1) Genetics, Biochemistry, Molecular biology of model plants 2000 - 2005 Millennium project Director: Tatsuo Sugiyama

Final Goal after 15 years of PSC2Contribution for the improvement of plant productivity

for more and better food and materials

Research Goal of RIKEN PSC2

Research goal (2010.4 - 2013.3)

・Construction of omics platform for the integration of metabolomics with transcriptomics, proteomics and phenomics ・Discovery of important regulatory networks that control plant growth, metabolic pathways and environmental responses ・Integration of bioinformatics and systems analysis of model plants ・Translational research for the application to crops, trees and medicinal plants ・Promotion of cooperative research for the application of research outputs

Plant Science Center (PSC2) 2005 - 2020 Director: Kazuo Shinozaki Metabolome, Gene discovery, Systems biology, Comparative genomics, GM technology

11

Director Kazuo Shinozaki Deputy Director Kazuki Saito

Metabolomics Research Division （Kazuki Saito GD , Masanori Arita DGD）

Functional genomics & Bioinformatics Research Area （Minami Matsui Kazuo Shinozaki Mentors） Growth Regulation & Productivity Systems Research Area (Hitoshi Sakakibara, Yuji Kamiya Mentors）

Environmental Response & Adaptation Research Area （Ken Shirasu Kazuo Shinozaki Mentors）

Advisor Tatsuo Sugiyama Coordinator Shigeo Yoshida Science communicator Machiko Itoh Assistant Tomoko Yoshimitsu Yokohama Office Hirohisa Imai Yukari Shindo Taichi Sato Osamu Ishizuka Yukiko Ohwada

Plant Immunity Group (K. Shirasu) Plant Proteomics Unit (Nakgami from April, 2010)

Gene Discovery Group (Shinozaki) Regulatory Network Unit (Shin) Signaling Pathway Unit (Tran)

Growth Regulation Group (Kamiya) Dormancy and Adaptation Unit (Seo) Cellular Growth & Development Team (Yamaguchi)

Plant Nutrition & Metabolism Team (Takahashi)

Biodynamics Group (Sakakibara) Cell Function Unit (Sugimoto)

Metabolic Function Group (Saito) Metabolic Systems Team (Hirai) Advanced NMR Metabolomics Unit (Kikuchi) Metabolomics Informatics Unit (Arita)

Visiting Scientists： K. Okada, H. Fukuda, T. Yamaya, Y. Ogihara S. Kanaya, D. Shibata M. Tomita, A. Soga K. Matsuoka, T. Wada T. Muranaka, E. Nambara

Plant Functional Genomics Group (Matsui) Plant Genomic Network Team (Seki)

Research Organization in 2010 (the 2nd phase of PSC2)Budget: 1.48 billion yen (2009) 1.25 billion yen (2010) PSC members: 100 +40 by other funds 6 group directors14 team and unit leaders

Integrated Genomics Team (Shimada) Integrated Genome Informatics Unit (Sakurai) R&D Program (Shinozaki)

PSC Research Platform and Collaboration 　

(1)  Metabolome Platform (Kazuki Saito) 　　 GC-MS, LC-MS, and NMR in Yokohama, and CE-MS in Tsuruoka Branch

(2) Hormonome Platform (Hitoshi Sakakibara, Yuji Kamiya)　　 Highly sensitive high-throughput hormone analysis (3) Transcriptome and Epigenome Platform, Full-length cDNA collection (Kazuo Shinozaki and Motoaki Seki) 　　 Contribution to AtGenExpress (Arabidopsis expression profile database) 　　 Affymetrix GeneChip ATH1 and Tiling array, Agilent oligoarray Next generation high-speed sequencer

(4) Mutant collection and Phenomics: FOX lines, Ds tagging FST lines (Minami Matsui, transferred from GSC to PSC)　　　 Functional Genomics and Comparative Genomics (5) Proteomics Platform (Ken Shirasu)　　 Protein complex analysis, Phospho- and ubiquitino-proteomics 　

High Citation Rate of PSC publications

Data from ‘Essential Science Indicators’ by Thomson Reuters (Nov 2008)

