Validation Analysis of Transcriptome Data by qRT-PCR

The reliability of transcriptome data was verified by quantitative real-time PCR (qRT-PCR) experiments. The complementary DNA (cDNA) reverse transcription of total RNA was performed using PrimeScript^TM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). A total of twelve flowering-related candidate genes were selected randomly, and Protein Phosphatase 2A (PP2A) was used as a reference gene to evaluate the expression stability of the candidate genes (Zhang et al., 2019). The primers of a reference gene and candidate genes were designed by PrimerQuest Tool (http://sg.idtdna.com/Primerquest/Home/Index) (Supplementary Table 2).

The qRT-PCR was performed using an SYBR Premix Ex Taq^TM II kit (TaKaRa, Dalian, China) on a Roche LightCycler480 quantitative PCR instrument. The final reaction volume was 20 μl, and each reaction mixture contained abm^® EvaGreen qPCR MasterMix-no dye 10 μl, forward and reverse primers 0.8 μl, cDNA 1 μl, and ddH₂O 7.4 μl. The following reaction procedures were used the enzyme activation at 95°C for 10 min, denaturation at 94°C for 15 s, annealing at 60°C for 1 min, denaturation and annealing for 40 cycles, and then the melting curve analyzed. The candidate gene expression level was calculated by the 2^−ΔΔCt method.