CBT in Malaria and Cassava Research addresses 6/8 MDGs

Thomas G. Egwang, PhD

ED, African Academy of Sciences, Nairobi, Kenya

DG/SD, Med Biotech Laboratories, Kampala, Uganda

Science and Technology for Sustainable

Development in Africa

AAAS Boston 17th Feb 2008

Cassava Food to> 500 million

Roots high in caloriesLeaves :Vitamins A and BStalks : propagation

Hardy and drought-resistantPoor soils, marginalized landNo fertilizers/pesticides

Animal feed Industrial applications

Sugar, acetone, alcohol

Food security to the poor

Cassava Family:EuphorbiaceaeGenus: ManihotGenus: Manihot esculentaPloidy: Allotetraploid (n=9)Genome size: 770MbpCassava Genetic Resources: -5700 acc. (CIAT Gene Bank) -2,000 (IITA Gene bank) -2,500 (EMBRAPA)98 wild Manihot relativesMonoecious Dicot

Source: FAOSTAT

Cassava: A ‘Shining Example’ of African Agriculture

Production in Tons/Ha of the three principal producing regions

2004

Important issues

• Diseases– Cassava Mosaic Disease (CMD)

• Starch content– Amylose versus amylopectin content

• Bitter taste: bitter strains, sweet strains• Glycoside of HCN• V. bitter forms > 100 pmm cyanide

S&T and cassava issues

• Molecular markers for breeders

• Transgenic cassava– modified starch content: low amylopectin

The Cassava Mosaic Disease (CMD)

A viral disease endemic in SS-Africa

Vectored by the white fly (Bemisia tabaci)

Est > $1 B/y Control: development of CMD

resistant varieties: 6-10 years Molecular markers

CMD-resistant CMD-susceptibleMultiple resistance to diseases and pests

Improved multiple disease and pest resistant clones showing high levels of resistance to CMD

Multiple Sources of Resistance to CMD

Targeted training in Cassava Genomics

Elizabeth Balyejusa KizitoElizabeth Balyejusa KizitoYona BagumaYona Baguma

Scientists at NARO (Uganda)-developingSLU

CIAT (Colombia) -advanced

Cassava Biotechnology Research: CBT needs

Tissue Culture

Genetic Transformation

Embryo rescue

Micro-propagation

Tissue Culture

Conservation and exchange

Molecular Markers

BAC Libraries

BAC Clone screening and fingerprinting

Genetic Markers Positional Cloning

Genomic/cDNALibraries

Genetic Mapping

QTL Mapping

Molecular Breeding

Applications of Molecular Markers in Cassava

BreedingIncrease heritability in the identification of

superior genotypes Follow specific chromosome segments from

unadapted wild relatives in breeding schemesReduce population sizes for more precise

field evaluationsDelineate heterotic pools or to choose

parents that maximize additive varianceAccelerate inbred line development

Molecular Marker Resources for Cassava at

CIAT 5 small insert (1-2kb) genomic libraries3000 low-copy RFLP marker probes3 genomic SSR sequence enriched libraries16 partial and full-length cDNA libraries 850SSR markers from genomic and cDNA libraries Two diversity array technology (DArT) chip with 1500

and 10,000 polymorphic clones respectivelyUnigene chip of 6,000 ESTsWeb-based data base www.cassavagenome.org

being prepared for easy access to these global public goods (GPG)

