New tools for the characterization and improvement of cassava
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Transcript of New tools for the characterization and improvement of cassava
New tools for the characterization and
improvement of cassava
Contract Review
15 April 2009
I Ingelbrecht
IITA, Ibadan, Nigeria
Outline
1. Work plan achievement
2. Quality of science
3. Communication
4. Capacity building
5. Resource mobilization
6. Project management
7. Other professional activities
8. Personal effectiveness
Cassava
Root crop; ranks 6th as source of
carbohydrates globally (163 M ton/yr)
Allopolyploid with disomic inheritance;
2n=36
C=700-800 Mbp
Vegetatively propagated
Grown in (sub)tropics of South Am.,
Asia, and Sub Saharan Africa;
introduced in SSA in 16th century
Cassava
• Resilient to adverse growth conditions (soil, drought)
• Adaptable to range of agroecologies
• Low maintenance
• High yield potential (80 ton/ha)
• Pest & disease: virus (CMD & CBSD), whitefly, other
• Highly heterozygous, vegetatively propagated
• Root: main use, has low nutritional value
• Long breeding cycle, shy flowering
+
-
1. Workplan achievement
2. Quality of science
3. Communication
4. Capacity building
5. Resource mobilization
6. Managing resources
7. Other professional activities
8. Personal effectiveness
Work plan achievement
- Contribute to IITA mission & community effort to enhance cassava R4D
Developing new tools for cassava
- Develop tools that will be useful to IITA for various applications and also by other
groups; reduce dependence
- Strengthen local skills & capacities; balance outsourcing and in house research
- Regeneration and genetic transformation protocols for African landraces
- A new vector for Agro-mediated transformation of dicots with derivatives
- A cassava-specific DNA microarray: a tool for reverse genetics/gene discovery
- EST-derived SSR markers for cassava
Why?
How?
What?
Uganda
Kenya
Tanzania
Mozambique
Zambia
DRC
ROC
Equatorial
Guinea
Malawi
CBSD reportedCBSD damagingCBSD devastating
1.Transformation of farmer-preferred cassava
for CBSD resistance
A transgenics approach for resistance to Potyviruses previously used and
grown commercially: eg papaya resistant to Papaya ringspot virus
All current transformation protocols are for ‘model’ genotypes, not
used by farmers or breeders in Africa: excellent research tool but
limited application in the field
Bottleneck since current protocols are highly genotype-
dependent
Develop protocol for (African) farmer-preferred lines
Why target cassava landraces?
Ongoing efforts on cassava landrace transformation
(unpublished):
- DDPSC, USA using FEC (based on Schopke et al., 1996)
- KU, Denmark using cotyledons (based on Li et al., 1996)
- CIAT, OSU, others?
- Produce SEs for cassava landraces (IITA)
- Develop new protocol for genetic transformation of cassava landrace
(using GUS reporter gene) (IITA)
- Determine CBSV sequence from viral isolates from different countries
(public domain; DSMZ)
- Make R-gene constructs; multiple constructs based on RNAi to aim for
resistance to different viral isolates (IITA; DSMZ)
- Test R-genes in N benthamiana (DSMZ)
- Transform farmer-preferred cassava using CBSD resistance gene(s)
(IITA)
Experimental Approach
Three basic steps in genetic transformation protocol:
1. In vitro shoot regeneration method
2. Gene transfer method: Agrobacterium-mediated
3. Selection and/or screening for transgenic shoots
Ideally, all steps are efficient (high % of success) and applicable to a range
of genotypes
Multiple shoots
Shoot org
anogenesis
Plantlets
Adventitious shoots
Primary SE
Em
bry
ogenesis
Em
bry
ogenesis
Explants; e.g. immature leaf lobes
Cotyledonary
stage SE*
Embryos
Friable embryogenic
Callus*
Embryogenic
suspensions
Protoplasts
Secondary
SE*
Cassava regeneration/transformation
Fig. modified from Zhang et al. 2006
cultivar CBSD OriginIn use by
farmers
Albert S Tanzania ?
