96 well optical reaction plates

All qRT-PCR reactions were carried out in Realplex (Eppendorf, Germany) Real Time PCR system using SYBR Green in 96 well optical reaction plates (Axygen, USA) sealed with ultra-clear sealing film (Platemax, USA). The PCR reaction was performed in a total volume of 10 µl, containing 1 µl of RNA (100 ng), 400 nM of each primer, 5 µl of 2× one step SYBR RT-PCR buffer 4 and 0.4 µl of prime script one step Enzyme Mix 2 (Takara, Japan) and made to 10 µl with Rnase-free H 2 O. The qRT-PCR cycling conditions were as follows: 42 °C for 5 min and 95 °C for 10 s (reverse transcription) followed by 40 cycles of 15 s at 95 °C, 15 s at 62 °C with fluorescent signal recording and 15 s at 72 °C. All samples were collected from three independent plants and tested in three technical replicates. The raw Cq values of each gene were taken as the input data for estimating relative and average expression of candidate gene using qBase plus software (ver: 2.4; Biogazelle) (Hellemans et al. 2007) .