Induction of Variation in the Petunia Mlo Gene for Resistance to Powdery Mildew

To develop genetic resistance to powdery mildew in Petunia hybrida, the mildew locus O gene (Mlo) was identified and characterized. The genomic sequence of petunia Mlo was determined  using petunia EST sequences with homology to tomato SlMlo1. A single copy of PhMlo was identified and its expression pattern in petunia was established by RT-PCR. Two approaches were taken to examine whether eliminating PhMlo expression can confer powdery mildew resistance. First, an EMS-mutagenized population of Petunia hybrida ‘Mitchell’ was developed after optimizing EMS exposure levels. High-resolution melting (HRM) analysis was used to screen DNA from M2 plants for PhMlovariation. Two SNPs were identified that resulted in the missense mutations S130L and G176E. The G176E substitution was predicted by Provean software analysis to be deleterious to PhMlo function. Plants with the S130L substitution may have originated from seed stock contamination. The second approach involves the reduction of PhMlo expression through RNA interference. A PhMlo RNAi construct was developed and is being introduced into petunia by Agrobacterium-mediated transformation.