RNA extraction and qRT-PCR analysis
Total RNA was extracted from apple trees, Arabidopsis seedlings, tomato
seedlings, and apple seedlings using an RNA Plant Plus Reagent Kit
(TIANGEN, Beijing, China). The RNA was used as the template to
synthesize cDNA with a PrimeScript RT Reagent Kit (Takara, Shiga,
Japan). The qRT-PCR analysis was conducted on a StepOnePlus Real-Time
PCR System (Thermo Fisher Scientific, USA). The reaction solution
contained 10 μL SYBR Green I Master Mix (CWBIO, Beijing, China), 0.5
μmol·L−1 primers (SANGON BIOTECH, Shanghai, China),
and 1 μL each template in a total volume of 20 μL. The PCR program was
as follows: 95°C for 3 min; 40 cycles of 94°C for 15 s, 60°C for 20 s,
and 72°C for 15 s. All the samples were analysed with three biological
replicates, each comprising three technical replicates. Relative gene
expression levels were calculated in accordance with the
2−ΔΔCt method (Livak & Schmittgen, 2001). The primers
used for qRT‐PCR (Table S4)
were synthesized by the Sangon Biotechnology Co. Ltd. (Shanghai, China).