1. 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

2. Cassava Food to> 500 million Hardy and drought-resistant
Roots high in calories
Leaves :Vitamins A and B
Stalks : propagation
Hardy and drought-resistant
Poor soils, marginalized land
No fertilizers/pesticides
Animal feed
Industrial applications
Sugar, acetone, alcohol
Food security to the poor

3. Cassava Family:Euphorbiaceae Genus: Manihot Genus: Manihot esculenta
Ploidy: Allotetraploid (n=9)
Genome size: 770Mbp
Cassava Genetic Resources:
-5700 acc. (CIAT Gene Bank)
-2,000 (IITA Gene bank)
-2,500 (EMBRAPA)
98 wild Manihot relatives
Monoecious Dicot

4. Cassava: A ‘Shining Example’ of
African Agriculture
Production in Tons/Ha of the three principal producing regions
Source: FAOSTAT

5. 2004

6. Important issues Diseases Starch content
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

7. S&T and cassava issues Molecular markers for breeders
Transgenic cassava
modified starch content: low amylopectin

8. 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

9. Multiple Sources of Resistance to CMD
Multiple resistance to diseases and pests
CMD-resistant CMD-susceptible
Improved multiple disease and pest resistant clones showing high levels of resistance to CMD

10. Targeted training in Cassava Genomics
Elizabeth Balyejusa Kizito
Yona Baguma
Scientists at NARO (Uganda)-developing
SLU
CIAT (Colombia) -advanced

11. Cassava Biotechnology Research: CBT needs
Tissue Culture
Genetic
Transformation
Embryo rescue
Micro-propagation
Conservation
and exchange
Molecular Markers
BAC Libraries
BAC Clone screening
and fingerprinting
Genetic Markers
Positional Cloning
Genomic/cDNA
Libraries
Genetic Mapping
QTL Mapping
Molecular Breeding

12. Applications of Molecular Markers in Cassava Breeding
Increase heritability in the identification of superior genotypes
Follow specific chromosome segments from unadapted wild relatives in breeding schemes
Reduce population sizes for more precise field evaluations
Delineate heterotic pools or to choose parents that maximize additive variance
Accelerate inbred line development

13. Molecular Marker Resources for Cassava at CIAT
5 small insert (1-2kb) genomic libraries
3000 low-copy RFLP marker probes
3 genomic SSR sequence enriched libraries
16 partial and full-length cDNA libraries
850SSR markers from genomic and cDNA libraries
Two diversity array technology (DArT) chip with and 10,000 polymorphic clones respectively
Unigene chip of 6,000 ESTs
Web-based data base being prepared for easy access to these global public goods (GPG)

