*4.5. Verification of RNA-Seq by qRT-PCR*

To confirm the reliability of RNA-seq data, we randomly selected several genes and tested their expression profiles using qRT-PCR. Total RNA was isolated from petals as already described above. The first strand of complementary DNA (cDNA) was synthesized from total RNA using the cDNA synthesis kit (Vazyme Biotech, Nanjing, China). Finally, qRT-PCR was performed on the CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA) using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) according to the manufacture recommended protocol. The amplification reaction volume was 20 μL, containing 10 μL of 2 × SYBR qPCR Master Mix and 10 μM of forward and reverse primers. All reactions were run in triplicate assays using the following cycling conditions: initial denaturation at 95 ◦C for 30 s followed by 40 cycles of PCR consisting of denaturation at 95 ◦C for 10 s and annealing at 60 ◦C for 30 s. All gene-specific primers for qRT-PCR were designed using the online primer design software (https://www.ncbi.nlm. nih.gov/tools/primer-blast/ (accessed on 22 January 2022)), and all the primers used in this study are presented in Table S2. The relative gene expression levels were calculated using the 2−ΔΔCT method [60].
