Fig. 2

A schematic overview of the mutagenesis and TILLING procedure. M0 seeds from two sister plants of a single-seed recurrent inbred descendant of N. caerulescens accession Saint-Félix-de-Pallières were treated with 0.3 or 0.4% EMS to generate M1 seeds. These were sown in trays with 54 plants each. M1 plants were grown and allowed to self-fertilize to generate M2 seed, which was bulk-harvested per tray. A subset of the M2 seeds harvested per tray were sown to grow M2 plants in the greenhouse. In total 5520 and 7000 M2 plants were grown to obtain the 0.3 and 0.4% populations, respectively, the latter of which was used for TILLING. The M2 generation was phenotyped for plant morphological traits and their ionome profile. M3 seeds were harvested from individual M2 plants of the TILLING population. For TILLING by to High Resolution Melting curve analysis (HRM), the genomic DNA of individual plants was isolated using a Kingfisher DNA isolation robot. DNA of four plants was pooled in single wells of 96-well microtiter plates. Each pool was used for PCR amplification. PCR products per pool were subjected to HRM. The wells that contained mutations were selected and subjected to a second HRM screen. Positive samples were again PCR amplified and confirmed by DNA sequencing the PCR product.