1. Development of genomics pipelines and
its integration with breeding
hb d
Joe Tohme
Joe Tohme
Workshop in celebration of the lives of
Dr María Jesús ("Chusa") Ginés and Ms Verónica Mera
May 12 13, 2010
May 12‐13 2010
WWW.ciat.cgiar.org
Eco-Efficient Agriculture for the Poor

2. Development of genomics pipelines
and its integration with breeding
d hb d
• Relevant technical advances
Relevant technical advances
• What are the needs?
What are the needs?
• What do we have in cassava?
What do we have in cassava?
• What are the next steps?
What are the next steps?
Eco-Efficient Agriculture for the Poor

3. A Decade of Major Plant Sciences Breakthrough
Whole Genomes Sequencing
2000, July 2000 2002
p g y
Citrus pathogen Xylella ode p a t ab dops s
Model plant Arabidopsis ce ge o e seque ced
Rice genome sequenced
sequenced in Brazil
genome sequenced private public effort
Eco-Efficient Agriculture for the Poor

4. Revolution in sequencing technologies
Revolution in sequencing technologies
2009‐2010
Routine
Sequenci
ng of
plant
genomes
and
associate
d pest
Eco-Efficient Agriculture for the Poor

5. Most important plant genomes sequenced
p p g q
or about to be sequenced
Maize genome sequenced
End 2009‐ Cassava sequenced
d d
with funds from DOE on
a CIAT genotype
a CIAT genotype
Jan 2010, three cassava genotypes
sequenced in China in
seq enced in China in
collaboration with Brazil
Jan 2010, Soybean genome sequenced
Eco-Efficient Agriculture for the Poor

6. Sloped land Acid soils, Colombia
(Laos)
Nigeria
Fertile soils, Vietnam
Humid environments Amazon, Brazil
Dry environments
Brazil
Orinoco river, Venezuela
Eco-Efficient Agriculture for the Poor
HERNAN CEBALLOS

7. Cassava Biodiversity
Wild Manihot species are
Wild Manihot
ild ih i
unique sources of genes that
could be used through
g
breeding with markers assisted
selection,
and/or genetic transformation
and/or genetic transformation
How are going to
How are going to
speed up genes
discovery and alleles
discovery and alleles
mining for key traits?
M. Bonierbale Eco-Efficient Agriculture for the Poor

8. Development of genomics pipelines and
its integration with breeding
its integration with breeding
Exploit untapped genetic diversity in genebanks, and
breeding populations
breeding populations
Needs
– Access to genomics facilities for gene discovery and
screening
– Ph
Phenotypic characterizations platform
i h i i l f
– Development of targeted breeding populations
– Bioinformatics capacities
– Knowledge of how to design transgenic products and how
to manage the whole process (biosafety and IP)
h h l (bi f d )
Eco-Efficient Agriculture for the Poor

9. Cassava Genomics Tools
Cassava Genomics Tools
Mapping Tools
‐ Several mapping populations linked to breeding
l l l k d b d
‐ ~ 350SSRS, 301 AFLP‐RFLPs, 17 Genes
‐ MAS for CMD and Post Harvest Deterioration
BAC Libraries
BAC Libraries
‐ 3 libraries with 5X, 10X, and 11X coverage
‐ Deposited at Clemson Genome Center
Generation of cassava EST Collection ‐ 5,700 Unigene set available
− T id tif
To identify genes involved in starch metabolism
i l di t h t b li
− To study cassava ‐ Xanthomonas axonopodis, the causal agent of CBB
− Collaborators: University of de Perpignan and IRD, France, U. Nacional
Microarray platform developed for cassava
yp p
A full length cDNA collection of cassava – 20,000 clones sequenced
‐ To develop a large scale of full length cDNA collections
‐ Collaborators: RIKEN Plant Science and RIKEN Genomic Centers, Japan
Co abo ato s: a t Sc e ce a d Ge o c Ce te s, Japa
Eco-Efficient Agriculture for the Poor

10. Cassava germplasm
Unexplored Source of Novel Traits and Genes
Goal: > 30,000 unigenes identified in 2010
Genotype: MTAI16 (KU50)
MTAI16
Treatment (Abiotic stress):
( bi i )
Drought, Heat, PPD, Al toxicity, etc)
EST: 20,000 full length cDNA
EST: 20 000 full length cDNA
collection
Outcome: First FL cDNA from
Outcome: First FL cDNA from
cassava (BMC Plant Biology 2007)
Eco-Efficient Agriculture for the Poor