　 Ins$tu$on Cita$ons Per Paper Papers Cita$ons 1 JOHN INNES CTR PLANT SCI RES 33.79 1,164 39,332 2 RIKEN 24.17 856 20,691 3 UNIV CALIF SAN DIEGO 20.23 1,134 22,941 4 MAX PLANCK SOCIETY 19.44 2,686 52,217 5 UNIV CALIF BERKELEY 18.38 2,053 37,742 6 HARVARD UNIV 17.6 1,149 20,221 7 DUKE UNIV 16.25 1,140 18,527 8 UNIV ARIZONA 15.94 1,676 26,717 9 RUTGERS STATE UNIV 15.06 1,355 20,402 10 CNRS 12.96 2,175 28,186

2nd Place in ‘Plant & Animal Science’ in the world (Institutes with >500 manuscripts in 10 years)

2nd Place in RIKEN research fields　 Field Cita$on Rate (X/Y) Cita$ons Papers Cita$ons/Paper (X) World's Avg. Cita$ons/Paper (Y) 1 MULTIDISCIPLINARY 9.35 867 23 37.7 4.03 2 PLANT & ANIMAL SCIENCE 3.56 20,691 856 24.17 6.79 3 IMMUNOLOGY 2.04 11,267 270 41.73 20.41 4 CLINICAL MEDICINE 1.5 14,070 807 17.43 11.61 5 MATERIALS SCIENCE 1.39 2,937 388 7.57 5.45 6 ENGINEERING 1.29 5,782 1,190 4.86 3.77 7 PHYSICS 1.24 46,397 4,787 9.69 7.84 8 MOLECULAR BIOLOGY & GENETICS 1.22 52,070 1,734 30.03 24.54 9 NEUROSCIENCE & BEHAVIOR 1.2 22,095 1,046 21.12 17.58 10 BIOLOGY & BIOCHEMISTRY 1.17 44,630 2,390 18.67 15.93 11 CHEMISTRY 1.05 21,843 2,222 9.83 9.35 12 MICROBIOLOGY 0.85 5,866 473 12.4 14.63

High citation/paper

High citation rate

1. Outline of RIKEN and Plant Science Center (PSC)




2. Asia-Africa Science and Technology Strategic Cooperation Promotion Program (2009-2011) (Supported by the Special Coordination Funds for Promoting Science & Technology) “Towards food security in Asia and Africa by development and application of advanced molecular breeding technologies for the tropical crop, cassava”

3. Biomass Engineering Program (BMEP, 2010-2019)

Council for Science and Technology Policy (CSTP) 　　　　　　　　　　　　　　　　　　　　　　　　　　　　　

Function of CSTP:

•  Overlooks all of the nation’s Science and Technology policy in Japan –  Investigates & deliberates basic policy of science and technology –  Investigates & deliberates guidelines on Budgetary/Personnel Research

Allocation –  Evaluates the research and development of national importance

Chair: Prime Minister (June 2010-: Naoto Kan) Cabinet Members: 　 6 Executive Members 　(academia/industry): 7 Science Council : 　1

2

Asia-Africa S & T Strategic Cooperation Promotion Program

Objectives: To establish S & T Partnerships in Asia and Africa, especially focused on the activities for developing and/or expanding advanced technologies.

･By expanding an open and equal partnership with Asian & African 　　countries, Japan would like to participate in developing S&T in this 　　　area. ・Especially, by Tackling the Challenges, in dissolving the common regional issues, in creating S&T of the region and in securing S&T human resources, the superiority of the region in S&T can be established and maintained. ･By constructing a sustainable framework, the S&T community in Asia and 　　Africa will be strengthened.

6

Asia-Africa S & T Strategic Cooperation Promotion Program

Objectives : To establish S & T Partnerships in Asia and Africa, especially focused on the activities for developing and/or expanding advanced technologies.

Financial Support: ・ Approx. 20 ~ 30 million yen / project / year ・ For a periods of 3 years

・ By the policy of the program; the collaboration should be based on the spirit of equal partnership, the research expenses by foreign research organizations outside Japan should not be covered.

Funded expenses related research exchanges: ・ Travel expenses in bilateral mode ・ Expenses for holding meetings etc.

