Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium

Abstract

Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum. Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost-and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods.

A better way to discover gene function in the social amoeba Dictyostelium discoideum

“…However, loss-of-function mutations in a gene that acts in a positive fashion downstream from the disrupted process will never be detected. On the other hand, the technique described by Li et al (2016) can result in gain-of-function mutations in positively acting genes that suppress the original phenotype. The random mutations resulting from treatment with NTG can change amino acid codons, generate new start or stop signals, or alter regulation.…”

“…That is, not all developmental genes can be discovered in REMI knockout mutants because some are expected to be vital for growth as well as development. In this issue of Genome Research, Gad Shaulsky and his colleagues describe a next-generation technique that fills this gap (Li et al 2016). …”

“…The approach described by Li et al (2016) obviates genetic mapping by exploring a sufficiently large mutagenized population. When a gene is found to be mutated in several independently isolated mutant strains, it becomes a candidate for the function under scrutiny.…”

“…Using short read alignments against the 34-Mb reference sequence of the organism, significant base changes have to be flagged and inspected in the context of the affected gene. This process has been automated for Dictyostelium, and the code is available online (Li et al 2016). …”

“…The advantages of chemical mutagenesis Li et al (2016) show that the critical base changes in Dictyostelium discoideum mutants generated by chemical mutagenesis can be pinpointed by whole-genome sequencing as long as a sufficient number of mutants with the same phenotype are analyzed, an approach that has been used in different model species (Smith et al 2008;Irvine et al 2009;Haelterman et al 2014;Schneeberger 2014). Although random mutagenesis with NTG will affect many genes that have nothing to do with the selected trait, any gene directly responsible for the phenotype will be found to have been mutated in multiple independent mutant strains.…”