Fast Isogenic Mapping-by-Sequencing of Ethyl Methanesulfonate-Induced Mutant Bulks

Mapping-by-sequencing (or SHOREmapping) has revitalized the powerful concept of forward genetic screens in plants. However, as in conventional genetic mapping approaches, mapping-by-sequencing requires phenotyping of mapping populations established from crosses between two diverged accessions. In addition to the segregation of the focal phenotype, this introduces natural phenotypic variation, which can interfere with the recognition of quantitative phenotypes. Here, we demonstrate how mapping-by-sequencing and candidate gene identification can be performed within the same genetic background using only mutagen-induced changes as segregating markers. Using a previously unknown suppressor of mutants of like heterochromatin protein1 (lhp1), which in its functional form is involved in chromatin-mediated gene repression, we identified three closely linked ethyl methanesulfonate-induced changes as putative candidates. In order to assess allele frequency differences between such closely linked mutations, we introduced deep candidate resequencing using the new Ion Torrent Personal Genome Machine sequencing platform to our mutant identification pipeline and thereby reduced the number of causal candidate mutations to only one. Genetic analysis of two independent additional alleles confirmed that this mutation was causal for the suppression of lhp1.In Arabidopsis (Arabidopsis thaliana) research, ethyl methanesulfonate (EMS) mutagenesis is a powerful tool that has been widely explored to uncover the functionality of many genes in a broad spectrum of pathways (Page and Grossniklaus, 2002). Recent advances in sequencing technology have greatly reduced the time required to pinpoint induced mutations. In a proof-of-principle experiment, mapping-by-sequencing (SHOREmapping) was first demonstrated on a mutant in the background of the Arabidopsis reference accession Columbia (Col-0) crossed to the diverged accession Landsberg erecta. A pool of DNA isolated from bulked segregants was sequenced and used for the simultaneous mapping and mutant identification (Schneeberger et al., 2009b). This first application was followed by other studies successfully applying similar methods (Cuperus et al., 2010; Austin et al., 2011).Although all described approaches are straightforward and extremely fast, their application is hindered by the requirement for interaccession crosses that impedes the success rate of screens based on quantitative traits, such as screens for genetic modifiers. The major obstacle is that the considerable phenotypic variation in F2 populations from crosses between diverged accessions impairs the recognition of mutants with subtle phenotypic alterations. In addition, if genetic screens involve modifiers of a preexisting mutant, the mapping depends on the availability of the primary mutant in another suitable accession, the introgression of the mutation in such a background, or the laborious additional genotyping for the presence of the first-site mutation.Avoiding these disadvantages, Ashelford et al. (2011) have demonstrated that the isolation of a causative EMS-induced change is possible by direct resequencing of a complete mutant genome. However, their approach initially resulted in 103 putative causal mutations that had the potential to change the amino acid sequences of 48 putative proteins. In addition, the mutations were clustered in two separate regions of the genome, even though the mutant had been backcrossed four times to the parental line.Recently, Abe et al. (2012) reduced the large number of candidate mutations by backcrossing mutant genomes to their nonmutagenized progenitor, followed by sequencing bulk segregants from these crosses. This drastically reduced the number of causal candidates, although it was not possible to pinpoint the causal change from the sequencing data alone. The main problem remains the short-read coverage at each of the candidate mutations, which is typically lower than the number of individuals combined within the bulked DNA. This hinders accurate allele frequency estimations based on the whole-genome sequencing data alone and thus makes it impossible to distinguish between causal and closely linked mutations.In this study, we combined isogenic bulk segregant analysis with deep candidate resequencing (dCARE) to facilitate the mutation identification of genetic modifiers based on bulked DNA and sequencing data alone. Our approach relies on the assumption that in pools of bulked segregants, the causative change occurs with the highest frequency among all EMS-induced changes (Fig. 1). Using resequencing data alone, it is not possible to distinguish between the subtle allele frequencies of EMS changes that are closely linked. However, dCARE of all candidate mutations using the new Ion Torrent sequencing technology enables quick and cost-effective detection of subtle allele frequency differences between closely linked mutations and thus allows the identification of causal candidates.Open in a separate windowFigure 1.Schematic illustration of the fast isogenic mapping approach. Chemical mutagens typically introduce hundreds of novel mutations. Within the M2 generation, mutants are screened for phenotypes. Selected plants are backcrossed to the nonmutagenized progenitor. The F2 offspring of such a cross forms an isogenic mapping population, as only novel mutations are segregating. Backcrossed individuals that display the mutant phenotype are selected, bulked, and their DNA is prepared as a pool and whole-genome sequenced. If the parental line is genetically different from the reference line Col-0, it needs to be resequenced in order to control for naturally occurring differences that need to be differentiated from novel mutations. Thus, all novel EMS-induced mutations can be selected for SHOREmap analysis by filtering for mutations that do not reside in the parental line. Candidate mutations (gray box) that show high mutant allele frequencies and linkage are selected for dCARE to pinpoint the causal mutation.The mutant identified by this fast isogenic mapping approach was isolated as a suppressor of developmental aberrations caused by defects in LIKE HETEROCHROMATIN PROTEIN1 (LHP1), which participates in the Polycomb Group (PcG) gene regulatory pathway in Arabidopsis. Enhancer/suppressor screens have been successfully used to identify genes that play a role in chromatin-mediated gene repression and activation in Drosophila melanogaster. For example, many components of the repressive PcG pathway were isolated as genetic enhancers or suppressors of homeotic mutations, whereas components of the Trithorax Group protein pathway were originally identified as suppressors of PcG-related mutations (Landecker et al., 1994; Gildea et al., 2000; Alonso et al., 2007).