7

Cassava (Manihot esculenta) An important tropical crop for food security and poverty reduction

in many Asian and African countries (an important source for a billion people’s food and income generation).

Indonesia = 20 Mt/yr

Thailand = 27 Mt/yr

China= 5 Mt/yr

Brazil= 27 Mt/yr

Colombia= 2 Mt/yr

Paraguay= 5 Mt/yr

Democra$c Republic of Congo = 15 Mt/yr

Ghana= 10 Mt/yr Nigeria= 43 Mt/yr

Southeast Asia= 73 Mt/yr

Sub-‐Saharan Africa= 114 Mt/yr

La$n America = 35 Mt/yr

Improvement of poor farmers

Food security

Improvement of poor farmers 18

Vietnam = 8 Mt/yr

(from 2007 data)

Growth in unfavorable environment (Dry, Acid soil & Oligotrophy soil)

Leaf: Livestock feed

“Alfalfa in the tropics”

Stem: Use for propaga$on

Root: Tapioca starch Use for foods and biomass

Cassava： A tropical crop without waste

Growth without fer$lizer

19

source for a billion people’s food and income generation in tropical countries

Problems that should be solved urgently in Africa and Asia 1. Development of the cassava cultivars that can adapt to global climate changes

2. Development of the cassava cultivars with high values added (high content of starch and sugars) 　＝　towards income generation

Integrated international-standard genomic analysis platform for the global cassava research community

Advancing the breeding efficiently and rapidly is indispensable.

20

White flies, Mealybug and Cassava mosaic disease (CMD)　　・CMD is a virus disease that is carried by white flies and is observed in Africa and India. ・Outbreak might occur in Southeast Asia. ・Loss: bigger than one billion US dollars per year

Principal Cassava Research Organization

Danforth PSC (USA）

Genome analysis, Transformation etc.

Ohio State Univ. (USA）

Wageningen Univ （Netherlands）

ETH （Swiss）

IITA （Nigeria）

Witswatersrand Univ. （Republic of South Africa）

Shanghai Cent.

（China）

EMBRAPA （Brazil）

Bath Univ (UK）

RIKEN（Japan） Establishment of genome analysis

platform

CIAT （Colombia）

Largest genetic resources ,

Molecular breeding, Identification of

useful genes and markers

Mahidol Univ. （Thailand）

Starch and sugar metabolism、

Marker breeding

21

1. Establishment of genomic analysis platformRIKEN PSC Motoaki Seki (Project Leader)

Tetsuya SakuraiMinami Matsui

RIKEN OSC Ri-ichiro Manabe a) Full-length cDNA resourceb) Large-scale ESTs and SNPs using next generation sequencersc) Agilent Oligoarray analysis systemd) Database of international-standarde) Transformation system in elite cultivars

Research Organization of Cassava International Collaborative Project (2009~)

3. Marker breeding of the cassava cultivars with high value addedThailand・Mahidol Univ.Jarunya Narangajavana

a) Breeding materialsb) Molecular markers c) Establishement of marker breeding

・Advancing the global breeding efficiently and rapidly.・Contribution to food security and poverty reduction

in Asia and Africa.

13

2. Molecular breeding of usefulcassava cultivarsColombia・CIAT・Manabu Ishitani

a) Useful genetic resourcesb) Molecular markers for useful

traits

Asia-Africa Science and Technology Strategic Cooperation　Promotion Program (2009-2011) (Supported by the Special Coordination Funds)

“Towards food security in Asia and Africa by development　and application of advanced molecular breeding technologies for the tropical crop, cassava”

Yoshinori Utsumi

Unique point of this project 　 High-standard genome technology (RIKEN PSC):　S & T diplomacy 　 Useful genetic resources (CIAT)：Positive use of useful resources　　　　　　　　　　　　 Useful breeding materials (Mahidol Univ)：Study with near gateway　　　　　　　　　　　　　

