*2.6. Expression Analysis of the HhMYB Genes by qRT-PCR*

Total RNA was extracted with a total RNA extraction kit (Shanghai PuDi Biotech Co., Ltd., Shanghai, China), and cDNA was synthesized with HiScript® III 1st Strand cDNA Synthesis kit (Vazyme, Nanjing, China), both procedures following their respective manufacturer recommendations. The Genscript tool (https://www.genscript.com/tools/ pcr-primers-designer, accessed on 20 March 2021) was used for designing primers for qRT-PCR (Table S1). qRT-PCR was performed using a StepOnePlus TM Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The reaction mixture contained 2 μL diluted cDNA, 10 μL 2 × SYBR Green Master Mix (Bimake, Houston, TX, USA), 0.4 μL ROX (Dye I), 1 μL each of forward and reverse primer, and ddH2O to a total volume of 20 μL. The PCR procedure utilized an initial denaturation step set as 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 s, 60 ◦C for 30 s, and 72 ◦C for 30 s. Melting curve analyses of the amplified products were conducted at 60–95 ◦C. The *Actin* was selected as a reference gene: forward primer: 5 -GGCACCTCTCAACCCCAAGG-3 , reverse primer: 3 -GAGAGAACGGCCTGGATGGC-5 [5]. Three technical replicates were prepared for each extract, and the quantitative results were analyzed using the 2−ΔΔCT method.
