Quantitative Real-Time PCR Analysis

qRT-PCR was performed using a TB Green Premix Ex Taq II kit (Tli RNaseH Plus) (Takara) according to the manufacturer’s instructions for gene expression analysis. The template cDNA fragment was synthesized from the extracted total RNA and was diluted to 1/10 for use in the qRT-PCR system. All the primers are detailed in Supplementary Table S1. The procedure was performed with a LightCycler 480II machine (Roche, Switzerland) using a 45-cycle program (95°C for 5 s and 60°C for 30 s). The relative expression of genes was calculated based on the threshold cycle according to the _{Δ Δ} Ct method (Schmittgen and Livak, 2008 (link)). The result was normalized to the expression of the reference gene actin in mulberry (Chen et al., 2018 ) or L25 in tobacco (Liu et al., 2018 (link)). Expression was determined for at least three replicates per treatment.