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 SAMtools²⁰ and annotated using snpEFF.²¹ 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.