*4.4. Measurement of Flower Anthocyanin*

Sample preparation and extraction methods were as follow, the freeze dried tissues were crushed using a mixer mill (MM 400, Verder Shanghai Instruments and Equipment Co., Ltd., Shanghai, China) with a zirconia bead for 1.5 min at 30 Hz. A 100 mg sample of the powder was weighed and extracted overnight at 4 ◦C with 1.0 mL 70% aqueous methanol. Following centrifugation at 10, 000× *g* for 10 min, the extracts were absorbed and filtered. Then the sample extracts were analyzed using a UPLC system, the analytical conditions were as follow, HPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm × 100 mm); solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program, 95:5 *v*/*v* at 0 min, 5:95 *v*/*v* at 11.0 min, 5:95 *v*/*v* at 12.0 min, 95:5 *v*/*v* at 12.1 min, 95:5 *v*/*v* at 15.0 min; flow rate,. 0.40 mL/min; temperature, 40 ◦C; injection volume: 2 μL [32,50]. The mass spectrometry data was analyzed using the software Analyst 1.6.1 (AB Sciex Pte. Ltd., Concord, Ontario, Canada) and based on the Metware Database (MWDB, Metware Biotechnology Co., Ltd., Wuhan, China), a local self-built database, and a public metabolite information database, to identify anthocyanin compounds. The experiments were repeated three times. 1.8 μm