Kibaha S Tanzania Yes
TME 12
TMS 96/0160
TME 117
TME 1
Kibandameno
ND
S
T
S
S
WCA
IITA
Nigeria
Nigeria
ECA
Yes
Yes
Yes
Yes
Yes
Cassava genotypes
cv Albert TME12
SE produced for 7 genotypes: 3 landraces from ESA
3 landraces from WCA
1 IITA elite line
Somatic embryogenesis
Multiple shoots
Shoot org
anogenesis
Plantlets
Adventitious shoots
Primary SE
Em
bry
ogenesis
Em
bry
ogenesis
Explants; e.g. immature leaf lobes
Cotyledonary
stage SE*
Embryos
Friable embryogenic
Callus*
Embryogenic
suspensions
Protoplasts
Secondary SE*
Summary cassava regeneration responses
Fig. modified from Zhang et al. 2006
+/-
++++
++++
++
+/-+/- 0 - 40%
++ 20 - 40%
++++ >90%
++++
- Optimized using GUS reporter gene from pOYE153
- Using organogenesis pathway with selection on Geneticin, recovered transgenic
TME12 aka ‘Tokunbo’ (TE<0.1%); transgenics with uniform expression levels
obtained:
Genetic transformation
LEAF ROOT STEM
STRONG
WEAK
Transgenic ‘Tokunbo’ in greenhouse
GUS expressed in leaf, stem and petiole
GUS expression in tuber and fibrous roots
GUS expression in propagated ‘Tokunbo’ transgenic
~70 clones tested:
1
13
14
27
28
41
expression remains stable and high in all
plants after ratooning
Also chimeric transgenics?
2. Transformation vector with CsVMV promoter
cassette
Objective
Develop new Agro transformation vector with two different,
constitutive promoters; CaMV 35S and CsVMV
Characteristics
• Generic vector, can be used for various traits in various dicot species:
- CBSD resistance - cassava
- starch modification - tobacco (N benthamiana)
- herbicide tolerance, etc
• Promoters are oriented towards the border sequences to reduce
unwanted gene silencing effects
• No repeats within the T-DNA to reduce gene silencing effects
nptllpCsVMV
pING71
9.5 kb3’nos
p35S
LB RB
- pCAMBIA2300 backbone
- Km gene for selection
- pCsVMV promoter cassette with polylinker for cloning gene of interest
polylinker for cloning:
- GUS
- virus resistance
- starch
- etc
pOYE153
11.5
pCsVMV
GUS
3’nos
p35S
LB RB
nptll With GUS ORF for testing functionality
of the construct
pOYE153pCAMBIA2301
Cassava
Tobacco
pOYE153 pCAMBIA2301
F1 transgenic tobacco plants
pScVMV drives higher expression levels compared to
p35S of pCAMBIA2301
nptll
nptll
pRAJ42
11.1
pCsVMV
CBSV-IR
3’nos
p35S
LB RB
With CBSV Inverted Repeat
for CBSD resistance
(IITA)
pING71-IV
9.7
pCsVMV
Intron
3’nos
p35S
LB
RBWith intron sequence for RNAi constructs
(DSMZ)
+ 2 other constructs targeting different viral isolates
- Produce SEs for cassava landraces (IITA) OK
- Develop new protocol for genetic transformation of cassava OK
landrace (using GUS reporter gene) (IITA; KU) (TME12)
- Determine CBSV sequence from viral isolates from different OK
countries (public domain; DSMZ)
- Make R-gene constructs; multiple constructs based on RNAi OK
to aim for resistance to different viral isolates (IITA; DSMZ)
- Test R-genes in N benthamiana (DSMZ) ongoing
- Transform farmer-preferred cassava using CBSD resistance ongoing
gene(s) (IITA)
Status cassava landrace transformation for CBSD
CBSD resistance is strain specific:
eg miRNA CBSV-Kenya in N benthamiana
Transgenic
Infection
miRNA-Ke
CBSV-Ke
miRNA-Ke
CBSV-Moz
No
CBSV-Ke
No
No
Empty vector
CBSV-Ke
R S!
