Arias, V.B.

Bellotti, Anthony C.

Bohórquez Cháux, Adriana

Duque E., Myriam Cristina

Mba, C.

Tohme, Joseph M.

Trujillo, G.

Vargas Bonilla, H.L.

Vargas Daza, Jaime

CGSpace

A. Bellotti1 ,  A. Bohórquez1,  J. Vargas1, B. Arias1, H.L. Vargas2, C. Mba3, M.C. Duque1, J. Tohme11Centro Internacional de Agricultura Tropical (CIAT), AA 6713, Cali, Colombia2 CORPOICA, Espinal, Tolima, Colombia. 3Intern. Atomic Energy Agency. P.O Box 100, A-1400, Vienna, Austria. http://www.ciat.cgiar.org/ipm/index.htmRECENT ADVANCES IN HOST PLANT RESISTANCETO WHITEFLIES IN CASSAVACentro Internacional de Agricultura TropicalInternational Center for Tropical AgricultureRESULTS AND DISCUSSIONCONCLUSIONS 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 RESISTANCEThe molecular data are being analyzed using QTL packages (QTL cartographer Qgene) to determine linkages between the markers and the phenotypic characterization.  As preliminary analysis X2 at the 5% level was done using SAS. Putative associations were found between 43 SSRsmarkers and the resistance.MOLECULAR ANALYSISBoth 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 cDNAsequences (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 susceptiblesgenotypes 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. socialisfeeding on parental genotypes MEcu-72, MCol-2246 and clones from the family CM 8996 at CORPOICA, Nataima (Tolima, Colombia).00.511.522.533.54Damage and Population scaleMECU 72 MCOL 2246DAMAGE LEVELPOPULATION870 011401 015115350280204060801001201401601 1.1-  2 2.1 - 3 3.1 - 4 4.1 - 5 5.1 - 6No. ClonesDamage level PopulationFig. 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 MarkerFrac. cM Id Name(67) SSRY242( 7.7 %) 7.8(128) rSSRY4Linkage Group ARec Dist MarkerFrac. cM Id Name(99) SSRY50(12.8 %) 13.1(58) SSRY225( 7.7 %) 7.8(53) SSRY204(12.5 %) 12.8(143) rSSRY112Linkage Group BRec Dist MarkerFrac. 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) rSSRY152Linkage Group CRec Dist MarkerFrac. 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) rSSRY295Linkage Group D Rec Dist MarkerFrac. cM Id Name(170) rSSRY252(11.8 %) 12.0(35) SSRY84(10.3 %) 10.4(187) rSSRY330Linkage Group ERec Dist MarkerFrac. cM Id Name(84) SSRY324( 9.1 %) 9.2(88) SSRY331( 9.1 %) 9.2(50) SSRY195Linkage Group FRec Dist MarkerFrac. cM Id Name(41) SSRY103( 3.0 %) 3.0(42) SSRY106( 6.9 %) 6.9(52) SSRY203Linkage Group GRec Dist MarkerFrac. 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) rSSRY113Linkage Group HRec Dist MarkerFrac. cM Id Name(48) SSRY192(13.9 %) 14.2(3) SSRY54(17.1 %) 17.8(124) rSSRY60Linkage Group IRec Dist MarkerFrac. cM Id Name(29) SSRY69(11.3 %) 11.5(31) SSRY74( 8.5 %) 8.6(192) rSSRY146(12.9 %) 13.2(26) SSRY170Linkage Group KRec Dist MarkerFrac. cM Id Name(46) SSRY20(15.1 %) 15.6(168) rSSRY250Linkage Group LRec Dist MarkerFrac. 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) rSSRY155Linkage Group MRec Dist MarkerFrac. cM Id Name(9) SSRY31(15.1 %) 15.6(93) SSRY149Linkage Group NRec Dist MarkerFrac. cM Id Name(13) SSRY42( 6.0 %) 6.0(107) rSSRY6Linkage Group ORec Dist MarkerFrac. cM Id Name(15) SSRY51(14.2 %) 14.6(172) rSSRY268Linkage Group PRec Dist MarkerFrac. cM Id Name(47) SSRY117(12.2 %) 12.5(90) SSRY119Linkage Group QRec Dist MarkerFrac. cM Id Name(55) SSRY207(12.6 %) 12.9(89) SSRY341Linkage Group RRec Dist MarkerFrac. cM Id Name(63) SSRY238(15.8 %) 16.4(191) rSSRY135Linkage Group SRec Dist MarkerFrac. 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) SRY270Linkage Group JAFLPs AnalysisAn 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).INTRODUCTIONFIELD EVALUATIONField 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. ABCDFig.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 72CG 489 -23M Col 1505CG 489 -4CG 489 -31CM  C 40CG 489 -34M Bra 12807060504030201000 5 10 15 20 25 30 35RRSRRSRTFig. 2.  Whitefly (A. socialis) nymphal mortality on resistant (R), tolerant (T) and susceptible (S) cassava clones.MATERIALS AND METHODSPLANT MATERIALFor 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 ANALYSISWe 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).REFERENCESArias 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.

Recent advances in host plant resistance to whiteflies in cassava

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Recent advances in host plant resistance to whiteflies in cassava

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