qRT-PCR for determining transgene expression

Leaf tissues from each sample were frozen in liquid nitrogen or dry ice immediately after sampling. RNA was isolated using a RNeasy® Plant Mini Kit (QIAGEN, Chadstone Center, VIC, Australia) according to the manufacturer’s protocol. One to two microgram of RNA samples were used for first-strand DNA synthesis in 20 μL reactions using Superscript® III reverse transcriptase kit (Life Technologies, Mulgrave, VIC, Australia). After the reverse transcript reaction, 3 μL of 10× dilutions of synthesis product were used for qPCR reaction using a C1000 Touch^TM thermocycler with the CFX96^TM Real-Time System (Bio-Rad). qPCR conditions included an initial denaturation at 95 °C for 3 min; 40 cycles of denaturation at 95 °C for 10 s and annealing/elongation at 60 °C for 30 s, followed by a melt step range of 65–95 °C with an increment of 0.5 °C. We used the wheat housekeeping gene TaCON as a reference gene for each qRT-PCR experiment⁵². qPCR primers specific for Sr61 (Sr61GSPF1 and Sr61GSPR1) were used to measure relative gene expression (Supplementary Table ³). Experiments included three technical replicates for each of three biological replications. δCq mean values were calculated and standard errors were determined. Gene expression values were log (base 2)-transformed.