HDist MarkercM Name

GY8513.1

OJ1

20.4

SSRY521.6SSRY1211.2SSRY48

14.5

rGY19912.6

rGY17010.3

rGY1046.4

GY127

19.7

rGY211

19.7

rSSRY91

24.3

SSRY178

16.9

SSRY6311.2

SSRY164

rSSRY31

Dist MarkercM Name

rSSRY527.2

rSSRY12

12.4

rQ11

19.0

rGY99

11.1

rGY8-1

12.0

GY85-19.1

nGY116.0

rSSRY178

37.4

rSSRY164

H

ADist MarkercM Name

ACP-19.2

rGY208

15.7

SSRY98

11.4

rGY759.3

GY128.2

GY286.8

GY2135.1

GY209

rSSRY4

Dist MarkercM Name

rSSRY4

16.4

rGY22-1

19.5

rGY75-17.3

rSSRY988.8

rBEST-28.8

GY687.0

GY288.9

GY32

A

Dist MarkercM Name

rGY1553.9rGY192

9.5GY154

14.6

rSSRY111

15.3

SSRY591.8SSRY10

11.1

rGY215

19.3

L7

28.1

AC-1

M

CDist MarkercM Name

rGY1442.5rGY232.5GY816.2rGY89

14.0

rGY177

12.0

O11a

16.1

GY174

9.8

rSSRY1836.4

rSSRY454.4

rSSRY95

17.8

rSSRY1095.8

SSRY32

Dist MarkercM Name

rK9a

20.1

rNI1.C27.6

GY23-1

13.4

rGY182.8rGY26

8.2rGY89-1

8.2SSRY171

9.5

GY429.5

GY447.0

GY546.2

GY81-1

CDist MarkercM Name

rA16

13.2

rGY191

17.5

rV17a6.5

GY1976.7

rGY1475.6

rSSRY50

26.0

GY207

BDist MarkercM Name

rAi14a

17.2

GY72

10.9

rSSRY619.4

rGY655.0

rPASK1

17.1

SSRY1131.8SSRY21

BDist MarkercM Name

rF19b

15.0

rGY118

12.7

GY217

11.0

rS24.4

rSSRY161

11.6

rGY176

20.3

rGY190

E

SSRY84

Dist MarkercM Name

rSSRY180

17.2

GY83

28.7

GY1712.3

SSRY7211.0

nGY162

15.6

rAE15

23.7

GY58

21.1

CDY123a11.5

GY11810.0

rI4a

17.5

G13

E

SSRY84

DDist MarkercM Name

rGOT-28.8

rGY1675.0

rGY1801.2GY2221.2GY1811.2GY423.7GY2193.7GY503.7GY1253.7GY1793.8rON17.0SSRY30.0SSRY1203.6rSSRY40

SSRY9

D

rSSRY169

10.6

GY4

14.8

SSRY1087.6

rGY246.5

rGY597.3

GY78

Dist MarkercM Name

rGY67H

15.8

rI18b

20.1

rJ1a

19.0

rGY57

20.5

rGY25 SSRY9SSRY120SSRY23

SSRY3

Dist MarkercM Name

rSSRY55

10.7

rJ72.5rG11

20.7

GY1108.9

rSSRY1007.9

SSRY88

19.2

Ai11b

LDist MarkercM Name

rSSRY55

19.4

N15b

12.1

GY537.4

rSSRY888.0

rGY110

LDist MarkercM Name

rSSRY172

22.0

rE14b

11.9

rJ9c7.5

rCDY744.4

CDY767.5

AD1c

14.4

K2a

27.0

GY349.4

SSRY1018.6

SSRY2 SSRY5

J

SSRY47

Dist MarkercM Name

O20

11.3

AE10a

11.8

CDY76

17.1

rSSRY172

10.9

rM8b9.5

GY7

10.6

GY34-1

25.9

K10

J

Dist MarkercM Name

rGY93

16.9

GY781.2GY976.8SSRY1352.5rSSRY30

22.4

rSSRY387.8

rGY41

16.7

CYP79(152)

