Developing new tools for cassava,Cassava regeneration/transformation,Transcriptome Analysis

1. New tools for the characterization and
improvement of cassava
Contract Review
15 April 2009
I Ingelbrecht
IITA, Ibadan, Nigeria

2. 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

3. 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

4. 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

5. Work plan achievement
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

6. Developing new tools for cassava
Why?
- Contribute to IITA mission & community effort to enhance cassava R4D
- Develop tools that will be useful to IITA for various applications and also by other
groups; reduce dependence
How?
- Strengthen local skills & capacities; balance outsourcing and in house research
What?
- 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

7. 1.Transformation of farmer-preferred cassava
for CBSD resistance
Equatorial
Guinea Uganda
Kenya
ROC
DRC
Tanzania
Malawi
Zambia
Mozambique
CBSD devastating
CBSD damaging
CBSD reported
A transgenics approach for resistance to Potyviruses previously used and
grown commercially: eg papaya resistant to Papaya ringspot virus

8. Why target cassava landraces?
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
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?

9. Experimental Approach
- 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)

10. 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

11. Cassava regeneration/transformation
Explants; e.g. immature leaf lobes
Embryogenesis
Shoot organogenesis
Secondary
SE *
Primary
SE
Multiple shoots
Embryogenesis
Cotyledonary Friable embryogenic
stage SE * Callus *
Adventitious shoots
Embryos Protoplasts
Embryogenic
suspensions
Plantlets
Fig. modified from Zhang et al. 2006

12. Cassava genotypes
In use by
cultivar CBSD Origin
farmers
Albert S Tanzania ?
Kibaha S Tanzania Yes
TME 12 ND WCA Yes
TMS 96/0160 S IITA Yes
TME 117 T Nigeria Yes
TME 1 S Nigeria Yes
Kibandameno S ECA Yes

13. Somatic embryogenesis
SE produced for 7 genotypes: 3 landraces from ESA
3 landraces from WCA
1 IITA elite line
cv Albert TME12

14. Summary cassava regeneration responses
Explants; e.g. immature leaf lobes
Embryogenesis +/-
Shoot organogenesis
Secondary SE *
Primary
SE
++++ ++++
Multiple shoots
++++
Embryogenesis
++ Cotyledonary Friable embryogenic
stage SE * Callus *
Adventitious shoots
Embryos Protoplasts
+/- Embryogenic
+/- 0 - 40% suspensions
Plantlets ++ 20 - 40%
Fig. modified from Zhang et al. 2006 ++++ >90%

15. Genetic transformation
- 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:
LEAF ROOT STEM
STRONG
WEAK

16. Transgenic ‘Tokunbo’ in greenhouse

17. GUS expressed in leaf, stem and petiole

18. GUS expression in tuber and fibrous roots

19. GUS expression in propagated ‘Tokunbo’ transgenic
~70 clones tested:
1 14 28
13 27 41
expression remains stable and high in all
plants after ratooning

20. Also chimeric transgenics?

21. 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

22. - pCAMBIA2300 backbone
- Km gene for selection
- pCsVMV promoter cassette with polylinker for cloning gene of interest
p35S
pCsVMV
nptll
pING71 polylinker for cloning:
3’nos - GUS
9.5 kb
LB - virus resistance
RB - starch
- etc

23. p35S pCsVMV
GUS
nptll With GUS ORF for testing functionality
pOYE153 of the construct
3’nos
11.5
LB RB

24. pCAMBIA2301 pOYE153
Tobacco
Cassava

25. F1 transgenic tobacco plants
pOYE153 pCAMBIA2301
pScVMV drives higher expression levels compared to
p35S of pCAMBIA2301

26. p35S pCsVMV
Intron
3’nos
nptll pING71-IV RB
9.7 With intron sequence for RNAi constructs
LB (DSMZ)
p35S pCsVMV
CBSV-IR
pRAJ42 With CBSV Inverted Repeat
nptll 3’nos
11.1 for CBSD resistance
LB (IITA)
RB
+ 2 other constructs targeting different viral isolates

27. Status cassava landrace transformation for CBSD
- 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)

28. CBSD resistance is strain specific:
eg miRNA CBSV-Kenya in N benthamiana
Transgenic miRNA-Ke Empty vector miRNA-Ke No No
Infection CBSV-Ke CBSV-Ke CBSV-Moz CBSV-Ke No
R S!