11. Genomics Tools:
Sequencing and annotations of
Sequencing and annotations of
20000 full length cDNA cassava clones
Achievements
• A full‐length cDNA library of cassava transcripts to
understand the regulatory networks involved in
stress response
stress response
• Gene annotations to identify gene function related
to cassava unique physiological features
to cassava unique physiological features
• Assist the annotation of the genome sequencing by
DOE‐Joint Genome Institute
Eco-Efficient Agriculture for the Poor

12. Sequencing and annotations of
20000 full length cDNA cassava clones
20000 full length cDNA cassava clones
Starch Biosynthesis
Eco-Efficient Agriculture for the Poor

13. Cassava germplasm
Unexplored Source of Novel Traits and Genes
Goal: > 30,000 unigene identified in 2010
Year: 2009‐2010 Whitefly infestation
Genotype: MEcu72 (whitefly resistant)
Treatment (Biotic stress): Whitefly, Green mites, Mealybugs,
Hornworm, Bacterial blight and pesticide
EST: 20,000 FL cDNA collection and 1 m 454 sequence
Expected outcome: novel genes associated with whitefly MEcu72
resistance
Mealybug treatment
Year: 2010
Genotype: M. peruviana (wild species), insect resistance
Treatment (Biotic stress) Mealybug, Whitefly, Bacterial
( ) y g, y,
blight and Root rots
EST: 20,000 FL cDNA collection
Expected outcome: novel genes related to insect
resistance
CMC40 M. peruviana
Eco-Efficient Agriculture for the Poor

14. Global Cassava Genomics Tool Development
Sarah Ayling Motoaki Seki Mahidol University
Umemura Yoshimi Tetsuya Sakurai Jarunya Narangajavana
Manabu Ishitani Minami Matsui (CASS MOL BIOTECH C di t )
M b I hit i Mi iM t i (CASS‐MOL‐BIOTECH Coordinator)
2009 2010 2011
December: 30,000 FL cDNA February: First Agilent
September: 30,000 gene March: Marker
sequenced from MEcu72 oligoarray developed
Agilent oligoarray developed for trait of
developed interest in Thailand
March: 20,000 FL cDNA constructed
from M. peruviana (MPER417‐003)
March: 1 million read of 454 sequence for
Ecu72 generated
E 72 t d
Cassava database constructed
Eco-Efficient Agriculture for the Poor

15. Xanthomonas axonopodis pv. manihotis
Foliar and systemic pathogen
F li d t i th
Previous studies (Valerie Verdier
Causal agent of Cassava
Bacterial Blight team IRD – CIAT and Camilo
Lopez, U. Nacional)
Lopez U Nacional)
• Molecular diversity of populations
in LA and Africa
in LA and Africa
• Mapping of R genes in cassava
cultivars
• Initiate plant‐pathogen interactions
Eco-Efficient Agriculture for the Poor

16. Solexa and 454 Sequencing of
Xanthomonas axonopodis pv. Manihotis
X h di M ih i
Team leader: Adriana Bernal, U. Los Andes, Colombia
Team leader: Adriana Bernal, U. Los Andes, Colombia
Solexa and 454 Sequencing
– Coverage 69,83% with 454 and
33,41% with Solexa
– Assembly used is pseudomolecule
y p
(not the real order of the 33
scaffolds)
– Still major gaps
Still major gaps
– 4086 protein‐coding genes
– Many pathogenicity genes common
with other pathovars
Eco-Efficient Agriculture for the Poor

17. Gene expression analysis of
Cassava Whitefly interaction using
Cassava‐Whitefly interaction using
the Cassava Unigene set microarray
Tissues from infested and non-infested cassava
non infested
leaves, from susceptible (CMC40) and resistant
(MEcu-72) genotypes at different time during whitefly
(
(Aleurotrachelus socialis) attack
)
MEcu-72
Resistant genotype
Time course experiment
Time-course
Infested vs non-
infested
Non-infested Resistant
resistant genotype (replica 1, 2 3)
Cassava
Unigene set
micorarray
Time-course experiment
Eco-Efficient Agriculture for the Poor

