Development of F2 and Backcross populations
The broccoli DH lines HX-16 with KGLT and HX-11 with TPLT were selected as paternal and maternal lines, respectively, and were crossed to develop the F1 hybrid. The BC1 was obtained by pollinating the F1 hybrid with HX-16, and the individuals with TPLT were selected to cross with HX-16 again to produce the BC2 population. In the BC2population, the individuals with TPLT were selected and selfed to construct the BC2F2 segregation population. An F2 segregation population was obtained by selfing the hybrid F1 derived from a mutant GS with green buds and the wild line PS with purple buds at low temperature. Plug-seedlings were obtained and seedlings were transplanted in a tunnel greenhouse at Yangdu experimental farm of Zhejiang academy of agricultural sciences in the autumn of 2017 (HX-11, HX-16 and their F1, the mutant GS and wild DH line PS), 2018 (HX-11, HX-16 and their F1, BC1, the mutant GS and the wild DH line PS), 2019 (HX-11, HX-16 and their F1, BC1 and BC2, the hybrid F1 of the mutant GS and the wild lines PS and germplasm materials in Brassica oleracea (Table 1), 2020 (HX-11, HX-16 and their F1 and BC2F2, the mutant GS and the wild lines PS and their F2 segregation population,) and 2021 (HX-11, HX-16 and their F1, the mutant GS and the wild lines PS, purple broccoli (PB)). Meanwhile, the wild line PS plants were cultured in an artificial chamber with 20℃/25℃ night/day temperature and a 14-hour photoperiod for the experiment of the temperature effect on the bud color in 2021 in order to check the effect of temperature on anthocyanin. Phenotyping of the buds in the segregation populations, respective parents, and the germplasm materials was conducted after experiencing cold air (0-50 C) for 10 days as the temperature of this range could make the buds of the maternal line HX-11 turn purple completely.
Table 1 Germplasm materials in Brassica oleracea