3. a 14K custom cassava long oligo array
a tool for gene discovery and
transcriptome analysis
Generic toolreverse genetics (genotype phenotype)
complements QTL & association mapping approaches
cassava genome sequencing effort
trait improvement through genetic transformation
Applications
1. Understanding function of genes/alleles/gene networks
2. Understanding allelic differences between gene families/varieties
3. Diagnostics
Target traits
drought response
plant-virus interactions
cyanogenesis
other
- Normalized cDNA libraries produced from control and water
stressed tissues (leaf, root and stem tissue)
- 18,166 ESTs sequenced (5’end) and assembled in 8,577
unigene set with functional annotation
Previously
10.2%
4.0%
2.1%
6.1%
3.5%
4.7%
3.2%
3.1%
3.1%
6.1%6.4%8.7%
1.6%
0.2%
11.2%
25.7%
0.1%
Metabolism
Energy
Cell growth, division DNA synthesis
Transcription
Protein synthesis
Protein destination
Transport Facilitation
Cellular transport
Cellular Biogenesis
Cellular communication/signal
transduction
Cell rescue, defense, death and
ageing
Ionic homeostasis
Cellular Organization
10.2%
4.0%
2.1%
6.1%
3.5%
4.7%
3.2%
3.1%
3.1%
6.1%6.4%8.7%
1.6%
0.2%
11.2%
25.7%
0.1%
Metabolism
Energy
Cell growth, division DNA synthesis
Transcription
Protein synthesis
Protein destination
Transport Facilitation
Cellular transport
Cellular Biogenesis
Cellular communication/signal
transduction
Cell rescue, defense, death and
ageing
Ionic homeostasis
Cellular Organization
- long oligo array: Agilent platform
- Why Agilent?
* flexibility
* accessibility
- Workflow:
Protocol
Probe
Selection
Microarray
order
Design ?Microarray
selection
Sample
preparation
Hybrid-
ization
Feature
extraction
Informatics
Microarray
scanning
Biological
question
Data
- Design and probe selection
Input: ~ 40,000 cassava sequences:
* 18,177 in house ESTs
* ~ 5,000 ESTs from root specific library (unpublished)
* remainder from public databases (EST, genomic, etc)
* ACMV and CBSV ORFs; Km ORF
Unigene set established, orientation determined
Input Targets 14113
Targets with Probe 13865
Probe Length
Shortest Probe 60.0
Length Mean 60.0
Length SD 0.0
BC Scores (1 = good; 4= bad)
BC_1 13473
BC_2 360
BC_3 13
BC_4 19
BC_poor 0
Total Probes 13865
Output: 13,865 unique probes ~ 14K
or ~ 25-50 % of cassava
transcriptome
Design summary
- Array architecture uploaded (eArray)
- 8x15K array format
- Microarray selection
- Hybridization and scanning
- Analysis: R Bioconductor
GENOTYPE CHARACTERISTICS
TME 3
TME 117
Landrace, CMD resistant, parent of mapping population
Landrace, source of majority of ESTs
TMS 96/0160 IITA breeding line, adopted in DR Congo, CBSD suscep.
TMS 30572 IITA breeding line, widely adopted in SSA, CMD tol.