High-level genome technology (RIKEN) 1.  Functional genomics using full- length (FL) cDNAs 　　・The largest collection of Arabidopsis FL-cDNAs（Seki et al. 2002. Science Yamada et al. 2003. Science) ・FL-cDNAs of useful crops and trees 　　　（Wheat, Soybean, Poplar etc.） 2.  Transcriptome analysis using DNA microarrays　　　・Many publications (Many knowhow) 3. Various useful database 　　・Arabidopsis, Soybean etc. (Great experience) 4. Plant Transformation 　　・Fox-hunting（Arabidopsis, Rice） (Great experience) 　　

Useful genetic resources (CIAT) More than 6,000 cassava genetic resources ・KU50 (High yield) ・ECU72 (White fly-resistant) ・MPER417-003 (Mealybug-resistant) ・M. flabellifolia (Cassava Bacterial Blight-resistant)

Useful breeding materials (Mahidol Univ.) ： Breeding materials ・F1 populations developed by cross between Ha-Natee (Low starch) X Huay Bong 60 (High starch) Studies on cassava cultivars with high values added ・Studies on starch metabolism

23

ATG STOP

Genome DNA

mRNA

Partial cDNA

Full-length cDNA

Can’t produce proteins 　

Can produce proteins (Applicable to transgenic plants)

Exon Intron

Full-length cDNA is an useful tool for basic and applied sciences.

Collection of Arabidopsis full-length cDNAs and its application to microarray

1) Construction of 19 full-length cDNA libraries using biotinylated cap trapper method in collaboration with

Hayashizaki group (RIKEN GSC) (Seki et al. Plant J. 1998, Science 2002)2) Collection of ~ 18,000 independent full-length cDNAs, ~70% of Arabidopsis genes (RAFL cDNAs) (Seki et al., Science 2002)3) Full-length Sequencing of ~13,000 RAFL cDNAs and their

pUNI clones in collaboration with SSP group (Ecker, Theologis & Davis) (Yamada et al., Science 2003)

4) RAFL cDNA clones are available from RIKEN BRC.5) Construction of microarray with ~7,000 full-length cDNAs6) Expression profiling of drought-, cold- and high salinity- stress inducible genes using the microarray (Seki et al., Plant Cell 2001, Plant J 2002, Fun Int Genomics 2002)

Promoter database of 14,000 Arabidopsis genes

プロモーター領域 (約1 kb)の塩基配列情報

Cis-acting elements found in Arabidopsis promoters

1000 bp 5’ flanking sequences of Arabidopsis expressed genes

http://pfgweb.gsc.riken.go.jp/index.html

a. Separate predicted genes correspond to a single experimentally identified gene.

AGI code 1 AGI code 2

5‘-end seq. of RAFL cDNA1


RAFL cDNA1

Genome

AGI code 1 AGI code 2



RAFL cDNA1

Genome

c. New identified genes which have not been predicted so far by AGI.

AGI code 1



RAFL cDNA1

Genome

b. Single predictions correspond to more than 2 experimentally identified genes. 5‘-end seq. of RAFL cDNA2


RAFL cDNA2

201 RAFL cDNA clones

297 AGI codes

837 RAFL cDNA clones Seki et al. (2002) Science 296: 141-145.

FL- cDNA collection from various plants and its application to microarray analysis

Wheat(Dr. Ogihara,

Yokohama City Univ.)Single-pass seq. of

40,000 clones,FL-seq. of ~12,000

Custom Oligoarray

Poplar(Dr. Shinohara, FFPRI)

Single-pass seq. of40,000 clones

Soybean(Soybean Japan Consortium)

Single-pass seq. of 40,000 clones,FL-seq. of 2,500 clonesCustom Oligoarray

Barley(Dr. Sato,

Okayama Univ.)Single-pass seq. of

40,000 clones

Cassava(Dr. Ishitani,

CIAT,Colombia)Single-pass seq. of

20,000 clonesCustom Oligoarray

Cryptomeria(Dr. Shinohara, FFPRI)

Single-pass seq. of20,000 clones

Morning glory

(Dr. Iida, NIBB)1 library

Thellungiellahalophila

(Dr. Taji, Tokyo Univ. of Agr.)Single-pass seq. of

20,000 clones,FL-seq. of 1,250

Custom Oligoarray

Physcomitrella(Dr. Hasebe, NIBB)Single-pass seq. of

40,000 clonesCustom Oligoarray

Porphyra(Dr. Saga,

Hokkaido Univ.)2 FL-cDNA

libraries

(Ishida, Morosawa, Sakurai UL and many collaborators)

Brassica napus(Dr. Nambara,

Univ. of Toronto;Dr. Watanabe,Tohoku Univ.)

