Ultrasybr mixture with rox 1

The total RNA was isolated using an RNAprep PurePlant Kit (Polysaccharides & Polyphenolics-rich) (Tiangen, China). The cDNA was synthesized using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan). qRT-PCR was performed to identify transcript levels using a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and UltraSYBR Mixture (With ROXI) (CWBIO, China) in a 20 μl volume: 2×UltraSYBR Mixture (With ROXI) 10 μl, forward primer 0.5 μl, reverse primer 0.5 μl, template cDNA 1 μl, and RNase-free water 9 μl. The PCR procedure was as follows: a pre-denaturation step at 95°C for 10 min; 40 cycles of 95°C for 10 s, 60°C for 30 s and 72°C for 32 s; and a melting curve of 95°C for 15 s, 60°C for 1 min, 95°C for 15 s and 60°C for 15 s. The 2^−ΔΔCT method was applied to calculate the relative expression of genes [50 (link)]. Figures for qRT-PCR were drawn using GraphPad Prism software [51 (link)].

Total RNA was isolated from cucumber and Arabidopsis plants using an RNAprep pure Plant Kit (TianGen, Beijing, China) following the manufacturer’s instructions. Subsequently, the RNA was reverse transcribed using the PrimeScript^® 1st Strand cDNA Synthesis Kit (Takara, Japan). qRT-PCR was performed using the UltraSYBR Mixture (with ROX I; Cwbiotech) in the iCycler iQ5 system (BioRad, CA, USA). The results were normalized to those of the cucumber ACTIN gene. Three biological replicates were performed for each analysis. The primers used in this study are provided in Table S1.

The deep-rooted ‘12 − 2’ (hybrid seedlings of Ralls and Malus spectabilis) self-rooted apple stock and ‘M9T337’ stock, an apple dwarf rootstock were screened. The seedlings of M. spectabilis were obtained from the Beijing Botanical Garden (Beijing, China) and those of Ralls and ‘M9T337’ stock were obtained from Shandong Institute of Pomology. The roots of ‘12 − 2’ were used for qRT-PCR analysis. The growth conditions used were those described by [39 (link)]. Modified Hoagland medium was used in self-rooted apple stock hydroponic cultivation as described by [18 (link)]. An RNAprep pure plant kit (Vazyme, Nanjing, China) was used to isolate total RNA from the samples according to the producer’s instructions. Reverse transcription was then performed with a EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (Trans, Beijing, China). Applied Biosystems 7500 real-time PCR system (Applied Biosystems) was used to perform qRT-PCR reactions using UltraSYBR Mixture (with ROX I; Cwbiotech). The results were standardized with apple 18 S gene. Three biological replicates were used for each test. Table S4 showed the primers used in this study.