Quantitative Real-Time PCR Protocol

Total RNA was extracted using Total RNA Rapid Extraction Kit (Zomanbio, Beijing, China). Removal of genomic DNA contamination and first strand cDNA synthesis were conducted using PrimeScript^TM RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Quantitative RT-PCR was carried out in a total reaction volume of 20 μL containing 100 ng of template cDNA, 0.4 μM of each primer, 1 × ROX reference dye, and 10 μL of 2 × SYBR premix Ex Taq II (TaKaRa). The qRT-PCR program was as follows: one cycle of 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 34 s at 60°C. A translation elongation factor gene PpTEF2 was selected as an internal control according to the previous report (Tong et al., 2009 (link)). All analyses were repeated three times using three biological replicates. The primers used for qRT-PCR are listed in Supplementary Table S2.