A shortcut in forward genetics: concurrent discovery of mutant phenotype and causal mutation in Arabidopsis M2 families via MAD-mapping

Forward genetics is a powerful tool to establish phenotype-genotype correlations in virtually all areas of plant biology and has been particularly successful in the model plant Arabidopsis. This approach typically starts with a phenotype in an M2 mutant, followed by identifying a causal DNA change in F2 populations resulting from a cross between the mutant and a wildtype individual. Ultimately, two additional generations are needed to pinpoint causal DNA changes upon mutant identification. We postulated that genome-wide allele frequency distributions within the mutants of M2 families facilitate discrimination of causal versus non-causal mutations, essentially eliminating the need for F2 populations. In a proof-of-principle experiment, we aimed to identify signalling components employed by the executor-type resistance () protein, Bs4C, from pepper (). In a native setting, is transcriptionally activated by and mediates recognition of the transcription activator-like effector AvrBs4 from the bacterial pathogen . Arabidopsis containing an estradiol-inducible transgene was used in a conditionally lethal screen to identify second-site suppressor mutations. Whole genome sequencing was used for 2 mutant llele-frequency istribution (MAD) mapping in three independent M2 families. MAD-mapping uncovered that all three families harboured mutations in , a novel component of executor R protein pathways. Our work demonstrates that causal mutations observed in forward genetic screens can be identified immediately in M2 families instead of derived F2 families. Notably, the timesaving concept of MAD mapping should be applicable to most crop species and will advance the appeal of forward genetics beyond applications in fundamental research.