A. Bellotti 1 , A. Bohórquez 1 , J. Vargas 1 , B. Arias 1 , H.L. Vargas 2 , C. Mba 3 , M.C. Duque 1 , J. Tohme 1 1 Centro Internacional de Agricultura Tropical (CIAT), AA 6713, Cali, Colombia 2 CORPOICA, Espinal, Tolima, Colombia. 3 Intern. Atomic Energy Agency. P.O Box 100, A-1400, Vienna, Austria. http://www.ciat.cgiar.org/ipm/index.htm RECENT ADVANCES IN HOST PLANT RESISTANCE TO WHITEFLIES IN CASSAVA Centro Internacional de Agricultura Tropical International Center for Tropical Agriculture RESULTS AND DISCUSSION CONCLUSIONS  Field evaluations in the family CM 8996 and their parentals confirm resistance of the genotype MEcu-72 and susceptibility of the parental MCol-2246; this allows us to do preliminary selection of F1 genotypes.  Using SSR markers, putative association with the parental lines were found.  A linkage map is being constructed using the SSR data, a RGA and the field phenotypic characterization. ASSOCIATION BETWEEN MOLECULAR MARKERS AND RESISTANCE The molecular data are being analyzed using QTL packages (QTL cartographer Qgene) to determine linkages between the markers and the phenotypic characterization. As preliminary analysis X 2 at the 5% level was done using SAS. Putative associations were found between 43 SSRs markers and the resistance. MOLECULAR ANALYSIS Both parents, MEcu-72 and MCol-2246, were evaluated with 343 cassava SSR markers (Mba et al, 2001), including 156 cDNA SSRs developed by Mba et al (submitted). Approximately 155 of the SSRs were polymorphic in the parentals and were evaluated in the F1 (286 individuals). For the construction of the linkage map, 103 SSRs were analyzed, of which 71 were anchored and segregating from the heterozygous female parent (MEcu-72) of an interspecific cross. The map consists of 19 linkage groups; which represent the haploid genome of cassava (Fig. 4). These linkage groups span 550,2 cM and an average marker density of 1 per 7,9 cM. The position of the 71 SSRs markers is shown in figure 5 of the cassava molecular genetic map (LOD = 25 and tetha ( θ ) = 25). Map distances are shown in Kosambi map units. So far, 26 SSRs markers (shown in green, Fig. 4) have been previously placed on the cassava framework map (Fregene et al, 1997), the other 45 SSRs are new. Thirty one of the 71 SSRs were cDNA sequences (Mba, in preparation) and the others were genomic DNA. ON GOING WORK - Saturation of Linkage map of Ecu-72, using AFLPs. - Isolation, cloning, sequencing and mapping of AFLPs polymorphic bands between resistants and susceptibles genotypes and design of SCARs for marker assisted selection. - QTLs analysis for resistance to whitefly. - The whitefly resistance will be the target for map-based cloning using the BAC libraries as tools. - Isolation of expressed sequences during the defense response of MEcu-72 to white fly attack. - In order to identify differentially expressed sequences, a new technology known as DNA chips or microarray is available to scan a significant number of clones. Microarray expression profiling detailed experiments will be used to identify putative early-response regulatory and/or signaling genes and to test the function of selected candidate genes using reverse genetics. Fig. 3. Cassava damage and whitefly population ratings due to A. socialis feeding on parental genotypes MEcu-72, MCol-2246 and clones from the family CM 8996 at CORPOICA, Nataima (Tolima, Colombia). 