14. Cassava Map 2004: 202 SSRs, 307 RFLPs, 100 RAPDs, 3 isozymesH
Dist
Marker cM
Name
GY85
13.1
OJ1
20.4
SSRY52
1.6
SSRY12
11.2
SSRY48
14.5
rGY199
12.6
rGY170
10.3
rGY104
6.4
GY127
19.7
rGY211
rSSRY91
24.3
SSRY178
16.9
SSRY63
SSRY164
rSSRY31
rSSRY52
7.2
rSSRY12
12.4
rQ11
19.0
rGY99
11.1
rGY8-1
12.0
GY85-1
9.1
nGY11
6.0
rSSRY178
37.4
rSSRY164 A
ACP-1
9.2
rGY208
15.7
SSRY98
11.4
rGY75
9.3
GY12
8.2
GY28
6.8
GY213
5.1
GY209
rSSRY4
16.4
rGY22-1
19.5
rGY75-1
7.3
rSSRY98
8.8
rBEST-2
GY68
7.0
8.9
GY32
rGY155
3.9
rGY192
9.5
GY154
14.6
rSSRY111
15.3
SSRY59
1.8
SSRY10
rGY215
19.3 L7
28.1
AC-1 M C
rGY144
2.5
rGY23
GY81
6.2
rGY89
14.0
rGY177
O11a
16.1
GY174
9.8
rSSRY183
rSSRY45
4.4
rSSRY95
17.8
rSSRY109
5.8
SSRY32
rK9a
20.1
rNI1.C2
7.6
GY23-1
13.4
rGY18
2.8
rGY26
rGY89-1
SSRY171
GY42
GY44
GY54
GY81-1
rA16
13.2
rGY191
17.5
rV17a
6.5
GY197
6.7
rGY147
5.6
rSSRY50
26.0
GY207 B
rAi14a
17.2
GY72
10.9
rSSRY61
9.4
rGY65
5.0
rPASK1
17.1
SSRY113
SSRY21
rF19b
15.0
rGY118
12.7
GY217
11.0
rS2
rSSRY161
11.6
rGY176
20.3
rGY190 E
SSRY84
rSSRY180
GY83
28.7
GY17
12.3
SSRY72
nGY162
15.6
rAE15
23.7
GY58
21.1
CDY123a
11.5
GY118
10.0
rI4a
G13 D
rGOT-2
rGY167
rGY180
1.2
GY222
GY181
3.7
GY219
GY50
GY125
GY179
3.8
rON1
SSRY3
0.0
SSRY120
3.6
rSSRY40
SSRY9
rSSRY169
10.6
GY4
14.8
SSRY108
rGY24
rGY59
GY78
rGY67H
15.8
rI18b
rJ1a
rGY57
20.5
rGY25
SSRY23
rSSRY55
10.7
rJ7
rG11
20.7
GY110
rSSRY100
7.9
SSRY88
19.2
Ai11b L
19.4
N15b
12.1
GY53
7.4
rSSRY88
8.0
rGY110
rSSRY172
22.0
rE14b
11.9
rJ9c
7.5
rCDY74
CDY76
AD1c
14.4
K2a
27.0
GY34
SSRY101
8.6
SSRY2 SSRY5 J
SSRY47
O20
11.3
AE10a
11.8
rM8b
GY7
GY34-1
25.9
K10
rGY93
GY97
SSRY135
rSSRY30
22.4
rSSRY38
7.8
rGY41
16.7
CYP79(152) G
rSSRY135
23.4
rGA-131
10.4
rGY94
AM18
rK16d
13.3
20.8
rCPY79(152)
rGY137
23.5
AB9a
24.8
SKDH-1
rAGPaseB
16.0
rSSRY106
rSSRY103
15.2
SSRY83 F
GY196
GY203
GY218
GY122
CDY107
GY37
GY204
GY194
GY186
SSRY179
17.0
SSRY68
Z11b
12.5
GY21
rCDY107-1
10.1
CDY123b
4.6
GY80
21.0
rV20a
28.3
H14c
GY16
23.0
rGY55 I
rAC1
GY130 R1
13.6
rW3
rGY201-1a
rD5a
rB3a
H14b
GY128
17.4
GY143
CDY29
22.6
CDY68
CDY128
rGY19
rSSRY8
rS12c
23.8
rU2a
rSSRY122
37.0
nrGY67
rGY36
3.0
rSSRY86
SSRY105
rGY87
W19b
GY105
5.9
SSRY177
8.4
GY5
5.7
SSRY99
rK3 U
K17
18.0
SSRY6
SSRY122
9.0
SSRY42
18.7
34.6
GY138
rGY164
GY223
4.5
SSRY170
CDY131-1
rGY113H
6.3
rGY92
SSRY185
2.3
SSRY20
21.5
U2c X
rM5a
CDY131
13.5
nCBB
rSSRY20
rAF14a
SSRY110 N
rCDY99
rGBSSII
SSRY13
rCDY44
rSSRY51
GY20
6.1
rGY10
GY108
GY52
5.3
GY13
Z18b
GY88
17.3
rGY47
4.0
nGY143
rGY109
rGY148
rGY145
2.4
GY201-1b
18.8
AD4b
SSRY35
rGY108
GY182
GY 124
rAF17
SSRY107
GY171
rGY210
5.5
ri4b
rA18
GY131
13.9
rSSRY83
GY66
rSSRY34
9.9
SSRY102
2.6
SSRY182
5.4
GY39
28.6
AM6
rGY115
rGY9
rGY1
rSSRY28
Ai19
PO2
16.8
SSRY7
F19a
AC11a
GY71
GY183
12.8
i16b R V
J13c
8.7
GY51
25.0
F15b
18.6
rSSRY19
rG4
rK11c
rAC5
rR13b
rP1a
rGY156
21.2
GY134
rGY220
GY63
rAC9a
GY100
31.4
rGY48
AE2 U1
rGY163
rGA-127
CDY106
27.8
rSSRY49
rGY119 K
rGY117
rSSRY175
1.5
rSSRY85
6.9
GY82
8.3
W16b
14.1
AC9b
GY119
rI2
SSRY174
rSSRY82

