*4.7. Expression of the Flavonoid Biosynthesis Related Genes During Di*ff*erent Growth Stages*

Nine unigenes that were related to flavonoid biosynthesis following Zhang et al. [51] and Park et al. [52] were selected for validation using qRT-PCR. The RNA was reverse-transcribed to cDNA using PrimeScriptTM RT reagent Kit (TaKaRa, Dalian, China). The qRT-PCR was performed using the SYBR®Premix ExTaq TM (TaKaRa, Dalian, China) on the LightCycler®480 II Real-Time PCR System (Roche, Carlsbad, CA, USA). Three biological replicates and three technical replicates were performed, and all the primer names and corresponding sequences are listed in Table S3. The qRT-PCR was performed in a 20 μL reaction volume containing SYBR Premix Ex Taq II (10 μL), forward prime (10 μM, 0.8 μL), reverse primer (10 μM, 0.8 μL), cDNA template (5 ng/μL, 2 μL), and ddH2O (6.4 μL). The PCR conditions were as follows: denaturation at 95 ◦C for 30 s, followed by 40 cycles of amplification (95 ◦C for 5 s, 60 ◦C for 30 s). The melting curves were measured at 95 ◦C for 5 s and 60 ◦C for 1 min. The elongation factor 1 alpha (*EF-1*α) gene of *A. roxburghii* was used as the internal control reference gene. Finally, gene expression was calculated using the 2−ΔΔCt method [53].
