Solanaceous crops that include tomato and potato are multi-billion dollar crops annually and potato and tomato are the most widely grown specialty crops in the U.S. In general, introgression of genes from wild species that confer resistances to various stresses has been the foundation of most plant breeding through the 20th century to the present. This strategy has been particularly effective in crops such as tomato where the gene pool is narrow and genetic variation is found in related wild species. A major problem with interspecific transfer can be difficulty in introgressing the trait of interest into the target species. Fertilization barriers may demand special techniques, or linkage drag may bring in deleterious traits. Thus, new approaches for (i) direct identification of novel resistance traits in cultivated species and (ii) facile transfer into related species are needed. Approach (i) depends on multi-generation screening of mutator tomato plants for novel resistance traits. Approach (ii) will use Cibus? Rapid Trait Development System (RTDS) to produce non-transgenic, specific mutations identified in approach (i), using chemically synthesized gene repair oligonucleotides (GRONs), in half the time of other breeding techniques. This technology harnesses the cell?s normal DNA-repair systems to correct and change specific targeted bases within the genome of a cell. The Gene Repair OligoNucleotide (GRON), a chemically synthesized oligonucleotide, is designed to create mismatched base-pairs compared to the target sequence within the host organism?s genome. The GRON hybridizes at the target region and the mismatched base-pairs work with the cells repair system at those sites to correct (replace, insert or delete) the designated base(s). Once the correction process is complete the GRON is degraded and the now-modified or repaired gene retains its normal pattern of expression and stability within the genome. Here we propose two linked proofs-of-concept for radically new ways to (i) select for novel crop variants resistant to different stresses and (ii) introduce novel resistance-conferring mutations into a related crop recalcitrant to breeding approaches. Approach (i) employs tomato as a prototype crop and Phytophthora infestans as a prototype pathogen and approach (ii) employs other solanaceous crops. OBJECTIVES: The proposed work will directly and substantially advance two new innovative technologies, (i) accelerated stepwise evolution in "mutator" plants and (ii) Cibus' Rapid Trait Development System (RTDS) for targeted mutagenesis. Specifically the technical objectives of Phase 1 are to 1) identify and sequence the mutator plant line that will serve as the initial parent for generating and identifying mutations associated with disease tolerance in tomato, 2) confirm in a blind reconstruction experiment that the disease screen can efficiently distinguish resistant from sensitive plants and 3) that a purely molecular screen can identify single nucleotide polymorphisms (SNPs) in unselected populations of cells. The long-term objective of this work is to enhance productivity of solanaceous plants
