Biogenetics and natural resources Dept.

IDENTIFICATION OF GENES AND GENEIDENTIFICATION OF GENES AND GENE--NEAR NEAR REGIONS RELATED TO ACTIVE COMPOUNDS REGIONS RELATED TO ACTIVE COMPOUNDS IN IN Warburgia ugandensis Warburgia ugandensis -- AN IMPORTANT AN IMPORTANT AGROFORESTRY TREE AGROFORESTRY TREE

-- Edward Muge (Ph.D student).Edward Muge (Ph.D student).- Ramni Jamnadass (ICRAF).Ramni Jamnadass (ICRAF).-- Kornel Burg (ARCSr).Kornel Burg (ARCSr).-- Joseph GlJoseph GlŐŐssl (BOKU).ssl (BOKU).

PICMEPICME

Currently 80% of people in sub-Saharan Africa rely onphytomedicines for health remedies.

Two- thirds of all medicinal plants are trees and the majority ofplant material is harvested unsustainably from the wild.

Little is known about the quality and effectiveness of plants thatinstitutions like ICRAF would wish to introduce into farminglandscapes.

Among the trees targeted for this evaluation is the East AfricanGreenheart (Warburgia ugandensis).

Common names:- pepper bark tree, East African greenheart, Ol-msogoni, muthiga, soget.

BiologyBiology

The genus Warburgia is one of the five genera of theThe genus Warburgia is one of the five genera of thefamily Canellaceae.family Canellaceae.

Has only three species, all African (W. salutaris, W.Has only three species, all African (W. salutaris, W.stuhlmannii, W. ugandensisstuhlmannii, W. ugandensis).).

Distribution Distribution

W.salutaris W.salutaris is found in Southern Africa, is found in Southern Africa, W.stuhlmanniiW.stuhlmanniiis endemic to coastal Kenya and Tanzania whileis endemic to coastal Kenya and Tanzania whileW.ugandensisW.ugandensis has a wider distribution within Easternhas a wider distribution within EasternAfrica.Africa.

WarburgiaWarburgia UgandensisUgandensis

ImportanceImportance

Used for treatment of malaria, an economicallyUsed for treatment of malaria, an economicallyimportant disease of the tropics.important disease of the tropics.

Have both antibacterial and antifungal medicinalHave both antibacterial and antifungal medicinalqualities. (Aqueous bark methanolic extracts andqualities. (Aqueous bark methanolic extracts andunique sesquiterpene 1unique sesquiterpene 1--4 dialdehydes).4 dialdehydes).

Curative of ailments such as stomachCurative of ailments such as stomach--ache,ache,constipation, toothache, common cold, cough, fever,constipation, toothache, common cold, cough, fever,muscle pains, weak joints and measles.muscle pains, weak joints and measles.

The dialdehydes possess potent antifeedant activity against AfrThe dialdehydes possess potent antifeedant activity against Africanicanarmyworms.armyworms.

Other minor uses include food, fodder, fuel, timber and gum.Other minor uses include food, fodder, fuel, timber and gum.

General ICRAF Objectives on Warburgia studyTo develop storage protocols for the otherwise recalcitrant seeds ofWarburgia ugandensis.

To asses population density in selected populations across the naturaldistribution range of the species within the genus Warburgia.

Resolve confusion over the present taxonomic status of the membersof genus Warburgia

Identification and establishment of their population structure andgenetic variation within its natural distribution range.

To determine the genetic relationship within and between the species ofand identify the type of mating system for W. ugandensis.

To develop marker system for breeding/selection purposes to obtain genotypeswith high anti-malarial effectivity and to further understand the effect of environmenton stability of the anti-malarial phenotype.

