Next-generation sequencing as a tool to quickly identify causative EMS-generated mutations

Abstract

The advent of next generation sequencing has influenced every aspect of biological research. Many labs are now using whole genome sequencing in Arabidopsis thaliana as a means to quickly identify EMS-generated mutations present in isolated mutants. Following identification of these mutations, examination of T-DNA insertional alleles defective in candidate genes or complementation of the mutant phenotype with a wild type copy of candidate genes can be used to verify which mutation is causative for the phenotype of interest. Here, we discuss the benefits and pitfalls of using this method to identify mutations underlying phenotypes.

Keywords: ABA, abscisic acid; AR, ABA Root Resistance; EMS, ethylmethane sulfonate; NGS, next-generation sequencing; PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; abscisic acid; ethylmethane sulfonate; mutation mapping; next-generation sequencing.

Figure 1.

Scheme for identifying potential causative mutations in Arabidopsis with NGS. In this example, we cross an ABA-resistant (AR) mutant created by ethyl methanesulfonate (EMS) treatment to wild type (Columbia-0). The resultant F₁ is self-pollinated to create a segregating F₂ population. Individuals displaying the phenotype of interest (ABA resistance in root elongation) are selected and allowed to self-pollinate to create F₃ progeny. These progeny are retested for the phenotype of interest, tissue from multiple retested F₃ lines are pooled, and bulk genomic DNA is sequenced using Illumina technology. The resulting reads are aligned to the reference sequence (TAIR v10) using Novoalign (Novocraft; http://novocraft.com) and SNPs identified by SAMtools and annotated using snpEFF. We then compare identified canonical EMS-induced changes (G-to-A or C-to-T) from our mutant to our lab wild type Col-0 strain to identify mutations unique to the mutant.