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


3. Communication



6. Managing resources



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(unpublished):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


79013282 gb_CL198Contig2.1.KVL45FFC7CB0000019B 2.8

79014250 CK640993.1 2.9


79006640 DV447666.1 3.0


79011755 CK652281.1 3.1

79015613 BI325199.1 3.1


79006299 gb_CBSV_CP 4.9

https://ukbc-tal15.biochemie.med.uni-goettingen.de/evibank/cgi-bin/clone_annot.cgi?79002281?













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



3. Communication






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)



3. Communication






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’



3. Communication






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



3. Communication






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)



3. Communication






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



3. Communication






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



3. Communication






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

New tools for the characterization and improvement of cassava

Technology

cassava genotypes

cassava landraces iita

cassava r4d

farmerpreferred cassava

target cassava landraces

cassava root crop

transformation of farmer

change79002281 gb