marker-assisted selection in wheat

156Marker-assisted selection – Current status and future perspectives in crops, livestock, forestry and fishest and the high cost of genotyping largepopulations has and will continue to limitthe use of MAS in most tomato breedingprogrammes.The potential of MAS to speed up thebreeding of tomato using molecular markerslinked to various resistance genes hasbeen examined in the authors’ laboratory.The two main goals of the research wereto find the most suitable markers, andto test the feasibility of MAS for pyramidingresistance genes in tomato “elite”lines selected for their good processingqualities.STRATEGIES FOR GENE TRANSFER ANDPYRAMIDINGSix tomato genotypes carrying variousresistance genes (Table 3) were crossed withtomato “elite” lines previously selected foryield and quality but lacking resistancetraits. Each resistant genotype was crossedinitially with each “elite” tomato line andvarious backcross schemes were then carriedout starting from different F 1 hybrids.At each backcross generation the screeningof resistant genotypes was performed usingmolecular markers linked to the resistancegenes and DNA extracted from youngleaves at seedling stage. Only resistantplants were then transplanted and grown inthe greenhouse. At flowering, crosses weremade with the recurrent parent to obtainthe subsequent generations.As the efficiency of MAS depends on theavailability of polymerase chain reaction(PCR)-based markers highly linked to theresistance gene to be selected, for eachresistance gene the most suitable markersystem was investigated. For this purpose,three different strategies were undertaken.The first involved searching PCR markersalready available in the literature andverifying their usefulness on the geneticmaterial used. The second consisted ofdesigning PCR primers from the sequenceof cloned genes reported in the GeneBankdatabase of the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov/Genbank), while the thirdinvolved designing PCR primers fromRFLP markers tightly linked to resistancegenes. This last strategy was made possibleby the availability of sequences of variousmapped tomato RFLPs in the SolGenesdatabase (www.sgn.cornell.edu).In most cases, the results were obtainedusing cleaved amplified polymorphicsequences (CAPS; Konieczyn and Ausubel,1993), which require one PCR reaction followedby restriction digest of the amplifiedfragment. In three cases (markers linked togenes Mi, Sw5 and Tm2a), the primers andenzymes used were those reported in the literature(Williamson et al., 1994; Folkertsmaet al., 1999; Sobir et al., 2000). In the caseof gene py-1, the procedure reported inthe literature (Doganlar et al., 1998) wasTable 3Tomato genotypes used as resistant parents in the backcross breeding schemes. For each genotyperesistant genes are reportedGenotype Resistance gene PathogenMomor Frl, Tm2a, Ve Fusarium oxysporum f. sp. radicis-lycopersici, TMV, Verticillium dahliaeMotelle I2, Mi, Ve Fusarium oxysporum f. sp lycopersici, Meloidogyne spp., VerticilliumdahliaeOkitzu I2, Tm2a Fusarium oxysporum f. sp lycopersici, TMVOntario Pto Pseudomonas syringaePyrella py-1 Pyrenochaeta lycopersiciStevens Sw5 TSWV