GGDist MarkercM Name

rSSRY135

23.4

rGA-13110.4

rGY948.8

AM1812.6

rK16d13.3

rSSRY38

20.8

rCPY79(152)7.8

rGY137

23.5

AB9a

24.8

SKDH-114.6

rAGPaseB16.0

rSSRY1068.2

rSSRY10315.2

SSRY83

rSSRY30

FDist MarkercM Name

GY1966.4

GY2035.1

GY2183.8GY122

7.5CDY1073.7GY372.5GY2042.5GY1945.0GY186

5.8SSRY179

17.0

SSRY68

rSSRY31

Dist MarkercM Name

Z11b

12.5

GY2111.5

SSRY6811.4

GY376.7

rCDY107-110.1

CDY123b4.6 GY80

21.0

rV20a

28.3

H14c

20.3

GY16

23.0

rGY55

F

I

rAC1

11.1

GY130

11.9

R1

13.6

rW3

19.4

rGY201-1a

13.1

rD5a

11.5

rB3a

H14b

15.0

GY128

17.4

GY143

cMDist Marker

Name

I

CDY29

22.6

CDY68

12.1

CDY128

rGY19

10.0

rSSRY8

14.5

rS12c

23.8

rU2a

MarkerNamecM

Dist

Dist MarkercM Name

rSSRY122

37.0

nrGY673.9rGY363.0rSSRY86

9.4SSRY105

13.3

rGY87

15.6

W19b8.8

GY1055.9

SSRY1778.4

GY55.7

SSRY99

14.6

rK3

UDist MarkercM Name

K17

18.0

SSRY67.3

SSRY1229.0

SSRY42

18.7

rSSRY86

34.6

GY1382.5rGY1643.7GY2234.5SSRY170

U

SSRY105

Dist MarkercM Name

CDY131-16.5

rGY113H6.3

rGY927.4

SSRY1852.3SSRY20

21.5

U2c

XDist MarkercM Name

rM5a7.0

CDY131

13.5

nCBB

10.0

rSSRY208.8

rAF14a

13.3

SSRY110

X

N

rCDY99

11.9

rGBSSII

8.6SSRY13

11.1

rCDY445.6

rSSRY518.4

GY206.1

rGY106.8

GY1086.2

GY525.3

GY13

14.6

Z18b

GY88

17.3

rGY474.0nGY143

cMDist Marker

Name

N

rGY109

9.4

rGY1485.3

rGY1452.4

GY201-1b

11.9

SSRY13

18.8

AD4b

SSRY35

6.7

rGY1085.1

GY182

GY 124

MarkerNamecM

Dist

Dist MarkercM Name

rAF17

19.4

SSRY1077.2

GY1717.6

rGY2105.5

ri4b

11.0

rA18

GY131

13.9

rSSRY83

MarkerNamecM

DistDist MarkercM Name

GY66

13.2

rSSRY34

9.9

SSRY1022.6SSRY1825.4GY39

28.6

AM6

Dist MarkercM Name

rGY1157.9

rGY9

15.6

rGY1

16.1

rSSRY28

11.3

Ai19

PO2

16.8

SSRY7

MarkerNamecM

Dist

F19a4.4

AC11a3.8

GY711.2

GY18312.8

i16b

RMarkerNamecM

Dist

V

J13c

8.7

GY51

25.0

F15b

18.6

rSSRY19

12.1

rG45.4

rK11c7.5

rAC57.6

rR13b

MarkerNamecM

DistV

rP1a

10.9

rGY156

21.2

GY134

9.9

rGY220GY63

6.0rSSRY19

16.0

rAC9a

MarkerNamecM

DistR

GY100

31.4

GY71

25.9

rGY48

12.3

AE2

24.8

U1

MarkerNamecM

Dist

rGY163

16.4

rGA-127

15.2

CDY106

27.8

rSSRY49

12.4

rGY119

KDistcM

MarkerName Dist Marker

cM Name

rGY117

17.3

rSSRY1751.5rSSRY856.9GY82

8.3CDY106

14.0

W16b

14.1

AC9b

10.0

GY119

19.0

rI2

K

SSRY174rSSRY82

Cassava Map 2004: 202 SSRs, 307 RFLPs, 100 RAPDs, 3 isozymes

RFLP Marker Segregation in an F1 Cross of Non-inbred

Parents

Three different CMD Resistance genes have been mapped and Can now be pyramided using molecular markers

