1. Biogenetics and natural resources Dept.

2. IDENTIFICATION OF GENES AND GENE-NEAR
REGIONS RELATED TO ACTIVE COMPOUNDS
IN Warburgia ugandensis - AN IMPORTANT
AGROFORESTRY TREE
- Edward Muge (Ph.D student).
- Ramni Jamnadass (ICRAF).
- Kornel Burg (ARCSr).
- Joseph GlŐssl (BOKU).
PICME

3. Currently 80% of people in sub-Saharan Africa rely on
phytomedicines for health remedies.
Two- thirds of all medicinal plants are trees and the majority of
plant material is harvested unsustainably from the wild.
Little is known about the quality and effectiveness of plants that
institutions like ICRAF would wish to introduce into farming
landscapes.
Among the trees targeted for this evaluation is the East African
Greenheart (Warburgia ugandensis).
Common names:- pepper bark tree, East African greenheart, Ol-
msogoni, muthiga, soget.

4. Warburgia Ugandensis
Biology
The genus Warburgia is one of the five genera of the
family Canellaceae.
Has only three species, all African (W. salutaris, W.
stuhlmannii, W. ugandensis).
Distribution
W.salutaris is found in Southern Africa, W.stuhlmannii
is endemic to coastal Kenya and Tanzania while
W.ugandensis has a wider distribution within Eastern
Africa.

5. Importance
Used for treatment of malaria, an economically
important disease of the tropics.
Have both antibacterial and antifungal medicinal
qualities. (Aqueous bark methanolic extracts and
unique sesquiterpene 1-4 dialdehydes).
Curative of ailments such as stomach-ache,
constipation, toothache, common cold, cough, fever,
muscle pains, weak joints and measles.
The dialdehydes possess potent antifeedant activity against African
armyworms.
Other minor uses include food, fodder, fuel, timber and gum.

6. General ICRAF Objectives on Warburgia study
To develop storage protocols for the otherwise recalcitrant seeds of
Warburgia ugandensis.
To asses population density in selected populations across the natural
distribution range of the species within the genus Warburgia.
Resolve confusion over the present taxonomic status of the members
of genus Warburgia
Identification and establishment of their population structure and
genetic variation within its natural distribution range.
To determine the genetic relationship within and between the species of
and identify the type of mating system for W. ugandensis.
To develop marker system for breeding/selection purposes to obtain genotypes
with high anti-malarial effectivity and to further understand the effect of environment
on stability of the anti-malarial phenotype.

7. Pilot study on the potential of different tissue extracts from W.
ugandensis trees as anti-malarial. (Dr Geoffrey M. Rukunga (KEMRI).
Sample Plant part IC50 Values
(µg/ml) Trends.
code The roots are by far more active
A1-T3 Young leaves >250
than the leaves.
A2-T3 Old leaves >250
A3-T3 Root bark 4.09±0.24
T3 roots had the highest activity
B1-T5 Young leaves 241.24±0.88 followed by T5, then T6.
B2-T5 Old leaves 33.70±2.94
B3-T5 Root bark 4.70±0.35 The work on the roots of T8 and
all stem barks is yet to be
C1-T6 Young leaves 162.74±43.08 completed.
C2-T6 Old leaves 212.74±16.0
C3-T6 Root bark 8.55±0.52
D1-T7 Young leaves 120.21±34.38
D2-T7 Old leaves 131.56±3.98
D3-T7 Root bark 14.88±2.65
E1-T8 Young leaves >250
E2-T8 Old leaves >250
CQ 0.0173 ± 0.0008

8. Sesquiterpenes
Plant secondary metabolites
They include:- Muzigadial,
Mukaadial, Polygodial,
Warburganal e.tc.

9. Goals of my Ph.D
Elucidate genes involved in sesquiterpene biosynthesis in Warburgia and
thereby identify and develop a marker system for breeding/selection
purposes. This marker would enable identification of Warburgia
genotypes with high anti-malarial affectivity using a procedure called CpG
island microarrays.
Finding genomic regions that are differentially methylated
(variation/diversity) in different individuals
Isolating gene and gene near sequences correlated to sesquiterpene
biosynthesis.
Is there any correlation of the identified gene(s) (marker) with altered
anti-malarial activity?

