Total RNA was extracted from different tissues of grape hyacinth, including roots, bulbs, and leaves, and from the flowers at five developmental stages using the Total RNA Kit (Omega, Norcross, GA, USA). The five stages (S1–S5) of petals of grape hyacinth were divided according to the petal pigmentation [30 (link)] (Figure 2 A). Next, 1 μg of RNA was used to synthesize cDNA by using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biotechnology, Dalian, China), following the manufacturer’s instructions. Then, qRT-PCR was carried out on the iQ5 RT-PCR instrument (Bio-Rad, Hercules, CA, USA) using AceQqPCR SYBR® Green Master Mix (Vazyme, Nanjing, China). The MaActin gene was chosen as the internal control gene for Muscari. Spp, and the NtTubA1 gene was used as the internal control gene for tabacum samples. Primers used for qRT-PCR are listed in Table S1 . The relative expression of MaDFR was quantified using the 2−△△Ct method [31 (link)]. Using the Correlationplot Rectanglel module of Qi MetA software (Qiji Biotechnology Co, Ltd, Beijing, China), the r value of MaDFR expression and anthocyanin accumulation of the five stages of flower development were recalculated using the Pearson method, not including the roots, bulbs, and leaves. All analyses were conducted with three independent experiments.
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