1. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org Melaku Gedil Molecular 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. The plunging cost of DNA sequencing has opened new applications in science and medicine. Source: Science 2006 Vol. 311. no. 5767, pp. 1544 - 1546 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. Completed Large-Scale Sequencing Projects [15] Arabidopsis thaliana (thale cress)Arabidopsis thaliana 5 chromosomes: 1, 2, 3, 4, 5, plastid, mitochondrion12345plastidmitochondrion Medicago truncatulaMedicago truncatula (barrel medic) 8 chromosomes: 1, 2, 3, 4, 5, 6, 7, 8 plastid, mitochondrionplastid [11] Oryza sativa (rice)Oryza sativa 12 chromosomes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, plastid, mitochondrion, mitochondrial plasmid B1, mitochondrial plasmid B2123456789101112plastidmitochondrionmitochondrial plasmid B1mitochondrial plasmid B2 [5] Populus trichocarpa (black cottonwood)Populus trichocarpa 19 chromosomes: I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, plastid, mitochondrionIIIIIIIVVVIVIIVIIIIXXXIXII In-progress Large-Scale Sequencing Projects - funded, genome sequence expected in GenBank Brachypodium distachyon 5 chromosomes: 1, 2, 3, 4, 5, plastid, mitochondrion Glycine maxGlycine max (soybean) 20 chromosomes: A1, A2, B1, B2, C1, C2, D1a, D1b, D2, E, F, G, H, I, J, K, L, M, N, O, plastid, mitochondrion Lotus japonicusLotus japonicus (lotus) 6 chromosomes: 1, 2, 3, 4, 5, 6, plastid, mitochondrionplastid Manihot esculentaManihot esculenta (cassava) 20 chromosomes: A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, plastid, mitochondrionABCDEFGHIJKLMNOPQRS Solanum lycopersicumSolanum lycopersicum (tomato) 12 chromosomes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, plastid, mitochondrion123456789101112plastid Solanum tuberosumSolanum tuberosum (potato) 12 chromosomes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, plastid, mitochondrion Sorghum bicolor 10 chromosomes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, plastid, mitochondrion Zea maysZea mays (corn) 10 chromosomes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, plastid, mitochondrion, mitochondrial 1.9 kb plasmid12345678910plastidmitochondrionmitochondrial 1.9 kb plasmid Plant Genome Projects

7. Cloned and characterized virus R genes in plants GeneHost speciesVirus2AVR Resistance mechanisms Cloning method Receptor structure Reference N N. tabacum TMV Helicase domain of replicase HR Transposon tagging TIR-NBS- LRR Whitham et al, 1994 Rx1 S. tuberosum PVX CPReplication Positional cloning CC-NBS- LRR Bendahmane et al, 1999 Rx2 S. tuberosum PVX CPReplication Positional cloning CC-NBS- LRR Bendahmane et al, 2000 Sw5 S. esculentumTSWV MPHR Positional cloning CC-NBS- LRR Brommonschenkel et al., 2000 HRT A. thaliana TCV CPHR Positional cloning LZ-NBS- LRR Cooley et al., 2000 RTM1 A. thaliana TEV nd Systemic movement Positional cloning Jacalin like seq Chisholm et al.., 2000 RTM2 A. thaliana TEV nd Systemic movement Positional cloning Jacalin like seq Whitham et al.., 2000 RCY1A. thaliana CMVCPHR Positional cloning CC-NBS- LRR Takahashi et al., 2001 Tm22S. lycopersicumToMV MPHR Transposon tagging CC-NBS- LRR Lanfermeijer et al., 2003 Pvr21/22 C. annuum PVYVPg Replication cell- cell movement, Approximation by homology eIF4E Ruffel et al., 2002 Mo11/2 L. sativaLMVnd Replication Tolerance Approximation by homology, eIF4E Nicaise et al., 2003 Sbm1P. sativumPSbMVndReplication Approximation by homology eIF4E Gao et al., 2004

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

14. Cloning/Sequencing Sequence Analysis Mapping PCR-Degenerate primer RPS2 N L6 LZ/T NBS LRR Conserved Sequence 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. Cloning TA-cloning, Invitrogen

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

26. SNPs identified by re-sequencing

27. BLAST output – Lineage report

28. BLASTBLAST output – Taxonomy report

31. CloneHitExampleLength (bp)Query coverage (%) Maximum Identity (%) C11Tme3 chloroplast1421394 C20-248-- C30-361-- C422RGC in solanum, Ty3/Gypsy- like retro 34520-85 (most>80)86-95 C50-181-- C623Ty3/Gypsy-like retro46220-86 (most>80)84-96 C720Ty3/Gypsy-like retro29522-93 (most>80)77-88 C90-166-- C100-308-- C110-217-- C120-326-- C130-425-- C1419RGA Saccharum and Vitis vinifera, Ty3/Gypsy-like retro 27747-87 (most ~60)80-89 C151linamarase=beta-glucosidase3044 (15 bp)100 C170-223-- 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 1.Survey of chromosome regions containing R genes and analogs 2.Molecular markers 3.SNP 4.Cloning (Seo 2006 viral gene in common bean isolated) 5.Physical mapping (Qiu 2007 leaf rust R in wheat) 6.NBS-profiling – Mantovani 2006, NBS profiling of genetic diversity – a modification of AFLP 7.cDNA-RGA-Budak 2006 Motif-directed RNA fingerprinting 8.Host-pathogen interaction/pathways e.g. ATP-binding or hydrolysis 9.Genome wide survey - Bioinformatics (Arabidopsis, Rice)

35. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org