Illumina HiSeq 2000 sequencing; Drought treatment and ABA feeding to ABA insensitive Populus tremula × tremuloides lines
Drought treatment and ABA feeding to ABA insensitive Populus tremula × tremuloides lines
A piece of stem was collected during the harvest and the bark was removed. The woody material was and shock frozen in liquid nitrogen and kept at -80 °C. Plants were multiplied by in vitro propagation and kept on 1/2 MS-medium for four weeks. Afterwards, rooted plants were potted in 1.5 L pots and kept in a CO2 chamber at a temperature of 22 °C, relative humidity of 80% and additional light (16 h, PAR of 100 μmol photons m-2 s-1). Soil mixed with 25 % v/v coarse sand (Ø 0.71 – 1.25 mm) and 25 % v/v fine sand (Ø 0.4 – 0.8 mm) was used. The chamber was supplemented with 1000 ppm CO2. The plants were grown there for six weeks and well irrigated before the treatment started. Three treatments were applied for about 4 weeks: well-watered (control), ABA feeding and drought. The control plants and ABA-fed plants were well-watered with tap water or ABA solution (ABA dissolved in tap water, 100 µM in the first two weeks and 200 µM from the 3rd week on) to keep the soil moisture around 0.4 m3 m-3. The soil moisture of drought-treated plants was gradually decreased from 0.4 to 0.1 m3 m-3 in the first two weeks and was kept constantly between 0.1 and 0.15 m3 m-3 afterwards. Nucleic acid was extracted according to CTAB extraction protocol (Chang et al., 1993). TruSeq mRNA Sample Prep kit v2 (Illumina, San Diego, CA, USA) according to manufacturer's instructions.