15. RFLP Marker Segregation in an F1 Cross of Non-inbred Parents

16. Gene Tagging of Multiple Sources of CMD Resistance Genes
Three different CMD Resistance genes have been mapped and
Can now be pyramided using molecular markers D
Y66
18.5
rI18b
20.5
rJ1a
20.0
rGY57
21.0
rGY25
21.2
SSRY9
23.9
SSRY3
16.2
SSRY23
CMD1 R
Dist
Marker cM
Name
rGY115
7.9
rGY9
15.6
CMD2
rGY1
16.1
rSSRY28
11.3
Akano et al. 2001
Ai19 M
SSRY102
24.4
SSRY230
16.6
Ns905
9.1
GY39
11.2
SSRY299
SSRY102
SSRY102
Fregene et al. 2000
24.4
24.4
SSRY230
SSRY230
16.6
16.6
NS170
CMD3
Ns905
Ns905
9.1
9.1
GY39
GY39
11.2
11.2
SSRY182
SSRY182
Hurtado et al. 2007
SSRY299
SSRY299

17. Positional Cloning of CMD2
High resolution map around CMD2
Contigs of BAC
clones around the
CMD2 locus
Candidate BAC Sequence

18. X Male Parent Female Parent TME3 TMS30555 1288 F1s Susceptible to CMD
High Starch
Resistant to CMD
(CMD2 gene)
1288 F1s

19. Cassava BAC Libraries
Purification and
cloning
HindIII partial
digestion
388kb
194kb
48.5kb
Mega base pair-sized
DNA in Agarose Plugs
Size selection
Cloning vector
Ligation and
Sizing of clones
3 BAC libraries with 5X, 10X, and 11X coverage of the genome respectively
97kb
6.9kb
48.5kb
Clone picking by
Robotics
Sizing of clones

20. BAC Clone Fingerprinting
Digestion with Hind III

21. Construction of a Contig Around CMD2
Contig construction and genetic mapping of the ends of a BAC cone (BAC-33b)
CMD2
BAC 33
SSRY-28
RME--1
NS158
RME--2
7cM
4cM

22. Bringing the Benefits of Modern Science to Farmer’s Fields
Molecular markers can help reduce breeding populations
Proof of concept: Farmer participation demonstrate quickly superiority of new germplasm
Social scientists/mol biologists

23. Two scientists Elizabeth Balyejusa: molecular markers
PhD
Grant awards
MBL and NARO
Yona Baguma: modified starch content
Heading Biotechnology Division, NARO

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

25. Hereditas 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.

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

27. Plant Science 164: , 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. .

28. Malaria

29. Malaria kills 110 Ugandan children <5 years old annually

30. Pregnant women (1st pregnancies) bear the brunt:
Poor pregnancy outcomes
Maternal anemia
Maternal deaths

31. Malaria control Drug treatment: RESISTANCE
Insecticide treated bed nets: RESISTANCE
Vector control IRS: RESISTANCE; LOGISTICS
Vaccines: future ray of hope

32. Targeted training Malaria drug resistance monitoring
Malaria vaccine studies

33. Malaria vaccines Development Clinical trials Total 13 4 27 37 9 46 2 1
Activity
phase
Vaccine
Type
Development
Clinical trials
Total
PreErythrocytic 13 4 27
Asexual 37 9 46
Transmission-
Blocking 2 1 3
Combination 6 8 58 16 74

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

35. Study Populations Apac, Uganda transmission : perennial
Longitudinal study
School children
Age 7-16 years
Case-control study of severe & mild malaria
Age 6-59 months
To study this, we have used from low and high malaria transmission areas

36. Before processing After processing Recombinant proteins 図1
Serine repeats (35) 23 S H N
989
Before processing I
III II
After processing
P47
P50 P6
P18
Recombinant　proteins 17
382
SE47’
まず、実験室レベルの精製レコンビナント蛋白質、SE47’とSE50Aで疫学調査を行った。
P50A
SE36 △ 図1

37. Correlation between anti-SERA IgG
and malaria symptom or blood parasitemia
Anti-SERA IgG and Fever
Anti-SERA IgG and blood parasitemia
Anti-SE47’
Anti-SE50A
Anti-SE47’
Anti-SE50A 4 2 . 1 4 1 4 1 2 1 2 3 1 . 5 1 1
Parasitemia (1000／μl blood)
Anti-E50A IgG1 ELISA OD 8 8
Anti-SE47' IgG3 ELISA OD 2 1 . 6 6 4 4 1 . 5 2 2 1 2 3 4 1 2 3 4
No Fever
< 37.5 ℃
Fever
> 37.5 ℃
No Fever
< 37.5 ℃
Fever
> 37.5 ℃
anti-SE47' IgG3
(OD at 405 nm)
anti-SE50A IgG1
(OD at 405 nm)