TMS 96/1089A IITA breeding line, resistant to CMD & CBSD*
Kibaha Tanzanian landrace, susceptible to CBSD
Albert Tanzanian cultivar, susceptible to CBSD
A. ‘Diversity’: expression profiling of different cassava genotypes
Transcriptome Analysis
B. Different growth conditions: greenhouse versus in vitro
(TMS 96/0160)
C. Healthy versus virus infected plant: ACMV and CBSV
Eg TME 4
CMD resist
CBSD suscept
Genotype vs TC: dot plot: fold change vs adjusted P value
Virus infected vs healthy
Setname Contrast Cut Off1
Short List TMS_96/0160_mitS-Control 0.05/2.0/1104
TMS_96/0160_ohneS-Control 0.05/2.0/168
TMS_96/0160_mitS-TMS_96/0160_ohneS 0.05/2.0/635
TME117_ohneS-Control 0.05/2.0/406
TME117_mitS-Control 0.05/2.0/937
1p-Value threshold/Contrast threshold/Number of candidates
Candidate gene lists
Description:
TMS 96/0160_ohneS - control
FDR=0.05; |Contrast|>=2
Clone ID Gene NameFold
change
79002281 gb_CL1576Contig1.1.KVL45FFC7CB0000... 2.6
79008123 gb_CBSV_6K2 2.6
79001735 BM260324.1 2.6
79011094 gb_CL1046Contig1.1.KVL45FFC7CB0000... 2.6
79013282 gb_CL198Contig2.1.KVL45FFC7CB0000019B 2.8
79014250 CK640993.1 2.9
79004173 gb_CL1734Contig1.1.KVL45FFC7CB0000... 3.0
79006640 DV447666.1 3.0
79004438 gb_CL1351Contig1.1.KVL45FFC7CB0000... 3.1
79011755 CK652281.1 3.1
79015613 BI325199.1 3.1
79009153 gb_CL1647Contig1.1.KVL45FFC7CB0000... 3.8
79006299 gb_CBSV_CP 4.9
Cassava Transcriptome Analysis - Summary
1. 14K Cassava-specific long oligo microarray developed
2. Microarray passed all QC, hybridization and detection limit is as expected
3. Results:
- Differential gene expression between varieties limited (~0.1% DEG)
- Profound effect of growth conditions on differential gene expression
- Sensitivity comparable or exceeds that of PCR: diagnostics tool
Number of markers for cassava limited; eg current map has
~ 400 markers; typically many 1000ds for non orphan crops
Contribute to the community effort to develop additional molecular
markers for cassava
• In silico identification of COS, SNPs and SSRs from EST unigene dataset
• 646 candidate EST-SSRs; duplicates with existing SSRs (CIAT collection)
eliminated; primers designed for 346 ESTs
• Candidate SNP markers + trace files provided to CBL colleagues
4. Marker development
Objective
Total number of EST sequences investigated: 18,166
Total number of unique SSR loci appropriate for primer modeling: 646 (3.3%)
Number of candidate SSR investigated : 346
PCR successful: ~ 90% Failed PCR: ~ 10%
Eliminate
PCR products with expected sizes Amplification of introns
Number of unigenes used for in silico identification of SSRs: 8,577
Workflow EST-SSR validation
Screen on diversity panel
> 500 bp
Two panels‘Africa’ panel: cassava elite lines and landraces from Africa
‘global’ panel: cassava from Africa, LA, Asia plus wild species,
and castor bean plus leafy spurge
Markers screened for polymporhism
Different levels of resolution:
SFR < PAGE < ABI3100 < DNA sequence
M1 2 3 4 5 6 7 8 9 10 1112 1314 15 16 1718 19 20 21 22 23 24
SFR
M 1 2 3 4 5 6 7 8 9 10 1112 1314 15 16 1718 19 20 21 22 23 24
Used for PCR optimization + screen for P using diverse panel:
All 346 primer pairs PCR optimized and screened on SFR gels
Polymorhism & cross species transferability
M 20 (23%)
P 66 (77%)
NA 3
NS 2
LONG 6
M 26 (33%)
P 53 (67%)
NA 11
NS 2
LONG 3
SET 1 – panel 2 SET 3 – panel 2
For set 1 and 3; a total of 119 markers are P
For set 3: 80 of 85 markers amplify wild Manihot species ~ 94%
13 of 85 and 9 of 85 amplify castor and leafy spurge resp. or 15 and 10%
TME117 TME419 M.
epruinosa
M.
brachyandra
M.