18. Genotype used for Sequencing cassava genome
CIAT AM 560‐2: line selfed from MCOL 1505
CIAT AM 560 2 li lf d f MCOL 1505
MCOL 1505 has been extensively used by CIAT breeding project
Several derived lines released in Colombia and Thailand
Several derived lines released in Colombia and Thailand
MCOL 1505
Self‐pollination
S1
Self‐pollination
S2
Self‐pollination
Hernan Ceballos
S3: AM 560‐2
Eco-Efficient Agriculture for the Poor

19. Cassava Database Development
Genotype
information
ts
onent Phenotypic
Ph t i
information
Genome
compo
information
EST
information
Genetic
Genetic
DB c
information
Next activity
Genome browser development http://cassava.psc.riken.jp/index.p
www.cassavagenome.org
www cassavagenome org
Contact: Tetsuya Sakurai (RIKEN PSC)
Sarah Ayling (CIAT)
Eco-Efficient Agriculture for the Poor

20. Cassava transcripts mapped with reference to the
castor bean, poplar, grape,
and Arabidopsis genome sequences
d A bid i
http://cassava.psc.riken.jp/
Eco-Efficient Agriculture for the Poor

21. Needs for a High throughput SNP platform
SNP markers of carotenoid biosynthesis genes for cassava breeding
SNP markers of carotenoid biosynthesis genes for cassava breeding
← increase [lycopene]
Cloning at Freiburg of carotenoid biosynthesis genes in cassava revealed
Cloning at Freiburg of carotenoid biosynthesis genes in cassava revealed Illuminsa SNP
bead Express
allelic variation in one (of the two) Phytoene synthase (PSY)
This variation was only found in yellow cultivars and was absent from the
white cassava. SNP ILLUMINA platform being implemented at CIAT
white cassava SNP ILLUMINA platform being implemented at CIAT
Eco-Efficient Agriculture for the Poor

22. Cassava Tolerance to Postharvest Physiological
Deterioration (PPD)
D t i ti (PPD)
In 2009, CIAT evaluated several potentially
tolerant genotypes, scoring the roots 5, 10,
20, and 40 days after harvest Susceptible
checks
Clones from two different groups of
genotypes showed no symptoms of PPD for
extended days
t d dd
Consolidates trials planned to provide G X E
data
Populations for mapping being developed
Eco-Efficient Agriculture for the Poor

23. CIAT Plant Phenotyping Platform
Trait Phenotyping Platform Field Phenotyping Platform
Discovery/ QTLs /Gene markers
Trait , Gene, QTLs
Lead Identification Promising lines
Proof of concept
Validation G X E
Feedback to Genomics
Back to
Environmental Multilocation trials
IS
Impact
Impact Environmental
GI
Feed B
Characterization
BMP
Eco‐efficient Products
Eco-Efficient Agriculture for the Poor

24. CIAT Plant Phenotyping Platform
Automation to identify genotypes under water stress
yg yp
(>500 lines in 30 minutes)
CIAT
Customised
software
High
resolution IR
camera
Barcode
reader
Eco-Efficient Agriculture for the Poor

25. CIAT Biotechnology
Empowering breeding
Empowering breeding
Strategy: Biotechnology strongly integrated with breeding and
characterization of germplasm bank
characterization of germplasm bank
Goal: Agronomical superior genotypes with desirable genes associated
with abiotic, biotic stress, and nutrition
Teams working on gene discovery, phenomics, markers assisted
selection, bioinformatics and transgenics
Strong focus on very few traits with well defined target
Strong focus on very few traits with well defined target
Eco-Efficient Agriculture for the Poor

26. From cassava genomic‐phenomics tools
to breeding
b di
Challenges now that the genome has been sequenced:
• Allele and allele combinations characterization of breeding
lines for mining allelic diversity and for complex traits
lines for mining allelic diversity and for complex traits
• High thorough put SNP screening
• Urgent needs for bioinformatics capacities and phenomic
platforms
Integration of breeding‐genomics tools to
Integration of breeding genomics tools to
improve cassava faster
Eco-Efficient Agriculture for the Poor

27. Agricultural Sciences for the 21 Century
Biotechnology, Precision
Precision
Agriculture
Systems Biology Breeding
and Synthetic Genomics‐
Biology
Bi l Phenomics
Bioinformatics
and
Computational
Biology
l
Talents, capital, IP management
and public private sector partnerships will be a must
Eco-Efficient Agriculture for the Poor