*2.1. Plant Materials*

Two broccoli inbred lines, including BT 126 (with purple heads) and SN 60 (with green heads), were used as parental lines in this study. To develop segregating populations for anthocyanin biosynthesis, the purple head plant BT 126 was crossed with the green head plant SN 60 (Figure 1). The F1 plants were self-pollinated to produce the two F2 populations with 302 and 280 individuals, respectively.

**Figure 1.** Phenotypes of the parents and F2 individuals and their frequency distributions. (**A**) Maternal line BT 126; (**B**) paternal line, SN 60; (**C**,**D**) the frequency distribution of the purple head trait in the F2 populations with 302 and 280 individuals planted in 2018 and 2019, respectively; (**E**–**I**) phenotype of two F2 populations with 1st to 5th grades of head color. The DNAs of 30 F2 individuals with extreme phenotypes (1st and 5th grade) were used to develop high anthocyanin and low anthocyanin bulks; scale bar = 1 cm.

Initially, a population of 302 F2 individuals, along with 10 plants from each parental line, were grown at Zhuanghang Experimental Base of Shanghai Academy of Agricultural Sciences, Shanghai in 2018. The phenotyping of each F2 individual was carried out on at least three separate days, after the flower head reached maturity in the field. Curd color, which showed a color distribution from green to purple, was visually scored from 1 to 5: 1–green, 2–slight purple, 3–light purple, 4–slightly darker purple, and 5–purple (Figure 1E–I). The total anthocyanin content has been determined by the high-performance liquid chromatography (HPLC) method at six biological replicates. The BT 126 is a purple broccoli variety, with an average content of anthocyanin 5.72 mg/g FW. It was crossed to SN 60, with very low anthocyanin content (0.64 mg/g FW), to generate F1 progeny. The average contents of anthocyanin, for the 5 phenotypic categories, were 5.38, 3.26, 2.29, 1.27, and 0.78 mg/g FW. All the above information was increased in the revised manuscript. Subsequently, two populations of 280 F2 and 580 F3 individuals were grown under routine management at Zhuanghang Experimental Base of Shanghai Academy of Agricultural Sciences, Shanghai in 2019 and 2020, respectively. We started the broccoli seeds in the plastic tunnel in August. Four weeks after germination, we transplanted them to field plots. The head color of each F2 and F3 plants was visually phenotyped

in 60 days, when grown from transplants, when the heads usually had a diameter larger than 2 <sup>1</sup> <sup>4</sup> inches. To avoid the sunlight and temperature effects, all the plants were grown in the same plastic tunnel. Leaf tissues were harvested and stored at −20 ◦C for DNA extraction. The head tissues of both parents (BT 126 and SN 60), at the full-size, mature stage, were collected from the same site of the top head, at three biological replicates, and used for RT-qPCR analysis. The anthocyanins in the broccoli heads were isolated and assessed, according to the method proposed by Liu C. et al. [4].

All materials were obtained from the Institute of Horticulture, Shanghai Academy of Agricultural Sciences.
