4.1. Plant Materials, Sampling, RNA Extraction, and RNA-Seq

ZS11 was from the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences (CAAS). ZS11 plants were grown under conventional field management and growth conditions at Yangluo Experimental Field (N:30°42′35.78″, E:114°30′49.05″), Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan. Axillary buds of different developmental states were collected from the bottom, lower, middle, and upper parts of the plant during the bolting period and named S1 (dormant axillary buds), S2 (temporarily axillary buds), S3 (being activated axillary buds), and S4 (already activated and elongated axillary buds), respectively. Samples were immediately frozen in liquid nitrogen and stored at −80 ℃ in a freezer for RNA-seq; there were three biological replicates for each type of axillary bud sample, with three plants in each replicate.

Total RNA extraction followed the instructions of the manufacturer of Plant RNA Kit (R6827-01, Omega, GA, USA). cDNA libraries were constructed using Poly-A Purification TruSeq library reagents and sequenced on an Illumina platform. The clean reads were mapped to the rapeseed (ZS11.v0) reference genome (https://yanglab.hzau.edu.cn/BnIR/genome_data, accessed on 17 November 2022) [32] using TopHat2 software (http://ccb.jhu.edu/software/tophat, accessed on 23 November 2022) [64]. The read counts of each gene were calculated using HTSeq 2.0 software (Python Software Foundation, VA, USA) [65]. Differentially expressed genes (DEGs) between two sample groups were analyzed using the DESeq R package. False discovery rate (FDR) <0.01 and fold changes (FC) ≥2 were set as the thresholds for significant DEGs. GO enrichment analysis of the DEGs was performed using the GOseq R package.