Quantitative Gene Expression Analysis

Total RNA was isolated from each organ using the RNAprep Pure Plant Plus Kit (DP441; TIANGEN Biotech Co. Ltd., Beijing, China) and reverse-transcribed to generate cDNAs. qPCR was performed using qPCR SYBR Green Premix (Vazyme, Nanjing, China) and CFX96 Touch Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The primer sequences for the ten CqTH genes were generated using Primer Premier 5.0 software [77 ] (Table S7). We used the GAPDH gene as the internal control because of its consistent expression across developmental stages in most plant organs [78 (link)]. qPCR was performed using three biological replicates and three technical replicates per sample. The relative expression of the target genes was determined using the 2^-ΔΔCT method [79 (link)].