Plants materials, cold treatment, and freezing tolerance assay
The wild-type B. rapa variety ‘Bre’ was grown under a 16-h light/8-h dark (22 °C) photoperiod for 3 weeks. To analyze the miRNAs abundance in ‘Bre’ under cold stress, some ‘Bre’ plants were moved to 4 °C condition for 7 days cold treatment and some were grown at 22 °C for additional 7 days as control. The aboveground parts of treated and untreated were then harvested, frozen in liquid nitrogen, and stored at −80 °C until extraction of total RNA. Small RNA sequencing (one biological replicate) was performed using the Illumina GAII sequencer at BIG (ShenZhen, China).
The wild-type B. napus variety ‘K407’, a Semi-winter ecotype, was grown under a 16-h light/8-h dark (22 °C) photoperiod with light intensity of 250 μmol m−2s−1. For the cold resistance assay, the plants were grown under standard conditions (22 °C) for 6 weeks before transferring to a cold environment (4 °C or 2 °C, 16-h/8-h light/dark photoperiod). The similar methods for studying cold stress in Brassica crops have been used in previous reports (Raza et al., 2021; Hussain et al., 2022). After cold treatment, the cold resistance of plants was determined. The freezing tolerance assay was performed in a freezing chamber, after cold acclimation for 24 h, in which wild-type and transgenic lines were subjected to a −10 °C treatment for 2.5 h. The plants were then allowed to recover at 22 °C for 7 days before determining the survival rate. The Pro content was measured immediately after the freezing treatment using a commercial assay kit (A107-1-1, Nanjing JianCheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions.
The wild-type B. rapa cultivar “Bre”, B. napus cultivar ‘K407’ are all inbred lines.