Dist MarkercM Name

rGY1157.9

rGY915.6

rGY116.1

rSSRY2811.3

Ai19

CMD2

R

Gene Tagging of Multiple Sources of CMD Resistance Genes

DY66

18.5rI18b

20.5rJ1a

20.0rGY57

21.0rGY25

21.2SSRY9

23.9

SSRY316.2

SSRY23CMD1CMD1

Fregene et al. 2000

Akano et al. 2001

M

SSRY182

SSRY102

24.4

SSRY230

16.6

Ns9059.1

GY3911.2

SSRY299

NS170

SSRY182

SSRY102

24.4

SSRY230

16.6

Ns9059.1

GY3911.2

SSRY299

SSRY102

24.4

SSRY230

16.6

Ns9059.1

GY3911.2

SSRY299

CMD3

Hurtado et al. 2007

High resolution map around CMD2

A C

250

R A G G G T C A A A

260

A C M A A A G C T T

270

C G A T C C C C G C

280

C A A A C C C T A T

290

T G T T G G T A C T

300

A G T T C A C G G G

310

T C A T T C T G C T

320

A T G C A G G A T

Candidate BAC Sequence

Contigs of BAC clones around theCMD2 locus

Positional Cloning of CMD2

Susceptible to CMDHigh Starch

TMS30555

Female Parent

1288 F1s

Resistant to CMD(CMD2 gene)

X

Male ParentTME3

HindIII partialdigestion

Purification andcloning

Ligation and Sizing of clones

Cloning vector

388kb

194kb

48.5kb

Size selection

Sizing of clones

3 BAC libraries with 5X, 10X, and 11X coverage of the genome respectively

Clone picking byRobotics

Mega base pair-sized DNA in Agarose Plugs

Cassava BAC Libraries

97kb

6.9kb

48.5kb

BAC Clone Fingerprinting

Digestion with Hind III

Construction of a Contig Around CMD2

CM

D2

NS

15

8

RM

E--

1

RM

E--

2

SS

RY

-28

BA

C 3

3

Contig construction and genetic mapping of the ends of a BAC cone (BAC-33b)

4cM7cM

Bringing the Benefits of Modern Science to Farmer’s

FieldsMolecular markers can help reduce breeding populations

Proof of concept: Farmer participation demonstrate quickly superiority of new germplasm

Social scientists/mol biologists

Two scientists

• Elizabeth Balyejusa: molecular markers– PhD – Grant awards– MBL and NARO

• Yona Baguma: modified starch content– PhD– Heading Biotechnology Division, NARO

CIAT-Outstanding Principal Staff Achievement Award (OPSA) granted to Segenet Kelemu

CIAT-Outstanding Young Scientist-of-the-Year Award (OYSYA)granted to Marcela Quintero.

CIAT-Outstanding Research Publication Award (ORPA) , granted to Elizabeth Balyejusa Kizito, Linley Chiwona-Karltun, Thomas Egwang, Martin Fregene, and Anna Westerbergh.

Achievement Award in Bean (Phaseolus) Improvementgranted by the Bean Improvement Cooperative (BIC) to Matthew Blair.

Hereditas. 2007 Sep;144(4):129-36

Quantitative trait loci controlling cyanogenic glucoside and dry matter content in cassava (Manihot esculenta Crantz) roots

Balyejusa Kizito E, Rönnberg-Wästljung AC, Egwang T, Gullberg U, Fregene M, Westerbergh A.

Genetic diversity and variety composition of cassava on small-scale farms in Uganda: an interdisciplinary study using genetic markers and farmer interviews.

Balyejusa Kizito E, Chiwona-Karltun L, Egwang T, Fregene M, Westerbergh A.

Genetica. 2007 Jul;130(3):301-18. Epub 2006 Nov 3.

Plant Science 164: 833-839, 2003

Expression patterns of the gene encoding starch branching enzyme II in the storage roots of cassava (Manihot esculenta Crantz)

Baguma Y, Sun C, Ahlandsberg S, Mutisya J, Palmqvist S, Rubaihayo PR, Magambo MJ, Egwang TG, Larsson H, Jansson C. .

