Rna kits

About 60 floral buds were harvested from the plants of each line (Ogu CMS, Nsa CMS, and their corresponding maintainer lines) at the same time. Samples collected from each line were pooled, frozen in liquid nitrogen, and stored at −70 °C for RNA preparation. Total RNA from two stages of floral buds (<2.5 mm and >2.5 mm) of pol CMS, Ogu CMS, Nsa CMS, and their corresponding maintainer lines were extracted by using RNA kits (Tiangen, Beijing, China) in accordance with the manufacturer’s protocol. The integrity of the total RNA was checked by 1% agarose gel electrophoresis. The concentration was detected by Nano-Drop (Thermo Scientific, Madison, WI, USA) and purity of RNA was determined by Agilent 2100 Bio-analyzer (Agilent, Waldbronn, Germany). RNA (10 μL) was sequenced using the Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) and 150 bp of data collected per run. After removing adapters and low-quality data, the resulting clean data was aligned to the B. napus reference genome [59 (link)]. Potential duplicate molecules were removed from the aligned BAM/SAM format records. FPKM (fragments per kilobase of exon per million fragments mapped) values were used to analyze gene expression by the software Cufflinks [60 (link)]. Three biological replicates were performed for each sample.

The total RNA of B2B cells was extracted according to the instructions of the RNA kits (Tiangen Biotech, Beijing, China). Then the RNA was used to synthesize the cDNA by reverse transcription kit (Thermo Scientific, United States). Subsequently, SYBR Premix Ex Taq II (Bio-red, United States) was used for real-time PCR. The following primers were used in our study: TFAM, F 5'-TTC​CAA​GAA​GCT​AAG​GGT​GAT​T-3' and R 5'-AGA​AGA​TCC​TTT​CGT​CCA​ACT​T-3'; PGC-1, F 5'-CAG​AGA​GTA​TGA​GAA​GCG​AGA​G-3' and R 5'-AGC​ATC​ACA​GGT​ATA​ACG​GTA​G-3'; COX4l1, F 5'-CCA​GAA​GGC​ATT​GAA​GGA​GAA​GGA​G-3' and R 5'-CCA​CAA​CCG​TCT​TCC​ACT​CGT​TC-3'; COX2, F 5'-CGC​ATC​CTT​TAC​ATA​ACA​GAC​G-3' and R 5'- TAG​GAG​TTG​AAG​ATT​AGT​CCG​C-3' (Sandon Biotech, Shanghai, China). PCR conditions were 1min at 95°C, followed by 40 cycles of 10 s at 95°C and 30 s at 60°C.