The effect of different nitrogen concentration treatments on soybean nodulation.

Sterilized seeds were germinated in sterilized vermiculite. After 7 days, seedlings were inoculated with rhizobia (USDA110, OD600=0.8) for 2 hours prior to transplanting into nutrient solution containing 5000 uM or 50 uM N supplied as nitrate and ammonium (3:1). Nutrient solution pH was checked daily and adjusted to 5.8-6.0 as necessary with H2SO4 or KOH. Plants transferred to hydroponics solution were reared in growth chambers (day/night: 14 h/10 h). Roots were harvested five days after inoculation by combining three seedlings as one biological replicate. At each sampling time point, three biological replicates were harvested and quickly immersed in liquid nitrogen prior to being subjected to total RNA extraction and then RNA-seq analysis.