*2.7. Quantitative PCR Analysis*

Primers were designed using QuantPrime (https://quantprime.mpimp-golm.mpg.de). cDNA was synthesized from 2 μg total RNA using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA), according to the manufacturer's protocol. qRT-PCR was performed using two independent biological replicates and three technical replicates. qRT-PCR was set up in 386-well PCR plates with 0.2 μM primers using SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA), following the manufacturer's protocol in a reaction volume of 10 μL via a Roche LightCycler 480 Real-Time system (Rotkreuz, Switzerland). Reaction conditions were as follows: denaturation at 95 ◦C for 5 min, 45 cycles of 95 ◦C for 10 s, 60 ◦C for 15 s and 72 ◦C for 8 s, heating from 65 to 95 ◦C. Two internal reference genes ELF and ATU were designed to normalize the relative gene expression levels for flag-leaf and panicle tissue, respectively, using the 2−CT method with ΔCT = CTgene − CTreference gene [57]. For comparison of fold change, scatterplots were generated using the log2 fold change determined between RNA-Seq and qRT-PCR, which is defined as ΔΔCT (for comparative threshold cycle).