glaziovii
Castor
bean
Leafy
spurge
97% 94% 85% 87% 91% 15% 11%
Fluorescent genotyping (ABI3100)
On a subset of P markers
To estimate # allele and their sizes
To develop fingerprinting kit
Primer
No
Primer
name
# alleles
per locus Allele sizes Predicted size
4 AT27 2 168, 170 166
5 AT45 2 210, 213 212
6 AT47 7 128, 132, 136, 153, 155, 157, 175 154
8 AT101 8 147, 149, 152, 157, 158, 163, 160 157
12 AT158 7 200, 202, 205, 209, 210, 211, 225 208
13 AAG54 8 157, 158, 159, 161, 167, 170, 173 165
15 AGA49 6 183, 190, 193,196, 199,204 198
16 AGA87 3 184, 198, 200 199
18 AGA157 4 249, 252, 260 250
20 CT19 4 169, 174, 184 183
21 CT22 5 207, 209, 214, 222 212
24 CT65 8 216, 243, 247, 249, 251, 253, 247
25 CT75 4 188,191,192,197 190
26 CT83 7 139, 145, 146, 142, 147, 148, 149, 152 150
27 CT109 3 164,168, 170 169
28 CT118 4 188, 192, 200, 202, 201
29 CT129 9 192, 198, 200, 204, 208, 210, 212, 216, 218 201
30 CAT46 4 220, 223, 226, 229 229
32 CTT15 3 170, 173, 177 173
34 GCA94 2 177, 182 179
Genotype # clones sequenced # alleles Allele sizes
Nachinaya 4 3 169, 171
CM6740-7 6 5 169, 171
TMS30572 3 3 167, 169, 171
M epruinosa 5 5 166, 171
DNA sequencing of alleles
Eg CT109
Overall size range: 166-171
Cassava only size range: 167-171
Overall unique alleles: 12
Cassava only unique alleles 7
Overall allele sizes: 166, 167, 169, 171
Cassava only allele sizes: 167, 169, 171
Genotype # clones sequenced # alleles
TME7 8 7
MTai7 4 4
TMS30572 3 3
M glaziovii 5 4 or 5
M brachyandra 9 8
M epruinosa 5 5
Eg TC31
Overall size range: 168-194
Cassava only size range: 168-182
Cassava only unique alleles 14
Nucleotide Substitutions (x100)
0
4.9
24
MTai3
MTai4
TME117
TMS30572_1
TMS30572_2
TMS30572_3
MTai2
MTai1
TME7_4
TME7_6
TME7_3
TME7_1
TME7_2
TME7_7
TME7_8
TME7_5
Mglaz4
Mglaz5
Mglaz3
Mglaz1
Mglaz2
Mepru1
Mepru2
Mepru3
Mepru4
Mbrach5
Mbrach3
Mbrach1
Mepru5
Mbrach8
Mbrach2
Mbrach4
Mbrach6
Mbrach7
cassava
wild
manihot
TC31 allele phylogenetic tree
- In total ~180 new polymorphic SSR markers (SFR)
- EST-SSRs transferable to other Manihot species but less to
other genera in Euphorbs
- More than 2 alleles/genotype in the marker/genotype
combinations examined so far! all multigene
families or ploidy in cassava higher than generally accepted
TME117 TME419 M.
epruinosa
M.
brachyandra
M.
glaziovii
Castor
bean
Leafy
spurge
97% 94% 85% 87% 91% 15% 11%
Conclusions
1. Workplan achievement
2. Quality of science
3. Communication
4. Capacity building
5. Resource mobilization
6. Managing resources
7. Other professional activities
8. Personal effectiveness
Quality of science
Bibliography
- Six articles published in refereed journals
- One article in R4D Review, 2nd Ed.
- Cowpea transposon sequences submitted to NCBI, USA with Acc No.
- Eleven abstracts (9 with poster) at various meetings
- Five manuscripts in preparation for refereed journals
Invited presentations
- Six invited presentations at (inter)national meetings in Uganda, Mozambique,
Tanzania, Belgium and USA (2).
