Gene-specific primers for soybean TCS genes were designed using the Primer3 software.23 (link) Primer specificity was confirmed by blasting each primer sequence against the soybean genome (Glyma1 model),24 (link) followed by analysing the melting curves and amplicon fragments. Primers were redesigned if the corresponding melting curve did not yield a single sharp peak and/or if they had an electrophoresis pattern that failed to produce a single amplicon of the correct predicted length. The CYP2 gene was selected as a reference gene in the expression profiling of soybean genes as recommended previously.25 (link) Quantitative real-time PCRs (RT-qPCR) were performed in 96-well plates on a Stratagene MX3000P system (Agilent Technologies, Santa Clara, CA, USA) using Thunderbird™ SYBR® qPCR Mix (Toyobo, Japan) reagents. Primer sets of 0.4 µM final concentrations for each primer were used in a final volume of 10 µl well−1. The thermal profile of the RT-qPCRs was at 95°C for 1 min, 40 cycles at 95°C for 15 s and at 60°C for 1 min. Dissociation curves were obtained using a thermal melting profile performed after the last PCR cycle: 95°C for 15 s followed by a constant increase in the temperature between 60°C and 95°C. Background-corrected raw fluorescence data were exported from the MX3000P system and analysed in LinRegPCR software with a built-in baseline correction and amplification efficiency calculation.26 (link),27 (link) Amplicon-based fluorescence thresholds were used to obtain the Ct values, and these values together with the amplicon-based mean efficiency were used for calculating the initial quantity of mRNA transcripts. Finally, the mRNA levels of each transcript were normalized with those of the corresponding CYP2 transcript.