Identification of the causative mutation (using Whole Genome Sequencing) in Arabidopsis EMS mutants showing subcellular trafficking defects

Goal of the study was to identify mutants resistant to auxin inhibition of endocytosis. Overall design: M2 generation of 5-day-old EMS-mutagenized PIN1:PIN1-GFP seedlings (progenies of 1, 920 M1) were co-treated with 10 µM NAA and 25 µM BFA for 1 hour and screened under the fluorescent microscope for individuals with PIN1- GFP signal accumulated in BFA bodies, therefore insensitive to auxin effect on endocytosis. In M3 generation, the candidates were confirmed, obtained alleles were back crossed to the parental line 3 times and the homozygous lines were selected showing clear cellular phenotype. For next generation sequencing, genomic DNA of 3 homozygous backcrossed individuals and the parental PIN1:PIN1-GFP plant were submitted to Vienna Biocenter Core Facility for whole-genome sequencing. Interactive tool, artMAP (Javorka et al., 2019) was used to map EMS induced mutations.