10. Study approach
DNA Approach
PCR amplification of sesquiterpene genes directly from the genomic DNA using
degenerate primers (based on known plant sesquiterpene sequences)
Preparing genomic libraries after methyl filtration for comparative purpose among
different tissues/plants on micro array
RNA Approach
RNA has been isolated from the root and stem cambia and leaves of the
different genotypes for establishment of cDNA libraries
About 1000 cDNA clones from each tissue will be picked and spotted on
microarray chip.
This array will be comparatively hybridized with RNA probes originating from
genotypes representing different anti-malaria efficiency.
The clones showing differential expression will be sequenced and annotated
by BLAST.

11. PCR with Degenerate primers.
A number of degenerate primers have been planned based on
conserved regions of sesquiterpene genes. These are still
being tried out on PCR as well as sequencing candidate PCR
products.
Degenerate house keeping genes primers have yielded
positive results.
Other techniques including genome walking is also being tried
out.
Chemical composition profiling has shown that most of the
compounds have more relatedness to triterpenes as opposed
to sesquiterpenes, Triterpene primers have also been
planned.

12. TECHNOLOGY
Methyl filtration.
Aimed at obtaining gene-near regulatory elements/CpG islands
Their role in gene regulation has been proven both in animals and plants
CG/CpG island is a short stretch of DNA in which the frequency of CG
sequence is higher than other regions.
CpG islands are found near or within genes (introns)
Cytosine residues of the DNA are generally methylated in eukaryotes and
extensively methylated in plants (CmG, CmCG)

13. Frequency of CpG islands in the isolated regions in rice
genome
Results based on the rice genome as model plant
Nearly 70 % of enriched clones contained CpG island sequences
About 1/3 of the clones originated from transposons
Similar results have been obtained in:
Sweet Potato
Banana
Warburgia

14. Flow diagram
C C
G G G G
C C 5m C C G G 5m
C
C 5mC G G C 5m
G G G G C 5m
C 5m C G C
G 5m G
C 5m G
C
Digest with HpaII+MspI and ligate adapter
C G G C
G G C C G G C5m
5mC C G G 5mC
G G C C5m G G5mC
5mC G G 5mC
C5mC G G C
Amplify small adapter-ligated fragments
Genomic representation

15. T8_Stem
LADDER
T8_Root
T8_Leaf
Genomic DNA from different tissues of
Warburgia ugandensis(T8).
KB
Material for cloning, colony picking, and
5000
PCR for printing on slides. 2000
800
400
100
Cloning

16. PCR amplification and validation on Agarose
_
+

17. DNA-Chips (DNA micro-arrays)
DNA

18. Hybridization on DNA chip
Methylation
fragments or
cDNA Mix eg.
Root + Stem

19. Scanning
Cy3 Cy5
532nm 635nm
Detection
Emisssion
Excitation
Relative Abundance
Cy3 / Cy5 (ratio) 1 2 3
Spot (gene)

20. Hybridized and scanned Microarray Chip
Statistical analysis of:
changes in gene
expression or
methylation

21. Progress
DNA Approach
PCR amplification of sesquiterpene genes directly from the genomic DNA using
degenerate primers (based on known plant sesquiterpene sequences)
Completed genomic libraries after methyl filtration and spotted the clones on
microarray chips.
Began initial hybridization standardizations and data analysis.
RNA Approach
RNA has been isolated from the root and stem cambia and leaves of the
different genotypes for establishment of cDNA libraries.
Completed cDNA library from leave tissue and picked 10,000 clones for spotting.

22. Conclusion and perspectives
New genes possibly involved in sesquiterpene biosynthesis
will be identified and isolated from yet poorly studied plant.
The research will bring first sequence data on W. ugandensis.
Partial sequences from some house keeping genes have so far
been obtained.
The relation of this particular secondary metabolites type to
anti malarial properties is expected to be clarified.
Comparative evaluation of the results obtained both in the
DNA based and in the RNA based approach will then be
used to try and identify genes involved in sesquiterpene
biosynthesis.

23. ACKNOWLEDGEMENTS
ICRAF (Trees and Markets team)
OÄD
ARCSr

24. THANK YOU