>250>2500.0173 ± 0.0008

Young leavesOld leaves

E1-T8 E2-T8 CQ

120.21±34.38131.56±3.9814.88±2.65

Young leavesOld leavesRoot bark

D1-T7D2-T7D3-T7

162.74±43.08212.74±16.08.55±0.52

Young leavesOld leavesRoot bark

C1-T6 C2-T6 C3-T6

241.24±0.8833.70±2.944.70±0.35

Young leavesOld leavesRoot bark

B1-T5B2-T5B3-T5

>250>2504.09±0.24

Young leavesOld leavesRoot bark

A1-T3 A2-T3 A3-T3

IC50 Values (µg/ml)

Plant partSample code

Pilot study on the potential of different tissue extracts from W. ugandensis trees as anti-malarial. (Dr Geoffrey M. Rukunga (KEMRI).

Trends.The roots are by far more activethan the leaves.

T3 roots had the highest activityfollowed by T5, then T6.

The work on the roots of T8 andall stem barks is yet to becompleted.

Plant secondaryPlant secondary metabolitesmetabolites

They include:They include:-- Muzigadial,Muzigadial,Mukaadial, Polygodial, Mukaadial, Polygodial, Warburganal e.tc.Warburganal e.tc.

SesquiterpenesSesquiterpenes

Goals of my Ph.DGoals of my Ph.D

Elucidate genes involved in sesquiterpene biosynthesis in WarburElucidate genes involved in sesquiterpene biosynthesis in Warburgia andgia andthereby identify and develop a marker system for breeding/selecthereby identify and develop a marker system for breeding/selectiontionpurposes. This marker would enable identification of Warburgiapurposes. This marker would enable identification of Warburgiagenotypes with high antigenotypes with high anti--malarial affectivity using a procedure called CpGmalarial affectivity using a procedure called CpGisland microarrays.island microarrays.

Finding genomic regions that are differentially methylatedFinding genomic regions that are differentially methylated(variation/diversity) in different individuals(variation/diversity) in different individuals

Isolating gene and gene near sequences correlated to sesquiterpeIsolating gene and gene near sequences correlated to sesquiterpenenebiosynthesis.biosynthesis.

Is there any correlation of the identified gene(s) (marker) witIs there any correlation of the identified gene(s) (marker) with alteredh alteredantianti--malarial activity?malarial activity?

Study approachStudy approach

DNA ApproachDNA ApproachPCR amplification of sesquiterpene genes directly from the genomPCR amplification of sesquiterpene genes directly from the genomic DNA usingic DNA usingdegenerate primers (based on known plant sesquiterpene sequedegenerate primers (based on known plant sesquiterpene sequences)nces)PreparingPreparing genomicgenomic libraries afterlibraries after methylmethyl filtration for comparative purpose amongfiltration for comparative purpose amongdifferent tissues/plants on micro arraydifferent tissues/plants on micro array

RNA ApproachRNA ApproachRNA has been isolated from the root and stem cambia and leaves oRNA has been isolated from the root and stem cambia and leaves of thef thedifferent genotypes for establishment of cDNA librariesdifferent genotypes for establishment of cDNA libraries

About 1000 cDNA clones from each tissue will be picked and spotAbout 1000 cDNA clones from each tissue will be picked and spotted onted onmicroarray chip.microarray chip.

This array will be comparatively hybridized with RNA probes oriThis array will be comparatively hybridized with RNA probes originating fromginating fromgenotypes representing different antigenotypes representing different anti--malaria efficiency.malaria efficiency.

The clones showing differential expression will be sequenced anThe clones showing differential expression will be sequenced and annotated d annotated by BLAST.by BLAST.

PCR with Degenerate primers.

A number of degenerate primers have been planned based onconserved regions of sesquiterpene genes. These are stillbeing tried out on PCR as well as sequencing candidate PCRproducts.

Degenerate house keeping genes primers have yieldedpositive results.

Other techniques including genome walking is also being triedout.

Chemical composition profiling has shown that most of thecompounds have more relatedness to triterpenes as opposedto sesquiterpenes, Triterpene primers have also beenplanned.

Methyl filtrationMethyl filtration..TECHNOLOGY

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 CGsequence is higher than other regions.

