Validation of Anthocyanin Biosynthesis Genes

For validating gene expression using qRT-PCR, 32 unigenes associated with anthocyanin biosynthesis and phytohormone metabolism were randomly selected (Supplementary Table 2). Total RNA isolation was conducted by using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). First-strand cDNA was synthesized with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Takara, Dalian, China), and the extracted RNA was used as template according to manufacturer’s instructions. A list of gene-specific primers is provided in Supplementary Table 1. The quantified expression levels of the tested genes were normalized against the house keeping genes TIP41-like protein (TIP41) according to previous study on L. aurea (Ma et al., 2016 (link)). qRT-PCR assays were conducted by the SYBR Premix Ex Taq™ II kit (Tli RNaseH Plus) (Takara, Dalian, China) in a Bio-Rad iQ5 Gradient RT-PCR system. Reaction conditions were: 30 s of denaturation at 95°C and 40 amplification cycles (5 s at 95°C, 30 s at 60°C). Calculation of relative target gene expression levels was done using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)). Experiments were conducted using three independent biological and three technical replicates.