29. 3. a 14K custom cassava long oligo array
a tool for gene discovery and
transcriptome analysis
Generic tool
reverse 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

30. Target traits
drought response
plant-virus interactions
cyanogenesis
other

31. Previously
- 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
Metabolism
10.2% Metabolism
10.2% Energy
Energy
25.7% 4.0%
25.7% 4.0% Cell growth, division DNA synthesis
Cell growth, division DNA synthesis
2.1% 2.1%
TranscriptionTranscription
6.1% 6.1%
Protein synthesis
Protein synthesis
Protein destination
3.5% 3.5% destination
Protein
Transport Facilitation
4.7% Facilitation
Transport
4.7% Cellular transport
11.2% Cellular transport
11.2% 3.2% Cellular Biogenesis
3.2% Cellular Biogenesis
3.1%
0.2% Cellular communication/signal
3.1% transduction
0.2% 3.1% Cellular communication/signal
1.6% transductionCell rescue, defense, death and
8.7% 3.1%
6.1% ageing
1.6% 0.1% 6.4% Cell rescue, defense, death and
8.7% 6.1% ageing
Ionic homeostasis
0.1% 6.4%
Ionic homeostasis
Cellular Organization

32. - long oligo array: Agilent platform
- Why Agilent?
* flexibility
* accessibility
- Workflow:
Design Microarray
selection
? Informatics Data
Probe Feature
Selection Biological extraction
question
Microarray Microarray
order scanning
Sample Hybrid-
preparation ization
Protocol

33. - 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

34. Design summary
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 Output: 13,865 unique probes ~ 14K
BC_poor 0 or ~ 25-50 % of cassava
transcriptome
Total Probes 13865

35. - Microarray selection
- Array architecture uploaded (eArray)
- 8x15K array format

36. - Hybridization and scanning
- Analysis: R Bioconductor

37. Transcriptome Analysis
A. ‘Diversity’: expression profiling of different cassava genotypes
GENOTYPE CHARACTERISTICS
TME 3 Landrace, CMD resistant, parent of mapping population
TME 117 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

38. 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

39. Genotype vs TC: dot plot: fold change vs adjusted P value

40. Virus infected vs healthy

41. Candidate gene lists
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

42. Description:
TMS 96/0160_ohneS - control
FDR=0.05; |Contrast|>=2
Fold
Clone ID Gene Name
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

43. 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

44. 4. Marker development
Objective
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

45. Workflow EST-SSR validation
Total number of EST sequences investigated: 18,166
Number of unigenes used for in silico identification of SSRs: 8,577
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%
PCR products with expected sizes Amplification of introns
> 500 bp
Eliminate
Screen on diversity panel

46. 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

47. SFR
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 21 22 23 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M
Used for PCR optimization + screen for P using diverse panel:
All 346 primer pairs PCR optimized and screened on SFR gels

48. Polymorhism & cross species transferability
SET 1 – panel 2 SET 3 – panel 2
M 20 (23%) M 26 (33%)
P 66 (77%) P 53 (67%)
NA 3 NA 11
NS 2 NS 2
LONG 6 LONG 3
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. M. M. Castor Leafy
epruinosa brachyandra glaziovii bean spurge
97% 94% 85% 87% 91% 15% 11%

49. Fluorescent genotyping (ABI3100)
On a subset of P markers
To estimate # allele and their sizes
To develop fingerprinting kit

50. Primer Primer # alleles
No name 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

51. DNA sequencing of alleles
Eg CT109
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
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

52. Eg TC31
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
Overall size range: 168-194
Cassava only size range: 168-182
Cassava only unique alleles 14

53. TC31 allele phylogenetic tree
MTai3
MTai4
TME117
TMS30572_ 1
TMS30572_ 2
TMS30572_ 3
MTai2
MTai1
TME7_4 cassava
TME7_6
TME7_3
TME7_1
TME7_2
TME7_7
TME7_8
TME7_5
Mglaz4
Mglaz5
Mglaz3
Mglaz1
Mglaz2
Mepru 1
Mepru 2
Mepru 3
Mepru 4 wild
Mbrac h5
Mbrac h3 manihot
Mbrac h1
Mepru 5
Mbrac h8
Mbrac h2
Mbrac h4
Mbrac h6
Mbrac h7
4.9
4 2 0
Nucleotide Sub stitutions (x100)

54. Conclusions
- In total ~180 new polymorphic SSR markers (SFR)
- EST-SSRs transferable to other Manihot species but less to
other genera in Euphorbs
TME117 TME419 M. M. M. Castor Leafy
epruinosa brachyandra glaziovii bean spurge
97% 94% 85% 87% 91% 15% 11%
- More than 2 alleles/genotype in the marker/genotype
combinations examined so far! all multigene
families or ploidy in cassava higher than generally accepted

55. Quality of science
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

56. 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)

57. Communications
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

58. • 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’

59. Capacity building
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

60. 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

61. Resource mobilization
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

62. 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)

63. Managing resources
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

64. • 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

65. Other professional activities
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

66. • 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

67. Personnel effectiveness
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

68. • 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

69. 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)

70. Acknowledgements
IITA, Nigeria
A Raji
O Oyelakin
B Odeseye
F Kolade
J Opabode
U Okechukwu
DSMZ, Germany
S Winter
KU, Denmark
S Bak
K Jorgensen
J Gorodkin
B Moller