*2.5. Analysis of the Candidate Gene*

To infer the gene regulation patterns of *BoF3'H* during anthocyanin biosynthesis, a qRT-PCR assay was performed. The RNA was isolated from BT 126 and SN60 at three head developmental stages, with both low and high anthocyanin contents. Reverse transcription was conducted using oligo-dT primers and PrimeScript™ RT Master Mix (Takara BIO, Inc., Shiga, Japan). The primer pair sequences for *BoF3'H*, *BoANS2*, and *BoTTG1* used in this study were previously designed for the RT-qPCR analysis. *BoLBD38.3*-specific primers were designed based on the sequences in NCBI (accession number: XM\_013739916). The qPCR primer pair sequences for *BoLBD38.3* were 5 -GCCCAAACGGAGACGATTAG-3 (forward) and 5 -AACCGTTCACTGGCGATGTG-3 (reverse), gene-specific primers that were confirmed to produce specific gene products by sequencing [15]. Real-time PCR was performed using TB Green® Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Dalian, China) on an ABI QuantStudio 5 real-time PCR system(Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. Actin was used as a reference gene [43]. The data were processed using the 2−ΔΔCt method [44]. Sequence data of *BoF3'H* can be found in the GenBank data libraries, under accession number XM\_013751545.
