*4.7. Cloning of MdSAPs and qRT-PCR Analysis*

We extracted total RNA from previously frozen apple tissues according to the CTAB method and from *Arabidopsis* leaves, using Trizol reagent (Thermo Fisher Scientific-CN; Shanghai, China; https://www.thermofisher.com/cn/zh/home.html) [53]. Two micrograms of total RNA were collected for synthesizing first-strand cDNA. For cloning *MdSAP*, complete open reading frames were obtained via RT-PCR from fully expanded leaves of "Golden Delicious" apple, using specific primers listed in Table 5. The 50 - and 30 -untranslated regions (UTRs) were obtained with a Rapid Amplification for cDNA Ends kit (TaKaRa, Dalian, China). For the qRT-PCR assays, reverse transcription was performed with 1 µg of total RNA from each sample, followed by PCR-amplification of 1 µL of the product. We conducted the qRT-PCR assays in 20-µL reaction mixtures that contained 10 µL of SYBR® Premix Ex Taq™ (TaKaRa; Beijing, China; http://www.takarabiomed.com.cn), and used an iQ5 instrument (Bio-Rad, Hercules, CA, USA) as described before [34]. Thermal cycling included an initial 3 min at 95 ◦C; then 40 cycles of 10 s at 95 ◦C, 30 s at 58 ◦C, and 15 s at 72 ◦C; followed by 3 min at 72 ◦C and then 81 cycles of 7 s each, increasing by an increment of 0.5 ◦C from 55 ◦C to 95 ◦C. Three biological replicates were tested in each assay, and ∆Ct values were calculated by using *MdMDH* as our endogenous control [54]. Relative quantification was calculated according to the 2 <sup>−</sup>∆∆*C*<sup>t</sup> method [55], and dissociation curve analysis was performed for determining the specificity of the amplifications.
