Rna plant plus reagent kit

Plant total RNAs were isolated using the RNA plant Plus Reagent Kit (Tiangen, Beijing, China) and TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using the PrimeScript™ RT reagent Kit with the gDNA Eraser (TaKaRa, Shiga, Japan). The quantitative RT-PCR assay was carried out using the Step One Plus™ Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) to examine the transcription level of the wax and disease resistance-related genes. The qRT-PCR was performed according to [47 (link)]. The primers used for qRT-PCR are listed in Additional file 1: Table S3.

Total pulp RNA was extracted using the RNAplant Plus Reagent kit (Tiangen, Beijing, China) based on the manufacturer’s protocol. Three biological replicates of each sample were kept at -80 °C and used for the extraction of RNA. The purity and integrity of the total RNA were determined by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Madison, WI, USA) and gel electrophoresis using the PrimeScriptTM RT Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s instructions; 2.0 µg of each RNA sample was used to generate first-strand cDNA for gene isolation. For miRNA reverse transcription, Mir-XTM miRNA first-strand synthesis and the TB Green Real-Time PCR (qPCR) kit were used (TaKaRa, China). The QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Singapore) was applied to perform quantitative reverse transcription PCR (qRT-PCR). The qRT-PCR patterns were performed at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 20 s. As an internal control U6 gene, DkActin (accession no. AB473616) was used. The forward miRNA primer was designed using the fasta sequence, and universal primer was used as a reverse primer (Table S1).

The different tissues of apple trees (new shoots, leaves, flower buds, flowers, and young fruit), Arabidopsis seedlings leaves, and apple seedlings leaves samples were ground to powders in mortars under frozen conditions. Then, 100 milligram (mg) from them were taken, respectively, for total RNA extracting by using an RNA Plant Plus Reagent Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. The RNA was used as the template to synthesise 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, United States). The reaction solution contained 10 μL SYBR Green I Master Mix (CWBIO, Beijing, China), 0.5 μmol⋅L^–1 primers (SANGON BIOTECH, Shanghai, China) (Supplementary Table 1), and 1 μL of each 1:5 diluted cDNA as a template in a total volume of 20 μL. The PCR programme 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. Apple MdActin (MD04G1127400) and Arabidopsis AtActin (AT2G37620) were used as reference genes, and the sequences can be found in GDR² and TAIR³ database, respectively. 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 and Schmittgen, 2001 (link)).