Dnase

Total RNA was extracted from tissue using the TRIzol reagent (catalog number: 15596026, Invitrogen, Carlsbad, CA, USA). The RNA integrity number (RIN) method was used to detect RNA quality, and RIN values are above 7.1. Only good-quality RNA was used for this experimental study. Total RNA was extracted from muscle tissue, and then DNA was digested with DNase (Tiangen, Shanghai, China). Subsequently, mRNA was broken into short segments by adding an interruption reagent, and a strand of cDNA was synthesized with six base random primers using the interrupted mRNA as a template. Double-strand cDNA was purified using a commercial kit (Promega Corp, Beijing, China); the purified double-strand cDNA was repaired, combined with an A-tail, and connected with a sequencing connector. The constructed library was qualified by an Agilent 2100 Bioanalyzer and sequenced by Illumina HiSeq 2500 (19 (link)).

1.5 × 10³ cells/well cells were cultured in 96‐well dish in the medium with a final concentration of 10μM BrdU. After incubation for 3 h, cells were fixed by 4% paraformaldehyde (PFA) for 15 min, then washed by PBS solution and treated with 0.2 TrtonX‐100, after which they were further washed and treated with DNase (Tiangen, China) for 20 min at room temperature. Then washed the cells with PBS and added BrdU primary antibody (Abcam, Cambridge, MA, USA) and incubated the cells at 4°C for 12 h. After washing the cell with PBS, cells were incubated with secondary antibody for 60 min. Washed the cells with PBS and detected the BrdU positive cells by fluorescence microscope.

Five-year-old ‘Kiyomi’ tangor and ‘Shiranui’ trees cultivated in a citrus research orchard located in Suining, Sichuan Province, China, were used. Fruit samples were harvested at two developmental stages, 150 and 210 days after flowering, assigned as first and second stages, respectively. At the first stage, fruit development occurred during the early expansion period, while at the second stage, fruit expansion had finished, and fruit reached their maximum size and began to mature. For each cultivar, fruit samples were collected from three different trees. Thus, each tree was an independent biological replicate. For RNA-seq, two independent randomly chosen biological replicates were used. Ten representative fruit from each tree were collected at each sampling stage. Segment membranes separated from fruit segments were immediately frozen in liquid nitrogen and kept at − 80 °C until use. Total RNA was extracted using an RNAprep Pure Plant Kit (TIANGEN, Beijing, China), which was specifically designed for materials rich in polysaccharides and polyphenolics. Extracted RNA was treated with DNase (TIANGEN, Beijing, China) to remove genomic DNA. RNA quality was surveyed by separation in 1.0% agarose gels, stained with ethidium bromide and observed by UV light.

Tissue samples from areas reflecting the silencing and overexpression phenotypes were collected to analyze the effects of target gene and anthocyanin biosynthesis genes expression. Tissues infected by Agrobacterium carrying vectors with no host gene fragment insert, or from non-infected plants were selected as control. Three independent biological replicates were analyzed. RNA plant plus reagent (TIANGEN BIOTECH CO., LTD., Beijing, China) were used to extracted total RNA from crabapple leaves and apple fruits. DNase (TIANGEN BIOTECH CO., LTD., Beijing, China) treatment was performed according to the manufacturer’s instructions. Total RNA was synthesized to first-strand cDNA by the Reverse Transcriptase M-MLV (RNase H^-) kit (TaKaRa, Ohtsu, Japan).