Plant Materials and RNA Sequencing

Cardamine insueta, C. amara, and C. rivularis plants used in this study were collected from Urnerboden. All plants were grown together in a plant cultivation room with 16 h light and 8 h dark cycle. The plants were planted in single pots, placed on trays, and watered from below.

Submergence treatment was started in the morning at 07:00. Two mature leaves were detached and submerged in water. We isolated RNA from the floating leaflets of the three species at nine time points after the start of submergence treatment (0, 2, 4, 8, 12, 24, 48, 72, and 96 h) using Qiagen RNeasy kit (Qiagen, Maryland, United States). RNA quality was assessed by Bioanalyser Nanochip (Agilent, Santa Clara, United States) and libraries quantified by Qubit (Thermo Fisher, Waltham, MA, United States). In total 27 libraries (3 species × 9 time points) were prepared according to NEBNext Ultra^textTM Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, United States) followed by paired end sequencing (100 bp × 2) on a HiSeq2000 with a HiSeq Paired-End Cluster Generation Kit and HiSeq Sequencing Kit (Illumina, San Diego, CA, United States). Trimmomatic (ver. 0.36) (Bolger et al., 2014) was used for discarding the low-quality reads with parameters of “PE -threads 4 -phred33 ILLUMINACLIP:adapters.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:50”.