Remove the map of Africa from this one. The audience knows well the regions

Remove the text and have a big chart then move the reference to the bottom of page (font size 13 or 14)

Again Show one table that gives evidence for a single message. Work on quality

Identification of specific markers linked to regional differentiation of Warburgia ugandensis in Kenya Ochieng’ Noel Onyango (BSc. General) I56/13839/05 Kenyatta University Dr. Alice Muchugi Biochemistry and Biotechnology Dept, Kenyatta University. Dr. Ramni Jamnadass Global Project 1 leader World Agroforestry Center Supervised by; Dr. Bonaventure Omondi Aman Department of Plant protection Sciences Swedish University of Agricultural Sciences 1 2 3

B C Warburgia ugandensis Sprague Other members: W. salutaris Chiov., W. stuhlmannii Engl. & W. elongata, Verdc. Common names: Pepper bark tree, Muthiga (Kikuyu), Soget (Kalenjin), Msokonoi (Swahili) General descriptions

Chemistry Uses: Expectorant, Chest infections, Sinusitis, Malaria, Venereal diseases, Stomach ulcers, Toothache and Dermatological disorders

INTRODUCTION 1,000- 3,000 m Steep ecological gradients Orographic (induce) Varied insolation Spatially/Temporally Restricted distribution in the Afro-alpine environment (Class VIII/IX) Species richness, endemism & internal consistency of the environment Plants in the belt are much associated with edaphic conditions rather than orographic

Problem statement The species population in Kenya have shown a high genetic differentiation in the Rift Valley thereby, a possible allopatric speciation Differentiation: Habitat fragmentation and decline in pop. size ~ genome disorders Adaption markers are important in climate change mitigation Research hypothesis There are clear diagnostic markers showing regional differentiation These markers are linked to the species regional differentiation Main objective Identify specific genetic markers linked to its regional differentiation within and across Kenyan Rift Valley. Justification Genetic disjunction studies are important for providing info on desired signatures in the genome that are linked to important traits. “Assists in the Germplasm development for plants management”

Sample collection

Genomics RAPD-PCR) Cloning pGEMT-E vector NCBI- BLAST Searches BSA PCR Colony screening DNA extraction Mix of 20 individuals, 9 populations ok. Sequencing Phylogenetics (CTAB) : Doyle and Doyle (1987)

An adaptation of BSA Technique for investigation of diagnostic markers closely linked to a specific trait (Michelmore et al., 1991) BSA compares 2 pooled DNA samples from individuals of segregating or those that contrast for a trait . Screening for pooled DNA samples derived from the populations to form a genotype

Cloning and transformation PCR recombinants Analysis 10X PCR buffer, 50 mM dNTPs;100 ng/μL M13,1DNA Taq (35 cycles). Plasmid DNA preparation QIAprep spin miniprep kit protocol cells resuspended Lysed Neutralized (cloudy) Spun at 10, 000 x g for 10 min (Spin column) PPT of Plasmid bound on silica gel PB buffer. Eluted in 10 μL mol. grade H2O

Represented populations to the Western Side of the Rift Valley Bands selection and recovery from the gels (QIAquick® gel extraction kit [QIAgen Inc., Valencia, CA]) Results

Bionformatics Tools General GenBank (USA): http://www.ncbi.nlm.nih.gov/ Genomes NCBI: http://ncbi.nlm.nih.gov/Genomes/index.html Similarity BLAST: http://www.ncbi.nlm.nih.gov/BLAST/ (BLASTX and TBLASTX) Multiple sequence alignment ClustalW: http://au.expasy.org/tools/scanprosite/ Phylogenetic analysis Mega4: http://www.megasoftware.net/

extreme low E-values (0.000-4e-33) at the stringent threshold limit of 0.0001 Data mining process

Phylogram derived from 3 amino acid sequence homologous of W. ugandensis sequences Pair wise distances for all sequences and their disparity pattern index Phylogenetic analysis for the sequences to obtain their evolutionary relationship by a uniform weighted parsimony method to analyze the sequences retrieved from MSA Phylogenetic Analysis

Phylogram derived from 3 amino acid homologous sequences to WaburgiaIC28W Pairwise distances for WarburgiaIC28/W and their disparity pattern index

The was clear association of RAPD-PCR showing repetable variation within W. ugandensis populations in Rift Valley (Muchugi et al., 2008) RAPD-PCR – amplicon unknown to scientist, could be plant DNA, endophytes, pathogens, contaminants etc. Seq analysis was the final proof, but found no link to W. ugandensis traits though it’s genome has not yet been sequenced, related species show low sequencing. Could be a case as of significant homologous results revealed Insertions inform of Transposable elements in the Chromosome (Dvorak, 2009) Most of the elements were along genome coding regions (mutagens), a possible cause alteration of gene function. Subject them to weak selection: TE efficacy varies with demographic factors, a driving force on their distribution in the genome (Lockton et al. 2008). Discussion

Conclusion There were no genetic markers that distinguish W. ugandensis spps E or W populations in the R. Valley; but probably elements T E’s in the genome. There were repeatable sequence variation between East and West pop. although we did not tie these to markers generated in this study. Therefore, we have provided a basis for targeted study of fine-scale diversity (eg by chloroplast DNA etc) The diversity revealed is useful in providing a basis for designing novel markers (we need to test them in two most divergent species) It has also indicate there is diversity of the species in the east to those in the west of the Great R. Valley (Muchugi et al., 2008) Evolutionary consequences can be due to transposition dynamics of TE families coupled to demographic histories of populations.

Recommendations Obtain sequence flanking of the putative genomic markers to assign biological functionality by genome walking amongst other techniques. Sequence Characterized Amplified Regions (SCARS) and Chloroplast DNA (cpDNA) to know the extent of the differentiation in the two regions. The conserved protein region observed should be studied by X rays to determine structure and functions.

Acknowledgements Staff of:

Thank you for the attention!!!