Mutagenomics: A facile method to identify causative mutations from a genetic screen

Genetic screens are remarkably powerful tools to dissect complex biological processes, but a rate-limiting step is generally the cloning of targeted genes. Here, we present a strategy, which we call "mutagenomics", for identifying causal mutations from a screen in a parallel fashion in the absence of backcrossing. Mutagenomics starts by sequencing the genomes of the mutant lines identified, which then are subjected to a three-stage pipeline. The first stage is the identification of sequence changes in genes previously linked to the targeted pathway. Next, genes are identified that are represented by multiple independent alleles more often than expected by chance, which was determined using a simulation strategy. For the remaining lines, candidate genes are identified by sequencing multiple lines of common descent. Our simulations suggest that sequencing as few as five sibling lines generally results in fewer than five candidate genes. This pipeline has the capacity to identify the genes corresponding to most mutants identified in a screen. We applied mutagenomics to a screen for mutants involved in the response to the phytohormone cytokinin. Mutagenomics identified likely causative genes for many of the mutant lines analyzed from this screen, including 13 alleles of the gene encoding the AHK4 cytokinin receptor. The screen also identified HY5, which encodes a basic domain/leucine zipper (bZIP) transcription factor that is a master regulator of photomorphogenesis. HY5 was found to mediate a subset of the transcriptional response to cytokinin. Mutagenomics has the potential to accelerate the pace and utility of genetic screens in Arabidopsis.