Next‐Gen Sequencing‐Based Mapping and Identification of Ethyl Methanesulfonate‐Induced Mutations in Arabidopsis thaliana

Abstract

Forward genetic analysis using ethyl methanesulfonate (EMS) mutagenesis has proven to be a powerful tool in biological research, but identification and cloning of causal mutations by conventional genetic mapping approaches is a painstaking process. Recent advances in next-gen sequencing have greatly invigorated the process of identifying EMS-induced mutations corresponding to a specific phenotype in model genetic hosts, including the plant Arabidopsis thaliana and the nematode Caenorhabditis elegans. Next-gen sequencing of bulked F2 mutant recombinants produces a wealth of high-resolution genetic data, provides enhanced delimitation of the genomic location of mutations, and greatly reduces hands-on time while maintaining high accuracy and reproducibility. In this unit, a detailed procedure to simultaneously map and identify EMS mutations in Arabidopsis is described.

Jasmonate signalling in Arabidopsis involves SGT1b–HSP70–HSP90 chaperone complexes

“…We used a genome resequencing-assisted approach 22,23 to map and identify the HSM1 gene. Computational analyses described in Methods showed that the SGT1b gene (At4g11260) was the most likely candidate corresponding to hsm1 .…”

“…The HSM1 ( SGT1b ) gene was cloned following a genome resequencing–assisted mapping and cloning procedure for EMS mutants 22,23 . Briefly, hsm1 was outcrossed to Ler to yield an F1 hybrid, followed by self-fertilization to produce F2 segregants.…”

GIPS: A Software Guide to Sequencing-Based Direct Gene Cloning in Forward Genetics Studies

“…The homozygous pho2 mutant (HNE8536) was prepared as described by Wang et al (2009). Hydroponic and soil pot experiments were performed as described by Zhou et al (2008).…”

“…An inorganic Pi content measurement was performed following the method described by Zhou et al (2008).…”

“…In general, methods for mapping the locations of phenotypeassociated mutations using genetic crosses and genome sequencing were well summarized by Schneeberger (2014). Previous sequencing-based forward genetics studies in plants (Schneeberger et al, 2009;Hong et al, 2010;Austin et al, 2011;Zhang et al, 2014) were typically based on bulked segregant analysis, whereby a mutant is crossed to a wild type to create an F2 (or a BC1F2) population. The F2 population is subsequently screened for the mutant phenotype.…”