1 FL-cDNAlibrary

Unique point of this project－The first case in cassava 　 High-standard genome technology (RIKEN PSC):　S & T diplomacy 　 Useful genetic resources (CIAT)：Positive use of useful resources　　　　　　　　　　　　 Useful breeding materials (Mahidol Univ)：Study with near gateway　　　　　　　　　　　　　

High-level genome technology (RIKEN) 1.  Functional genomics using full- length (FL) cDNAs 　　・The largest collection of Arabidopsis FL-cDNAs（Seki et al. 2002. Science Yamada et al. 2003. Science) ・FL-cDNAs of useful crops and trees 　　　（Wheat, Soybean, Poplar etc.） 2.  Transcriptome analysis using DNA microarrays　　　・Many publications (Many knowhow) 3. Various useful database 　　・Arabidopsis, Soybean etc. (Great experience) 4. Plant Transformation 　　・Fox-hunting（Arabidopsis, Rice） (Great experience) 　　

Useful genetic resources (CIAT) More than 6,000 cassava genetic resources ・KU50 (High yield) ・ECU72 (White fly-resistant) ・MPER417-003 (Mealybug-resistant) ・M. flabellifolia (Cassava Bacterial Blight-resistant)

Useful breeding materials (Mahidol Univ.) ： Breeding materials ・F1 populations developed by cross between Ha-Natee (Low starch) X Huay Bong 60 (High starch) Studies on cassava cultivars with high values added ・Studies on starch metabolism

30

Previous Research Achievements (~2008) 1. Construction of full-length (FL) cDNA library from KU50 (MTAI16)

cultivar with high yield (2007). About 20,000 cassava FL-cDNA clones (~11,000 groups). 　・Sakurai et al. (2007) BMC Plant Biol. 7:66. 　　　・ An invited lecture in Cassava International Meeting (2008, Ghent) There were several requests for the establishment of functional genome analysis platform.

Press release (Dec. 12, 2007）

2. Construction of cassava full-length cDNA and portal database (by Dr. Tetsuya Sakurai)

All mRNA seq. and its annotation in cassava FL-cDNA seq. and its annotation

31

Portal (Cassava Online Archive http://cassava.psc.riken.jp/

FL-cDNA DB http://amber.gsc.riken.jp/cassava/

Achievement goal of the project (2009-2012) 1. Identification of about 30,000 non-redundant expressed cassava genes a. Cassava FL-cDNA resources ・　1 normalized FL-cDNA library (from whitefly-resistant ECU72) 　・　5’-end sequencing of 30,000 FL-cDNAs (finished) ・　1 normalized FL-cDNA library (from Mealybug-resistant MPER417-003) (finished) ・　5’-end sequencing of 20,000 FL-cDNAs (finished) b. Identification of large-scale expressed genes (ESTs) and its SNPs from

useful cassava genetic resources (ECU72 and MPER417-003 etc.) by next-generation sequencers (454 and Illumina)

2. Cassava oligoarray analysis platform containing more than 30,000 genes 　・　Identification of useful genes (markers) 3. Cassava database of international standard 　・　Functional classification of genes, mapped position of genes and SNP information 　・　Centralization of all cassava expressed genes 4.  Transformation system of cassava elite cultivars ・　1 Asian one (KU50) and 2 African ones (1 Kenya and 1 Nigeria, TMS60444)

・Development of platform of integrated cassava functional genomics

・Establishment of molecular breeding for high-yield and high-value cassava

1. Identification of about 30,000 non-redundant expressed cassava genes a. Cassava FL-cDNA resources

Library feature

KU50- Abiotic

(Sakurai et al. 2007)

ECU72- biotic

(RIKEN,CIAT Unpublished)

MPER417- biotic

(RIKEN,CIAT Unpublished)

Total

Clones 19,968 29,952 19,968 69,888

Distinct genes (ESTstat)

