NextSeq 500 sequencing; Transcription profiling of Populus x canescens leaves to evaluate the effect of co-cultivation with ectomycorrhizal fungus Laccaria bicolor
Leaves were cut off and directly frozen in liquid nitrogen Populus x canescens plants were propagated and cultivated as described by Müller et al. (2013). Plants were in vitro multiplicated by micro cuttings and grown on half strength Murashige & Skoog medium for three weeks at 23 °C/24 °C, 60-85 μE×m−2×s−1 photosynthetically active radiation (PAR) and a 16 h/8 h day/night regime. Subsequently, the plants were transferred in an in vitro sandwich system (Müller et al. 2013) and either cultivated in the presence or absence of the ectomycorrhizal fungus Laccaria bicolor. After three weeks, the plants were potted into 0.65 L pots containing a mixture of washed and twice autoclaved sand (0.71-1.21 mm and 0.4-0.8 mm particle size and soil a ratio of 10:2 v/v). For acclimation the plants were covered with a transparent plastic beaker for one week, which got gradually lifted during the second week and ultimately removed after 14 days. All plants were treated with modified Long Ashton nutrient solution (Hewitt, 1952) containing 2 mM KNO3 excess nutrient solution ran off. The plants got transferred into 3 L pots containing the same substrate after 4 weeks of greenhouse cultivation. Plants were randomized regularly. The leaves were harvested for transcriptome analysis after 13 weeks of green house cultivation. Plants were cultivated in the presence or absence of the ectomycorrhizal fungus Laccaria bicolor (see growth protocol). RNA from Populus leaves was extracted using the innuPrep Plant RNA kit (Analytik Jena, Germany) according to the manufacturer's protocol Libraries were constructed using the TrueSeq RNA Library Prep Kit (Illumina) according to the manufacturer's instructions