Melaku Gedil Molecular Geneticist/Breeder

1. Melaku GedilMolecular Geneticist/Breeder The Use of Comparative Genomics to Tap Resistance Gene Analogs in Cassava and its Wild Relatives International Institute of Tropical Agriculture Ibadan, Nigeria

2. Arabidopsis 2000 Rice 2002

3. Cost of sequencing

4. Declining cost of sequencing Emerging technologies 454 Pyrosequencing technology Illumina's Solexa sequencing technology SOLiD technology Further substantial leaps in sequencing technology is expected x

5. Plant Genome Projects

7. Cloned and characterized virus R genes in plants

8. Predicted domain of R genes

9. Common domains in cloned R genes-1 • NBS (nucleotide binding site) Subdomains 1. Kinase - 1a - GPGGVGKT in RPS2 2. Kinase - 2 - LIVLDD 3. Kinase - 3a - DWFGxGSR 4. Membrane spanning domain - GGLPLAL

10. Common domains in cloned R genes -2 LRR (Leucine rich repeat) • tandem repeats of 20-30 aa, each with a regular spacing of Leucines and other hydrophobic residues • ligand binding domain - elicitor recognition • Consensus Sequence • LxxLxxLxxLxLxxLxxNxLxxIPxx • PxxaxxLxxLxxLxaxxxxaxxa - RPS2 • specificity determinant

12. Materials and Methods

13. Primers 1. Based on P-loop and NBS 2. Based on Arabidopsis NBS-LRR alignment (Meyers 2003) 3. Based on Manihot NBS-LRR sequence (RGH1 & RGH2)

14. LZ/T NBS LRR RPS2 Conserved Sequence N L6 PCR-Degenerate primer Cloning/Sequencing Sequence Analysis Mapping PCR-based cloning of Candidate R-Gene

17. RESULTS

18. PCR Amplification with degenerate primers Amplification with degenerate primers derived from At-NBS-LRR Three forward and 7 reverse primers (a total of 21 pairs) were tested on the TME3 clone (Fig. x). Different primer pairs yielded different pattern of banding with fragment sizes ranging from 500-1500 bp. 50F-470RL was considered for further analysis. Four DNA templates (TME3, TME7, and TME117-a, TME-117-b) were amplified (Fig. xx). Amplicon size ranged from 200 – 900 bp.

19. TA-cloning, Invitrogen Cloning

20. Sequencing • Sequencing was performed using the BigDye v3.1 chemistry on ABI310 and ABI 3100 Genetic Analyzer. • Sequencing and characterization of 17 randomly selected colonies containing the PCR fragment amplified by the degenerate primers resulted in 15 sequences.

21. Sequence Editing and Analysis - BioEdit

22. Sequence Editing and Analysis - CodonCode

23. Phylogenetic Relationships

24. Sequence Divergence • Pairwise analysis of 15 sequences conducted in MEGA4 [#781].

25. C6 C4 Clone C4 and C6 primer position Position 153 C vs T Position 115 G vs C/G het Position 297 A vs G Example of SNPs Re-sequencing Primers

26. SNPs identified by re-sequencing

27. BLAST output – Lineage report

28. BLAST output – Taxonomy report BLAST

31. RGA clones similarity search limited to Manihot esculenta (id 3983 on Entrez)

32. Cassva RGA matching NBS-LRR genes

33. Work in progress • More sequencing of RGA clones from Manihot esculenta • More sequencing of RGA clones from wild Manihot and castor bean • cDNA-RGA • Sequence analysis and characterization • STS marker design and assay • BSA analysis of potential primers • CAPS marker (WebCutter)

34. APPlCATIONS • Survey of chromosome regions containing R genes and analogs • Molecular markers • SNP • Cloning (Seo 2006 viral gene in common bean isolated) • Physical mapping (Qiu 2007 leaf rust R in wheat) • NBS-profiling – Mantovani 2006, NBS profiling of genetic diversity – a modification of AFLP • cDNA-RGA-Budak 2006 Motif-directed RNA fingerprinting • Host-pathogen interaction/pathways e.g. ATP-binding or hydrolysis • Genome wide survey - Bioinformatics (Arabidopsis, Rice)

35. THANK YOU