13,634 6,580 9,361 ~20,000 Non-redundant

genes

New cassava transcripts

7,817 (57%)

2,215 (34%)

4,089 (44%)

~12,340 Non-redundant

genes

KU50 ECU72

4,443 2,137 9,191

KU50 MPER417

5,465 3,896 8,169

ECU72 MPER417

3,437 5,924 3,143

(in collabora$on with CIAT group)

Total No. of Cassava distinct genes that have been identified so far as ESTs using Sanger method: 27,366

12,340 7,260 7,766

RIKEN & CIAT NCBI

T Treatments Age rom transfered to the soilf Tissue

Duration of treatment before RNA extraction

Biotic stress (9) Whitefly (Aleurotrachelus socialis) 2 month leaf, stem 7, 15, 25 days Mealybug (Phenacoccus herreni) 2 month leaf, stem 7, 15, 25 days Mites (Mononychellus tanajoa) 2 month leaf, stem 3, 7 days Whitefly and Mealybugs 2 month leaf, stem 15, 25 days Whitefly and Mealybugs and Mites 2 month leaf, stem 3, 7 days CBB (Xanthomonas axonopodis) 2 weeks leaf, stem 1, 3 days Root Rot (Phytopthora tropicalis) 6 weeks leaf, stem 1, 3 days Root Rot (Phytopthora palmivura) 6 weeks leaf, stem 1, 3 days Xanthmonas & Phytophthora 2 weeks leaf, stem 1, 3 days

Abiotic stress (14) DesiccaJon 1 month leaf, stem unJl 50 % water loss Fluorescent light 1 month leaf, stem 4 hours UV 1 month leaf, stem 15, 30 min Red light 1 month leaf, stem 0.5, 1, 6 hour Dark 1 month leaf, stem 24 hours Nitrogen gas 1 month leaf, stem 1 hour CO2 gas 1 month leaf, stem 1 hour O2 gas 1 month leaf, stem 1.5 hour heat at 42°C (0.5, 1, 6, 24 hour) 1 month leaf, stem 0.5, 1, 6, 24 hour Cold at 4°C (0.5, 1, 6, 24 hour) 1 month leaf, stem 0.5, 1, 6, 24 hour Water dipping (BQ lab) (1 hour) 1 month leaf, stem 1 hour 10 uM 2,4-‐D Auxin 1 month leaf, stem, root 0.5, 1, 6, 24 hour 100 uM ABA 1 month leaf, stem, root 0.5, 1, 6, 24 hour 20 mM SA 1 month leaf, stem, root 0.5, 1, 6, 24 hour 100 uM Gibberellin 1 month leaf, stem, root 0.5, 1, 6, 24 hour 200 uM AlCl3 1 month leaf, stem, root 0.5, 1, 6, 24 hour 200 mM NaCl 1 month leaf, stem, root 0.5, 1, 6, 24 hour 10 g/L Mannose 1 month leaf, stem, root 0.5, 1, 6, 24 hour Cu]ng 1 month leaf 10 min, 0.5, 3, 6, 24 hour Wounding 1 month leaf 10 min, 0.5, 3, 6, 24 hour round-‐up 1 month leaf, stem 30 min FerJlizer 1 month leaf, stem 30 min InsecJcide 1 month leaf, stem 30 min fungicide 1 month leaf, stem 30 min

un-treated (2) plant in soil 1 month leaf, stem In vitro plants 1 month leaf, stem, root

Conditions and tissues used for RNA extraction for FL cDNA library construction and next-generation sequencer analysis (MPER417-003)






Cassava Oligoarray Development

2010~: 30,000 gene Agilent oligoarray

developed

September: >15,000 gene

Agilent oligoarray developed

Agilent 20K oligoarray

experiments (in progress)


experiments will be done

2009 2010 2011

Contigs (11,423) Singlets (18,221) Distinct sequences: 22,481 Prove design example (Agilent eArray) 60 mer probes: 19,177

Make Fast file & design probes

Check orientation and compliment sequences

FL clones and other ESTs in genebank

1st custom 20K oligoarray (2009)

