Quantitative Real-Time PCR Analysis of Chinese Cabbage Development

Quantitative real-time PCR (qPCR) analyses were performed as detailed previously (Jiang et al., 2018 (link)), using primers compiled in Supplementary Table S1. Sampling of plants at the seedling and flowering stages was performed as published previously (Jiang et al., 2018 (link)), while sampling at the rosette and heading stages was performed at the 6^th and 8^th weeks, respectively. When rosette stage sampling was performed, three leaves were collected from each of nine individual ‘Chiifu’ plants (three biological replicates, three plants per replicate). With the oldest leaf numbered as leaf one, samples were collected from different positions (top, middle, bottom) on three different leaves (outer leaf, first leaf; middle leaf, 10^th leaf; inner leaf, 20^th leaf), designated as the RL1, RL2, and RL3 from the outer to the inner leaves. In the heading stage, three leaves (outer leaf, first leaf; middle leaf, 20^th leaf; inner leaf, 40^th leaf) were similarly collected from each of nine ‘Chiifu’ plants, with these samples being respectively designated as HL1, HL2, and HL3. For BR1 expression in bolting resistant or bolting non-resistant Chinese cabbage, ten inbred lines from heading stage were selected from our laboratory, the top point of short stem (GP) and the top point of inner leaf (TP) were sampled, TP was used as control. Three biological replicates with three plants of different lines per replicate were sampled. After collection, samples were snap-frozen with liquid nitrogen and stored at −80°C for subsequent RNA isolation. qPCR primers for the expression analyses of representative mark genes were listed in Supplementary Table S1.