Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing

Tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. EMS mutants of tomato have recently proved their utility for discovering new functions in plants leading to improved breeding stock for superior tomato varieties. However, until recently, the identification of causal mutations underlying remarkable phenotypes has been a very lengthy task that many labs could not afford due to spatial and technical limitations. Here, we describe a simple protocol for mapping-by-sequencing causal mutations in tomato, in which the phenotypes of interest are first isolated by screening a mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then generated by crossing the mutant with a wild type (non-mutagenized) genotype and the F2 segregants displaying the same phenotype are pooled. Finally, whole genome sequencing and analysis of allele distributions in the pools allows the identification of the causal mutation. The whole process takes 6 to 12 months, from the isolation of the tomato mutant to the identification of the causal mutation. This strategy overcomes many previous limitations, is of simple use and can be applied in most labs with limited facilities for plant culture and genotyping.