Goal: 30K Agilent Custom Array = 70% Estimated Genes in Cassava Genome

2nd custom 30K oligoarray (2010-2011) Futures: •  Available in 2010 ~ 2011 •  >30,000 gene proves on a chip •  More biotic-related genes •  Genes from wild species

Contact: Motoaki Seki (RIKEN PSC) Tetsuya Sakurai (RIKEN PSC) Manabu Ishitani (CIAT)

1. Expression profiling under desiccation treatment in KU50, ECU72 and MPER417-‐003 using 20K ver.1 array

MaterialsECU72 leaf, root, stemKU50 leaf, root, stemMPER417-‐003 leaf, root, stem

Condition MS medium (2% Sucrose + MS vitamine)30℃Light at 12 h and Night at 12 h

MethodsRNeasy Plant Mini kit (QIAGEN)

QIAGEN↓Sample (Max 〜100 mg F.W.)↓Follow manuscript protocol

ECU72-‐1 leaf ECU72-‐2 leaf

KU50-‐1 leaf KU50-‐2 leaf KU50-‐3 leaf

ECU72-‐1 Dry leaf ECU72-‐2 Dry leaf ECU72-‐3 Dry leaf

Quality cheak of total RNA (from leaves) using CE

Results 1

ECU72 (3/3cutting)

KU50(2/25cutting)

Desiccation: 60 min

KU50-‐1 Dry leaf KU50-‐2 Dry leaf KU50-‐3 Dry leaf

Untreatment

State of plant before and after the desiccation treatment

ECU72-‐3 leaf

(Yoshinori Utsumi, in collaboration with CIAT group)

Genetical difference among the cassava material and correlation coefficients of the signal intensities between experiments

Our Agilent custom cassava array can be applied to expression profiling in various Manihot species!

Venn diagram representing clones up-regulated or down-regulated by desiccation treatment among three genotypes.

up-regulated (P<0.001, fold change>2.0)

down-regulated (P<0.001, fold change>2.0)

M ECU72 M THAI16

M PER417-003

56 16

128

26 20

46

666

M ECU72 M THAI16

M PER417-003

246 61

332

5 4

21

332

Control Desiccation treatment (RT, 60 min)

Genes up-regulated or down-regulated by desiccation treatment among the three genotypes.

M THAI16

2. Expression profiling during root development in KU50 and WT cassava using 20K ver.1 array (Yoshinori Utsumi, in collabora$on with Mahidol Univ. group)

4-week-old fibrous roots

(KU50)

8-week-old roots (KU50)

Plant materials used for expression profiling:

1. KU50, 4-week, Fibrous roots 2. KU50, 8-week, Fibrous roots 3. KU50, 8-week, Storage roots 4. KU50, 8-week, Intermediate

storage roots

1.  WT, 5, 12 and 13-week, Fibrous roots

(preliminary data) 12-week-old roots (Wild Type

Local Cultivar)

Never form storage roots

Cassava Oligoarray Development

2010~: 30,000 gene Agilent oligoarray

developed

September: >15,000 gene

Agilent oligoarray developed


experiments (in progress)


experiments will be done

2009 2010 2011

Contigs (11,423) Singlets (18,221) Distinct sequences: 22,481 Prove design example (Agilent eArray) 60 mer probes: 19,177

Make Fast file & design probes

Check orientation and compliment sequences

FL clones and other ESTs in genebank

1st custom 20K oligoarray (2009)

Goal: 30K Agilent Custom Array = 70% Estimated Genes in Cassava Genome

2nd custom 30K oligoarray (2010-2011) Futures: •  Available in 2010 ~ 2011 •  >30,000 gene proves on a chip •  More biotic-related genes •  Genes from wild species

Contact: Motoaki Seki (RIKEN PSC) Tetsuya Sakurai (RIKEN PSC) Manabu Ishitani (CIAT)

Future expression profiling studies using 30K ver.2 array (Collabora$on with Mahidol Univ. and CIAT groups)

1. Expression profiling in Huay Bong 60 (High yield, High starch and High HCN contents) and Hanatee (Low yield, Low starch and Low HCN contents) on responses to Cassava anthracnose disease (CAD) (Collabora$on with Mahidol Univ. group)

