*4.8. Total RNA Extractions and Expression Analysis of RsAP2 Genes*

Total RNA extraction was conducted with a FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). The cDNA reverse-transcription process was conducted with HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China). The primers were designed with Primer Premier software (version 5.0) and synthesized by General Biol Company (Anhui, China). The quantitative real-time PCR process was conducted with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on the platform of ROCHE (LightCycler® 480 II, Swiss). The RT-qPCR reaction was conducted in a 96-well plate containing 10.0 μL SYBR Mix, 0.4 μL 10 μM solution of primer, respectively, 0.18 μL undiluted cDNA and 9.02 μL double distilled H2O. The qPCR program was conducted in 3 steps: pre-denaturation stage had 1 cycle of 95 ◦C for 30 s; recirculation reaction stage had 40 cycles of 95 ◦C for 10 s and 60 ◦C for 30 s; melting curve had 1 cycle of 95 ◦C for 15 s, 60 ◦C for 60 s and 95 ◦C for 15 s, in order. Three repeats were performed for each sample. Housekeeping gene *Actin* was taken as the internal reference gene [53]. Relative expression levels of the genes were calculated using the 2-ΔΔCT formula [54].
