Construction Of Molecular Genetic Map By AFLP Markers And Resistance Gene Multi-caps Assistant Selection In Tomato

Construction of a high density genetic map is the basis of gene localization, gene cloning, structural and functional genome research. In this study, the facts which affected on the AFLP amplified results of tomato were studied. Then, on the basis of the optimized system, An AFLP genetic linkage map was constructed using an REL population derived from the hybrid of Lycopersicon esculentum Mill. ' 99165' × Lycopersicon hirsutum f. typicum LA1777. Baed on the major diseases including root knot nematode, tomato mosci virus (ToMV), tomato spotted wilt virus (TWSV) in China for tomato production, a SCAR system was developed. Then a multiple PCR system could be used to simultaneousely identify both of genes including Mi gene with resistance to root knot nematode, Tm22 gene with the resistance to ToMV and Sw-5 gene with the resistance to TSWV. The established multi-CAPS could be used as an efficient tool for the selection assisted by molecular makers at the early stage for tomato breeding.The results are as follows:1. 22 polymorphic primer pairs with clear and repetitive amplification were obtained by screening 64 EcoRI/MseI primer combinations an average of 11.4 polymorphic bands were observed from each AFLP primer combination.2. By the analysis of total 274 AFLP molecular markers with software JoinMap 3.0, a total of 125 markers were mapped to 18 linkage LGs, covering 662cM with an average distance of 5.3cM cM between loci. The number of markers inevery linkage LG varied from 3 to 22, and the length is from 14 cM to 58 cM.3.Optimization on SCAR-CAPS system of tomato resistance gene:A standard SCAR-CAPS system fitting for tomato was established. The results showed that each optimized amplification reaction contained in 25μl: 30-50μg DNA, 0.2μmol ·L^-1 of each primer, 200μmol·L^-1 of dNTP, 1.5-2.5mmol ·L^-1Mg²⁺and 1.5unit Taq DNA polymerse. The optimized PCR cycles started at 94℃(3min), followed by 40cycles of 1 min at 94℃, 1 min at 56℃ and 2 min at 72℃, finally extended 5min at 72℃. 1.5% agarose gel in discriminating polymorphism.4.The PCR products were completely correspond to the amplified bands produced by single SCAR primer. Among them, co-dominant SCAR1 marker tightly linked with Mi gene produce 750bp fragment in both resistant and susceptible tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme TaqI and HindIII. Genotype with homozygous and heterozygous Mi gene could produce respectively 570bp, 160bp and 750, 570bp, 160bp bands. Susceptible genotypes still present 750bp fragment. The dominant SCAR2 marker tightly linked with SW-5 gene would produce only 400bp PCR product in resistant genotype. The co-dominant SCAR3 marker tightly linked with Tm2² gene would produce 950bp fragment in both resistant and susceptible tomato lines.