2. Expression profiling in MPER417-‐003 (Resistant), ECU72 (Resistant) and KU50 (Sensi$ve) aker inocula$on with white fly (Collabora$on with CIAT group)

Genes in involved in whitefly resistance

Genes in involved in High yield, High starch, High HCN and CAD resistance






Future prospects on our contribution to global cassava projects in other countries:

• 　Global development using platform of integrated cassava functional genomics (FL-cDNAs, microarray and database) in collaboration with the leading research organization. • 　Achievement development of useful cassava breeding materials selected into the neighbors (AGI, Vietnam; PI: Dr. Ham) of Thailand. ・　Achievement development of useful cassava breeding materials in Africa in collaboration with IITA (or AATF).

46

Cassava (Manihot esculenta) An important tropical crop for food security and poverty reduction

in many Asian and African countries (an important source for a billion people’s food and income generation).

Indonesia = 20 Mt/yr

Thailand = 27 Mt/yr

China= 5 Mt/yr

Brazil= 27 Mt/yr

Colombia= 2 Mt/yr

Paraguay= 5 Mt/yr

Democra$c Republic of Congo = 15 Mt/yr

Ghana= 10 Mt/yr Nigeria= 43 Mt/yr

Southeast Asia= 73 Mt/yr

Sub-‐Saharan Africa= 114 Mt/yr

La$n America = 35 Mt/yr

Improvement of poor farmers

Food security

Improvement of poor farmers 47

Vietnam = 8 Mt/yr

(from 2007 data)

(No GM cassava)

Tentative plan for the next phase for application of useful GM cassava into Vietnam

in collaboration with AGI (PI: Dr. Ham) , if funded

1. Importation of useful transgenic KU50 from CIAT

2. Test phenotype in multiple confined field locations in Vietnam (3 years)

3. Lay out all necessary regulatory framework in Vietnam

4. Regulatory information collected through field evaluation, such as environment assessment

5. Select elite line as pre-commercial variety

6. Training cassava farmer to deal with GM cassava

Program Director

Kazuo Shinozaki

Biomass Engineering Program (BMEP) (2010~2019)

R&D for the contribution to Energy and Environment

u  Development of new technology related to Environment and Energy programs

Global Programs

Sustainable society

For the development to larger programs in Green Innovation

Green & Material Science Program 1. Material science for the contribution to environmental program and clean energy 2. Contribution to Green Innovation

Biomass Engineering Program　 1. Plant Biomass production, Biomass processing, Bioplastic production 2. Contribution to Green Innovation to solve global programs, such as energy, food and environment

New Industry

u Interdisciplinary research among Biology, Chemistry and Engineering

Sustainable environemnt

Contribution to solve global problems for the survival of humankind

Water（水） Energy（エネルギー） Health（健康）　　　　　　　　　

Agriculture（農業） Biodiversity（生物多様性）

　　　　+　　　 Poverty（貧困）

R&D to solve global problems （Koffi Annan UN Secretaryl） R&D

Green Innovation

２

Fossil Fuel Biomass

Petrochemistry

Energy Chemical material

Refinement

Heavy oil

Light Oil

Kerosene

Gasoline

Naphsa

Bioprocess

Gas

Oil

Sugars

Innovative Technology

21st Century, Sustainable society 20th Century, Consumerism

New Industry

CO2 Fermentation

gas 　Extraction

CO2

CO2

CO2

Material Chemical

Oil

Energy

Recycle, Carbon Neutral

Green Technology for the sustainable society and life

・To reduce fossil fuel and to increase biomass for the sustainable society and life ・New technologies to develop novel materials and fuel based on biomass ・New industries for bioprocess and biomaterials based on new biotechnologies

Plant

3

Growth in unfavorable environment (Dry, Acid soil & Oligotrophy soil)

Leaf: Livestock feed

“Alfalfa in the tropics”

Stem: Use for propaga$on

Root: Tapioca starch Use for foods and biomass (bioplas$cs)

Cassava： A tropical crop without waste

Growth without fer$lizer

52

Source for a billion people’s food and income genera$on in tropical countries

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Cassava Japanese Initiativegcp21.org/Bellagio/13-MotoakiSeki.pdf · 2000 - 2005 Millennium project...

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