Analysis of gene expression
Total RNAs of apple shoots under different treatments were extracted using an OminiPlant RNA kit (CoWin Biosciences) per manufacture’s instruction. The first-strand cDNA was synthesized using the PrimeScript First Strand cDNA Synthesis Kit (TaKaRa) per manufacture’s instruction.
Quantitative real-time PCR (qRT-PCR) reaction solutions (20 μl) were assembled by combining the forward and reverse primers (0.25 μM for each), template cDNA (50 ng), and SYBR Green PCR Master Mix (10 μl). The reactions (95 °C, 5 min; 95 °C, 15 s, 60 °C, 1 min, for 40 cycles) were performed using the iCycler iQ5 Detection System (Bio-Rad). 18S rRNA served as internal control. The relative gene expression was calculated using the 2-ΔΔCt methods. Three biological repeats were performed for each individual experiments. Primers used for qRT-PCR were listed in Table S2.