CpG islands are found near CpG islands are found near or withinor within genes (genes (intronsintrons))

Cytosine residues of the DNA are generally methylated in eukaryotes andextensively methylated in plants (CmG, CmCG)

Frequency of CpG islands in the isolated regions in rice Frequency of CpG islands in the isolated regions in rice genomegenome

Results based on the rice genome as model plantResults based on the rice genome as model plant

Nearly 70 % of enriched clones contained CpG island sequencesNearly 70 % of enriched clones contained CpG island sequences

About 1/3 of the clones originated from transposonsAbout 1/3 of the clones originated from transposons

Similar results have been obtained in:Similar results have been obtained in:

Sweet PotatoSweet PotatoBananaBananaWarburgiaWarburgia

Flow diagramFlow diagram

G G C C

C C G G

C5mC G GG G C5m C

5mC C G G

G G C C5m

5mC 5mCG G

G G 5mC 5mCDigest with HpaII+MspI and ligate adapterDigest with HpaII+MspI and ligate adapter

G G C CC G G C

G G C5m

5mC C G GG G C C5m

5mCG G5mC

5mC G GC5mC

5mCG G C

Amplify small adapter-ligated fragmentsAmplify small adapter-ligated fragments

Genomic representationGenomic representation

2000

5000

400

800

100

KB

LAD

DE

R

T8_

Ste

m

T8_

Leaf

T8_

Roo

t

Genomic DNA from different tissues of

Warburgia ugandensis(T8).

Material for cloning, colony picking, and

PCR for printing on slides.

Cloning

PCR PCR amplificationamplification and and validationvalidation on on AgaroseAgarose

_

+

DNA-Chips (DNA micro-arrays)

DNA

Hybridization on DNA chipHybridization on DNA chip

Methylation fragments or cDNA Mix eg.Root + Stem

Scanning

Relative AbundanceCy3 / Cy5 (ratio)

Spot (gene)1 2 3

Excitation

Emis

ssio

n

Detection

Cy3532nm

Cy5635nm

Hybridized and scanned Microarray ChipHybridized and scanned Microarray Chip

Statistical analysis of: changes in gene expression or methylation

ProgressProgress

DNA ApproachDNA ApproachPCR amplification of sesquiterpene genes directly from the genomPCR amplification of sesquiterpene genes directly from the genomic DNA usingic DNA usingdegenerate primers (based on known plant sesquiterpene sequedegenerate primers (based on known plant sesquiterpene sequences)nces)CompletedCompleted genomicgenomic libraries afterlibraries after methylmethyl filtration and spotted the clones onfiltration and spotted the clones onmicroarray chips.microarray chips.Began initial hybridization standardizations and data analysisBegan initial hybridization standardizations and data analysis..

RNA ApproachRNA ApproachRNA has been isolated from the root and stem cambia and leaves oRNA has been isolated from the root and stem cambia and leaves of thef thedifferent genotypes for establishment of cDNA libraries.different genotypes for establishment of cDNA libraries.Completed cDNA library from leave tissue and picked 10,000 clonCompleted cDNA library from leave tissue and picked 10,000 clones for spotting.es for spotting.

New genes possibly involved in sesquiterpene biosynthesisNew genes possibly involved in sesquiterpene biosynthesiswill be identified and isolated from yet poorly studied plantwill be identified and isolated from yet poorly studied plant..

The research will bring first sequence data on The research will bring first sequence data on W. ugandensis.W. ugandensis.Partial sequences from some house keeping genes have so farPartial sequences from some house keeping genes have so farbeen obtained.been obtained.

The relation of this particular secondary metabolites type to The relation of this particular secondary metabolites type to anti malarial properties is expected to be clarified.anti malarial properties is expected to be clarified.

Comparative evaluation of the results obtained both in the Comparative evaluation of the results obtained both in the DNA based and in the RNA based approach will then be DNA based and in the RNA based approach will then be used to try and identify genes involved in sesquiterpene used to try and identify genes involved in sesquiterpene biosynthesis.biosynthesis.

Conclusion and perspectivesConclusion and perspectives

ACKNOWLEDGEMENTS

ICRAF (Trees and Markets team)

OÄD

ARCSr

THANK YOUTHANK YOU