0 0.5 1 1.5 2 2.5 3 3.5 4 Damage and Population scale MECU 72 MCOL 2246 DAMAGE LEVEL POPULATION 87 0 0 1 140 1 0 15 115 3 50 28 0 20 40 60 80 100 120 140 160 1 1.1- 2 2.1 - 3 3.1 - 4 4.1 - 5 5.1 - 6 No. Clones Damage level Population Fig. 4: Preliminary Cassava framework Map of MEcu-72 for Resistance to White Fly, consisting of SSRs and a RGA (Contig39) (Lod = 25 and theta = 25) Rec Dist Marker Frac. cM Id Name (67) SSRY242 ( 7.7 %) 7.8 (128) rSSRY4 Linkage Group A Rec Dist Marker Frac. cM Id Name (99) SSRY50 (12.8 %) 13.1 (58) SSRY225 ( 7.7 %) 7.8 (53) SSRY204 (12.5 %) 12.8 (143) rSSRY112 Linkage Group B Rec Dist Marker Frac. cM Id Name (66) SSRY241 (11.3 %) 11.5 (133) rSSRY79 ( 8.1 %) 8.2 (14) SSRY45 (12.2 %) 12.4 (132) rSSRY76 (12.5 %) 12.8 (139) rSSRY95 (18.0 %) 18.8 (74) SSRY274 (17.4 %) 18.1 (157) rSSRY222 ( 7.9 %) 8.0 (195) rSSRY151 (19.1 %) 20.1 (86) SSRY329 ( 3.6 %) 3.6 (56) SSRY219 (10.3 %) 10.4 (196) rSSRY152 Linkage Group C Rec Dist Marker Frac. cM Id Name (65) SSRY240 ( 2.9 %) 2.9 (2) SSRY40 ( 7.1 %) 7.1 (117) rSSRY3 (17.5 %) 18.3 (116) rSSRY9 (10.1 %) 10.3 (75) SSRY282 ( 8.6 %) 8.6 (169) rSSRY251 ( 5.2 %) 5.2 (178) rSSRY295 Linkage Group D Rec Dist Marker Frac. cM Id Name (170) rSSRY252 (11.8 %) 12.0 (35) SSRY84 (10.3 %) 10.4 (187) rSSRY330 Linkage Group E Rec Dist Marker Frac. cM Id Name (84) SSRY324 ( 9.1 %) 9.2 (88) SSRY331 ( 9.1 %) 9.2 (50) SSRY195 Linkage Group F Rec Dist Marker Frac. cM Id Name (41) SSRY103 ( 3.0 %) 3.0 (42) SSRY106 ( 6.9 %) 6.9 (52) SSRY203 Linkage Group G Rec Dist Marker Frac. cM Id Name (76) SSRY283 ( 4.4 %) 4.4 (4) SSRY77 (17.0 %) 17.7 (179) rSSRY296 ( 7.3 %) 7.3 (200) rSSRY21 ( 8.3 %) 8.4 (118) rSSRY113 Linkage Group H Rec Dist Marker Frac. cM Id Name (48) SSRY192 (13.9 %) 14.2 (3) SSRY54 (17.1 %) 17.8 (124) rSSRY60 Linkage Group I Rec Dist Marker Frac. cM Id Name (29) SSRY69 (11.3 %) 11.5 (31) SSRY74 ( 8.5 %) 8.6 (192) rSSRY146 (12.9 %) 13.2 (26) SSRY170 Linkage Group K Rec Dist Marker Frac. cM Id Name (46) SSRY20 (15.1 %) 15.6 (168) rSSRY250 Linkage Group L Rec Dist Marker Frac. cM Id Name (20) SSRY55 (11.5 %) 11.7 (182) rSSRY317 (15.6 %) 16.1 (12) SSRY39 (17.1 %) 17.8 (137) rSSRY88 ( 9.1 %) 9.2 (197) rSSRY155 Linkage Group M Rec Dist Marker Frac. cM Id Name (9) SSRY31 (15.1 %) 15.6 (93) SSRY149 Linkage Group N Rec Dist Marker Frac. cM Id Name (13) SSRY42 ( 6.0 %) 6.0 (107) rSSRY6 Linkage Group O Rec Dist Marker Frac. cM Id Name (15) SSRY51 (14.2 %) 14.6 (172) rSSRY268 Linkage Group P Rec Dist Marker Frac. cM Id Name (47) SSRY117 (12.2 %) 12.5 (90) SSRY119 Linkage Group Q Rec Dist Marker Frac. cM Id Name (55) SSRY207 (12.6 %) 12.9 (89) SSRY341 Linkage Group R Rec Dist Marker Frac. cM Id Name (63) SSRY238 (15.8 %) 16.4 (191) rSSRY135 Linkage Group S Rec Dist Marker Frac. cM Id Name (105) Cont39 (20.3 %) 21.5 (145) rSRY101 ( 8.3 %) 8.4 (127) rSRY2 (11.8 %) 12.0 (208) rSRY5 ( 8.9 %) 9.0 (165) rSRY229 ( 5.4 %) 5.5 (49) SRY193 ( 5.5 %) 5.5 (73) SRY270 Linkage Group J AFLPs Analysis An analysis was done of 128 combinations of primers with both parentals, MEcu-72 and MCol-2246, and both bulks of 10 whitefly resistant and 10 susceptible DNA. We obtained 53 polymorphic combinations, in which we found 425 polymorphic bands between the resistant and the susceptible (Fig. 5). INTRODUCTION FIELD EVALUATION Field evaluations carried out at Nataima (Tolima) demonstrate that there was considerable whitefly pressure as plant damage and pest populations were high (from 4 to 6 on the damage and population scales). However, some genotypes, in spite of the high pressure, had low damage levels. It can therefore be concluded that these genotypes have resistance levels similar to those of the resistant parent. Whiteflies are considered one of the world’s major agricultural pest groups, attacking a wide range of plant hosts and causing considerable crop loss. There are nearly 1200 whitefly species with a wide host range. As direct feeding pests and virus vectors, whiteflies cause