Paper review (external)
- Eight manuscripts for international Scientific Journals
- Two proposals for granting agencies (NSF, USA; AARI, Canada)
1. Workplan achievement
2. Quality of science
3. Communication
4. Capacity building
5. Resource mobilization
6. Managing resources
7. Other professional activities
8. Personal effectiveness
Communications
• Attended to CBL visitors (donors, collaborators, etc) with on
average one visit every 1 to 2 weeks
• Gave two interviews on agricultural biotechnology, to NTA and
BBC
• Wrote one article for ‘R4D review, 2nd Ed (2009); provided
inputs for a second
• Contributed to DVD on IITAs R4D program:
‘Award winning Research for Development’
1. Workplan achievement
2. Quality of science
3. Communication
4. Capacity building
5. Resource mobilization
6. Managing resources
7. Other professional activities
8. Personal effectiveness
Capacity building
Trained 4 undergraduate students and 6 graduate students (4 MSc and 2 Phd)
Trained 3 technical staff in various biotechnologies
Hosted 4 external professionals for hands-on capacity building
Resource person at workshop on ‘Recent Advances in the Applications of
Molecular Markers in Tropical Agriculture’ and invited the WABWS to jointly organize
this workshop with IITA
Lab Safety Training: 59 lab users attended the CBL Lab Safety Training
between June 2006 and March 2009; also used at NRCRI, Nigeria
Organized training course on ‘Working with radioactive chemicals’ at IITA with
external resource people from the NNRA
1. Workplan achievement
2. Quality of science
3. Communication
4. Capacity building
5. Resource mobilization
6. Managing resources
7. Other professional activities
8. Personal effectiveness
Resource mobilization
Projects funded
• BioCassava Plus. Supplemental Grant. Bill and Melinda Gates Foundation. With Dr Maziya-Dixon. 2008-2010
• Cassava genetic transformation for the longevity of cassava brown streak resistance in Tanzania. Partners: IITA-Tanzania; Mikocheni Agricultural Research Institute, Tanzania. RF. 2007-2009. With Drs Herron, Ndunguru
Linking phenotypes with genotypes: development and validation of a genome-wide DNA microarray as a reverse genetics tool in cassava (Manihot esculenta L Crantz). 2009 IITA Opportunity Grant. With Drs Gedil, Raji, Hearne and Franco
Proposal submitted
Enhancement of iron and zinc contents of cassava (Manihot esculenta Crantz) by soil bacteria and bacterial secondary metabolites. With ETH, Switzerland
(Additional 5 proposals or CN submitted; not funded/considered)
1. Workplan achievement
2. Quality of science
3. Communication
4. Capacity building
5. Resource mobilization
6. Managing resources
7. Other professional activities
8. Personal effectiveness
Managing resources
• Established charge back system in CBL through bench fee and
service charges for cost recovery as recommended by the IITA
admin ($ 67,861 recovered for 2007-2008)
• Streamlining of procurement and inventories in CBL; worked with
Supply Chain for inventory of the CBL chemical and supply
stores
• Balanced special projects budgets
1. Workplan achievement
2. Quality of science
3. Communication
4. Capacity building
5. Resource mobilization
6. Managing resources
7. Other professional activities
8. Personal effectiveness
Other professional activities
• Assistant Editor: In Vitro Cellular and Developmental Biology – Plant
• Ad hoc reviewer of papers for international scientific journals and
granting agencies such as NSF, USA.
• Member RDC
• Collaboration with IITA Genebank, MARI-Tanzania and WARDA, Benin
1. Workplan achievement
2. Quality of science
3. Communication
4. Capacity building
5. Resource mobilization
6. Managing resources
7. Other professional activities
8. Personal effectiveness
Personnel effectiveness
• Manage facilities, oversee procurement and inventory of common items and the CBL support staff:
• hold regular lab meetings with CBL scientists
• regular updating of booklet ‘Operational Guidelines of CBL’
• re-established a hot lab facility and renewed license for use of radiochemical at IITA with support from DDG-Support and IITA Safety committee
• jointly with colleagues, developed draft plan for CBL refurbishment
• CBL has been accident-free with enabling environment for biotech research
• Act for IITA admin when requested
• Timely response to requests for inputs by CGO, PPS, Supply Chain and IITA admin
Member of IITA’s procurement committee
Future planning
- Use tools for product development; move from ‘output’ to ‘outcome’
eg - cassava landrace with useful traits via genetic transformation
- microsatellite-based fingerprinting kit for the characterization of
cassava genetic resources
- Expand role of the Biotech Lab in Ibadan to serve as a research center
for national programs, other institutions (beyond traditional IITA
mandate crops)
IITA, NigeriaA Raji O OyelakinB OdeseyeF KoladeJ OpabodeU Okechukwu
DSMZ, GermanyS Winter
Acknowledgements
KU, DenmarkS Bak
K Jorgensen J Gorodkin B Moller