DETECTION OF THE TOMATO MI 1.2 GENE BY PCR USING NON-ORGANIC DNA PURIFICATION
Keywords:germplasm, lycopersicon, meloidogyne, nematode, resistance, root-knot, susceptibility
AbstractA PCR based method using DNA extracted with non-organic reagents was developed to detect the presence of the two alleles (Mi and mi) of the tomato Mi 1.2 resistance gene in plant material. Plant tissue from young leaves of tomato plants was blotted onto a FTA® Card (Whatman, Inc.) and processed according to the manufacturer's instructions. Individual samples from the FTA® Card containing the DNA template were used in PCR reactions. The forward primer (IMOF1) anneals to the 3' end of the conserved region and the beginning of the intron, and the reverse (IMOR1) anneals to the C terminal of the leucine-rich repeat (LRR), amplifying a 998 bp DNA fragment corresponding to the marker for the resistance allele (Mi) and a 900 bp fragment corresponding to the marker for the susceptible allele (mi). Tomato cultivars Motelle and Motaci (isogenic for the Mi 1.2 locus), L. peruvianium f. glandulosumcv. Canta, L. esculentum var. cerasiforme VFNT Cherry, Mossol, Motabo, G-178 and VFN-8 showed the marker for the homozygous resistant genotype (Mi/Mi). Tomato cultivars Alicante, Harzfeuer F1, Marglobe, Marmande and Rutgers showed the marker for the homozygous susceptible genotype (mi/mi) and cultivar Big Boy showed both markers, corresponding to the heterozygous resistant genotype (Mi/mi). The chi-square value using these markers was [chio= 1.2. Two advanced tomato lines P1 (Mi/Mi) and P2 (mi/mi) and 13 individual plants of their hybrid F[sub1] progeny were also analyzed, with the Mi and mimarkers present in the resistant (P1) and susceptible parent (P2), respectively, and both markers present in all the heterozygous F[sub1] individuals. This PCR and FTA® Card combination results in a fast, accurate, cost effective and reliable method for use in marker-assisted selection in breeding programmes and plant model studies of the host root-knot nematode interaction.