38. Mean ODs for IgG subclass responses to SE47’ and SE50A
as a function of age in Apac District, Northern Uganda
Anti-SE50A IgG
Anti-SE47' IgG
ELISA OD
ELISA OD
一般にマラリア抗原分子に対して、宿主であるヒトはかなりよく反応するが、それが即座に防御免疫につながる保証は全くない。
次に、抗SERA抗体価とマラリア免疫の相関を見る。
0 - 1
2 - 5
6 - 8
9 - 10
> 40
0 - 1
2 - 5
6 - 8
> 40
11- 14
9 - 10
11- 14
Age group
Age group I g G 1 2 3 4

39. Parasite growth inhibition by human serum
Hondurasｰ1
FCR3 K1
Patient Serum 75 50
Parasite culture
Parasite Growth inhibition (%)
Parasite Growth Inhibition Assay after 24 h. 25
102
103
104
105
IgG3 ELISA titer against SE47’

40. 8
Recombinant 47’ neutralizes the inhibitory effect of patient serum on in vitro parasite growth 50 40
SE50A 30
Parasite Growth Inhibition (%)
SE47’ 20
SE36
SE36の精製では、後々ヒトでのPhase I, Phase II の臨床試験に応用できるように、GMPに近い条件で精製した。 10 10 20 30 40
Concentration of recombinant protein
for neutralization (μg/ml)

41. Median SE36 titre compared with level of pigmented leucocytesMM SM SM
IQR 7, ,500 1,500

42. Production of malaria vaccine SE36 under
GMP environments for clinical trials
GMP production facilities for SE36 located in the Kanonji institute of
The Research Foundation for Microbial Diseases of Osaka University
(BIKEN)

44. P. falciparum: chloroquine resistance
Countries with at least one study indicating chloroquine total failure rate > 20%
Countries with at least one study indicating chloroquine total failure rate > 10%
No recent data available

45. 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%

46. 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%

47. P. vivax: chloroquine prophylactic or treatment failure
P. vivax prophylactic or treatment failure

48. 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
Sample ID
In addition to the techniques we also developed very simple methods for sample collection:
A few drops of blood from finger-prick are deposited on a small piece of filter paper cut into tiny rectangles at one end. The sample identification information are noted on the other end.
The paper is then air dried, it can be stored and shipped at room temperature and can even be sent out by regular mail.
Each blood-socked-tiny-rectangle can be cut out to extract DNA for molecular analysis.

49. Mutation Sites in the PfCRT Transmembrane Protein
NH2
COOH
R371
H97
K76
Q271
I356
N75
M74
C72
N326
Tom Wellems’ group at the NIH, discovered pfcrt, the parasite gene that is responsible for chloroquine resistance. pfcrt has a few point mutations, the critical one for resistance being the K76T mutation.
A220

50. Molecular Diagnosis of Chloroquine Resistance in the Field

51. PCR detection of pfcrt K76T polymorphisms
P4-w
P4-m D1 D2
Apo1
DIAGNOSTIC PCR PCR1: P1+P2
PCR2: P3+P4-w or P4-m
RESTRICTION PCR1: P1+P2
PCR2: D1+D2
Digestion with Apo1

52. Restriction digestion analysis of pfcrt K76T
M Dd2 3D7 C M
300bp
This is a sample gel of the restriction analysis. You can see that samples #1 and 2 are uncut and therefore are mutated while samples #4 and 5 are cut and of the wild type. Sample 3 is a mixte infection of mutant and wild tupe alleles. Dd2 is the resistant ctrl, 3D7 is the sensitive ctrl and C represent a negative ctrl. M is a 100bp DNA ladder.

53. Distinguishing Recrudescent from reinfection with Microsatellite ta99
Dd2 Hb3 C

54. dihydrofolate reductase
Mode of action of antifolates
GTP
Sulfadoxine (SD)
Pteridine
pABA
dihydropteorate synthase
(DHPS)
Pyrimethamine (PM)
Dihydrofolate
dihydrofolate reductase
(DHFR)
Tetrahydrofolate
Thimidine, Methionine synthesis

55. 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

56. 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

58. 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: DipBSc drug resistance
Best AMANET student paper 2006
Prossy Namuwulya
Short term training Plasmodium transfection: LUMC

59. Gender equality/equity issues addressed?
Not entirely

60. Other training in malaria
Group workshops
Bioinformatics 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

61. CBT in Malaria and Cassava Research addresses 6/8 MDGs
Science and Technology for Sustainable Development in Africa

62. 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

63. Thank you