Quantitative Analysis of Flavonoid, Ascorbic Acid, and Glutathione Metabolism

In total, twenty-seven differentially expressed flavonoid, ascorbic acid, glutathione, and ASA–GSH metabolism-related unigenes were selected for qRT–PCR analysis. The reverse transcription kit (Vazyme, Nanjing, China) reverse transcribes 1 μg of total RNA into cDNA for qPCR. The qRT–PCR reactions were carried out using a 20 μl reaction volume containing Chamq universal SYBR qPCR master mix (Vazyme, Nanjing, China), primer, cDNA template, and ddH₂O and were performed on a LightCycle 96 system (Roche, Switzerland). All the primers of unigenes were designed online by the Integrated DNA Technologies website (Supplementary Table 1). The relative expression levels of unigenes were calculated by the 2^–ΔΔCT method using the 18s gene of P. ternata as a reference gene (Xue et al., 2019 (link)).