Malaria

Malaria kills 110 Ugandan children <5 years old annually

Pregnant women (1st pregnancies) bear the brunt:

•Poor pregnancy outcomes

•Maternal anemia

•Maternal deaths

Malaria controlDrug treatment: RESISTANCE

Insecticide treated bed nets: RESISTANCE

Vector control IRS: RESISTANCE; LOGISTICS

Vaccines: future ray of hope

Targeted training

•Malaria drug resistance monitoring

•Malaria vaccine studies

Malaria vaccines Activity phase

VaccineType

Development Clinical trials Total

PreErythrocytic 13 4 27

Asexual 37 9 46

Transmission-Blocking

2 1 3

Combination 6 2 8

Total 58 16 74

Merozoite

RBC membrane

SERA

Localization of SERA (serine repeat antigen) in P. falciparum infected RBC by immuno-gold staining

Longitudinal study School children Age 7-16 years

Study Populations

Case-control study of severe & mild malaria Age 6-59 months

Apac, Uganda transmission : perennial

After processing

Before processing

NS H

Recombinant proteins

Serine repeats (35)23 191-225

I III II

17 382

△193-225

P47 P50 P18

SE47’

SE36

989

P6

図 1

P50A

0 1 2 3 4

0

2

4

6

8

10

12

14

0 1 2 3 4

0

2

4

6

8

10

12

14

0

1

2

3

4

0

0.5

1.0

2.0

1.5

Correlation between anti-SERA IgG and malaria symptom or blood parasitemia

No Fever < 37.5 ℃

Fever> 37.5 ℃

anti-SE47' IgG3 (OD at 405 nm)

anti-SE50A IgG1 (OD at 405 nm)

Anti-SERA IgG and Fever Anti-SERA IgG and blood parasitemia

Pa

ras

ite

mia

(1

00

0／

μl

blo

od

)

No Fever < 37.5 ℃

Fever> 37.5 ℃

An

ti-S

E4

7' I

gG

3 E

LIS

A O

D

An

ti-E

50

A I

gG

1 E

LIS

A O

D

Anti-SE47’ Anti-SE50A Anti-SE47’ Anti-SE50A

Mean ODs for IgG subclass responses to SE47’ and SE50A as a function of age in Apac District, Northern Uganda

IgG 1 IgG 2 IgG 3 IgG 4

11-

14

0

2.0

1.5

1.0

0.5

0

1.0

0.75

0.5

0.25

Anti-SE47' IgG Anti-SE50A IgG0

- 1

2 -

5

15 -

20

6 -

8

21 -

30

9 -

10

31 -

40

> 4

0

11-

14

0 -

1

2 -

5

15 -

20

6 -

8

21 -

30

9 -

10

31 -

40

> 4

0

Age group Age group

EL

ISA

OD

EL

ISA

OD

Parasite growth inhibition by human serum

75

50

25

0

Honduras ｰ 1

FCR3

Par

asit

e G

row

th i

nh

ibit

ion

(%

)

IgG3 ELISA titer against SE47’

105102 103 104

K1

Patient Serum

Parasite Growth Inhibition Assay after 24 h.

Parasite culture

Recombinant 47’ neutralizes the inhibitory effect of patient serum on in vitro parasite

growth

SE50A

SE47’

SE36

Concentration of recombinant protein for neutralization (μg/ml)

50

40

30

20

10

0Pa

ras

ite

Gro

wth

Inh

ibit

ion

(%

)

0 10 20 30 40

8

SMSM

MM

Median SE36 titre compared with level of pigmented leucocytes

IQR 7,000 6,500 1,500

GMP production facilities for SE36 located in the Kanonji institute of The Research Foundation for Microbial Diseases of Osaka University

(BIKEN)

Production of malaria vaccine SE36 under GMP environments for clinical trials

P. falciparum: chloroquine resistance

Countries with at least one study indicating chloroquine total failure rate > 20%

No recent data available

Countries with at least one study indicating chloroquine total failure rate > 10%

P. falciparum: mefloquine treatment failure

Countries with at least one study indicating mefloquine total failure rate > 20%

No failure reported

Mefloquine total failure rate < 10%

No recent data available

Countries with at least one study indicating mefloquine total failure rate > 10%

P. falciparum: sulfadoxine-pyrimethamine

resistance

Countries with at least one study indicating sulfadoxine-pyrimethamine total failure rate > 20%

No failure reported

Sulfadoxine-pyrimethamine total failure rate < 10%

No recent data available

Countries with at least one study indicating sulfadoxine-pyrimethamine total failure rate > 10%

