Virus resistance and molecular mutational analysis of tomato mutant lines

Tomato leaf curl Philippine virus (ToLCPV) is the causal agent of the damaging tomato leaf curl disease prevalent in the Philippines. Through the use of ethylmethane sulfonate (EMS) in inducing mutation in tomato, genetic materials for ToLPCV resistance breeding were generated. Pool 1 of these materials with known susceptible and resistant controls was screened for host resistance, of which 26 entries were selected as candidate resistant lines. The lines were re-evaluated using larger sample size with known disease progress parameters (disease scores and AUDPC). Nine individual plants with at least 0 (immune) to 1 (highly resistant) disease response rating were selected and were subjected to PCR analysis to detect possible infection and differential loads of ToLCPV. After PCR optimization to amplify just enough copies of the target virus genome sequence, the differential titer load of the virus among the selected plants and controls was determined. This is the first report applying standard PCR protocol but reduced amplification to screen for differential virus infection in the host plant. To validate resistance, field trial is being conducted consisting of the progenies of all the sister lines of the nine selected plants. TILLING (Targeting Induced Local Lessions IN Genome) and DCode (DGGE, SSCP) mutation analysis is also being optimized to characterize the induced resistance through EMS mutagenesis. The samples used for preliminary analysis of natural SNPs are the selected resistant mutants, controls Hawaii 7996 and wild relative Solanum pimpinellifolium, and DCode control kit containing wild-type and mutant DNA samples. Four SNP markers with adaptor 40-base pair GC clamps are initially in mutation screening. For TILLING, tomato positive controls are established using commercial Surveyor Nuclease sup(Tm) S1 endonuclease. Digesting heteroduplexes of Hawaii 7996 and S. pimpinellifolium PCR products for the 15 CG genes has confirmed the natural SNPs inherent at CG1 and CG8 genes.