major damage in agroecosystems based on cassava in the Americas, Africa and to a lesser extent, Asia. The most damaging species on cassava in northern South America is Aleurotrachelus socialis. Typical damage symptoms include curling of apical leaves, yellowing and necrosis of basal leaves and plant retardation. The ”honeydew” excreted is a substrate for a sooty-mold fungus that interferes with photosynthesis (Fig. 1). The rate reduces root yield by 4 to 79% depending on the duration of attack (Bellotti, 2002). More than 5,000 cassava genotypes have been evaluated at CIAT and CORPOICA for whitefly resistance. At present, the major source of host resistance in cassava is the genotype MEcu-72 (Bellotti and Arias, 2001) (Fig. 1). When feeding on MEcu-72 A. socialis had less oviposition, longer development periods, reduced size and higher mortality than when feeding on the susceptible genotype, (Fig, 2). Due to the importance of whiteflies as a pest and virus vector, it is important to understand the nature of genes that confer resistance in the genotype MEcu-72. To study the genetics of this resistance, a cross was made between MEcu-72 (resistant genotype) x MCol-2246 (susceptible genotype), to evaluate F1 segregation, using molecular markers. This will accelerate the selection of whitefly resistant germplasm and isolate resistant genes. Fig. 5. Silver stained polyacrylamide gel showing: combination ACA-CTT of AFLP of both parents (R resistant, S susceptible) and Bulks resistants and susceptibles, show the polymorphic band # 50 unique in the resistants. A B C D Fig.1. A: Nymphal Stages of A. socialis, on a cassava leaf. B: Leaf curling on a cassava plant with high populations of A. socialis. C: Presence of sooty mold fungus on a cassava leaves attacked by A. socialis. D: Resistant genotype MEcu-72 and a susceptible genotype. % Mortality (n=200) Develop time (days) M Ecu 72 CG 489 -23 M Col 1505 CG 489 -4 CG 489 -31 CM C 40 CG 489 -34 M Bra 12 80 70 60 50 40 30 20 10 0 0 5 10 15 20 25 30 35 R R S R R S R T Fig. 2. Whitefly (A. socialis) nymphal mortality on resistant (R), tolerant (T) and susceptible (S) cassava clones. MATERIALS AND METHODS PLANT MATERIAL For the present work we have used the cross MEcu-72 (as the resistant parent) x MCol-2246 (as the susceptible parent). A total F1 offspring of 286 genotypes (family CM8996) was produced from this cross. These materials were sowed and evaluated in the field during 2002 and 2003 at two different locations in Colombia: Espinal- Tolima, (CORPOICA-NATAIMA) at 350 m.a.s.l. and Santander de Quilichao, Cauca, at 990 m.a.s.l. With this evaluation we will identify gene segregation in the offspring and we will be able to select the resistant and susceptible materials. The evaluation was performed in the field using population and damage scales. MOLECULAR ANALYSIS We are using Simple Sequences Repeat (SSR) and AFLPs to find markers associated with resistance for mapping the resistant gene(s). We are using RGAs sequences (isolated from cassava previously). REFERENCES Arias B. 1995. Tesis de Maestria. Universidad Nacional de Colombia, Palmira, Valle. Bellotti, A.C. and B. Arias, 2001. Crop Protection 20:813-823. Bellotti, A.C. 2002. (Eds. R.J Hillocks, J.M. Thresh and A.C. Bellotti). CAB International 2002. 332pp. CIAT, 1995. Cassava program annual report. 54p. Fregene, MA, Angel, F, Gomez, R, Rodriguez, F, Roca, W, Tohme, J. and M. Bonierbale. 1997. Theor. Appl. Genet. 95: 431-441. Mba, R.E.C., Stephenson, P., Edwards, K., Melzer, S., Mkumbira, J., Gullberg, U., Apel, K., Gale, M., Tohme, J. and Fregene, M. 2001. Theoretical and Applied Genetics Journal. 102:21-31. March 8-14, 2004 – CBN-VI.