P. vivax: chloroquine prophylactic

or treatment failure

P. vivax prophylactic or treatment failure

Sample ID

Molecular monitoring of drug resistance

-Finger prick blood spotted onto filter paper

-Sample was air-dried

-Storage at room temperature

-Shipment in luggage or by regular mail

-DNA extracted by simple methods for molecular analysis

Mutation Sites in the PfCRT Transmembrane Protein

NH2

COOH

K76

N75M74

C72

H97

A220

N326

Q271 R371

I356

TCRP1

TCRP2

TCRP3

TCRP4-wTCRP4-m

Nested Mutation Specific PCR of pfcrt

Primary amplification PCR1: TCRP1 + TCRP2

Diagnostic PCR PCR2: TCRP2 + TCRP4-w or TCRP4-m

K76T

M S R S R S R S R S R

ctrl H2ODd2 3D7 106/1

pfcrt diagnostic PCR

Molecular Diagnosis of Chloroquine Resistance in the Field

PCR detection of pfcrt K76T polymorphisms

P1

P2

P3

P4-wP4-m

D1

D2

Apo1

DIAGNOSTIC PCR PCR1: P1+P2 PCR2: P3+P4-w or P4-m

RESTRICTION PCR1: P1+P2PCR2: D1+D2Digestion with Apo1

Restriction digestion analysis of pfcrt K76T

M 1 2 3 4 5 Dd2 3D7 C M

300bp

Distinguishing Recrudescent from reinfection with Microsatellite ta99

6 9 19 33 35 53 Dd2 Hb3 CM

Pteridine

Dihydrofolate

Tetrahydrofolate

pABA dihydropteorate synthase(DHPS)

Thimidine, Methionine synthesis

dihydrofolate reductase(DHFR)

Pyrimethamine (PM)

Sulfadoxine (SD)

GTP

Mode of action of antifolates

Relevance of Malaria Studies to MDG

•Change in CQ/SP drug policy

•Phase I-II trials of SERA5

•IPTp assessment of Coartem-on-going

•Collaboration with Control Pgm

Targeted CBT to meet MDGs• Cassava: Molecular markers

– Elizabeth Bayejusa Kizito

• Cassava: Modified starch content– Yona Baguma

• Malaria: Vaccine studies– Brenda Okech

• Malaria Drug resistance– Anne Nalunkuma– Connie Agwang

Female scientists trained• Elizabeth Balyejusa Kizito

– PhD Ag biotech– Best CIAT paper 2006– Woman science fellowship-USA 2007

• Brenda Okech– PhD Malaria vaccine studies– Career development award TDR/GSK– Management of malaria vaccine clinical trials– UK and Japan

• Anne Kazibwe: MSc malaria drug resistance– PhD drug resistance

• Connie Agwang: DipBSc drug resistance– Best AMANET student paper 2006

• Prossy Namuwulya– Short term training Plasmodium transfection: LUMC

Gender equality/equity issues addressed?

Not entirely

Other training in malaria• Group workshops

– Bioinformatics 2002 HHMI– Drug resistance genotyping MIM/DTR, IAEA– Molecular biology primer WHO /TDR– > 70 Africans from> 12 countries

• IAEA training for Latin America– Panama, Colombia, Peru

• Malaria/HIV interactions– Mentored clinical fellow under ILA EGPAF– International Scholars Award EGPAF

• Media malaria advocacy and outreach– Uganda Media for Health (UM4H)– Malaria Consortium

CBT in Malaria and Cassava Research addresses 6/8 MDGs

Science and Technology for Sustainable

Development in Africa

Acknowledgement• HHMI• Elizabeth Glaser

Pediatric AIDS Fdn• WHO/TDR• SIDA/SAREC• IAEA• MIM/TDR• NIH• EU• Govt of Japan• Malaria Consortium

• NARO• CIAT• MBL staff • Martin Fregene• Ann Westerbergh• Urban Gullberg• Christian Johannson• LindleyChiwona-Karltun• Eleanor Riley• Lisa Ranford-Cartwright• Cally Roper• Toshihiro Horii• Andy Waters• Christine Clayton• Alan Thomas

Thank you