4.9. qRT-PCR Analysis

Twelve variegation-related genes were subjected to qRT-PCR on the ABI 7500 Sequence Detector (Applied Biosystems, Foster, CA, USA) using a SYBR Premix Ex Taq Kit (Takara; Otsu, Japan) according to the manufacturer’s protocol. Specific primers were designed using the Primer Premier software (Table S11). The cDNA synthesis and qRT-PCR were performed as described previously [17]. The relative expression levels of genes in the petals at the different opening stages were normalized to the TUB gene expression level in the same sample and calibrated to the transcription level in the PR petals at stage 1. All reactions were conducted in three biological replicates and three technical replicates. A two-sided t test was used to compare the expression levels.