*4.6. Quantitative Real-Time PCR (qRT-PCR)*

The cuttage seedlings of *R. chinensis* 'Old Blush' and the grafted seedlings of *R. beggeriana* were used for qRT-PCR, and the cold treatment of the two species is the same as in Section 2.6. Total RNA was extracted using the Quick RNA Isolation Kit (Huayueyang Biotech, Beijing, China). The first-strand cDNA was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio Inc., Dalian, Liaoning Province, China). The qRT-PCR reactions were performed using TB Green™ Premix Ex Taq™ II (TaKaRa, Japan) and carried out on a CFX96 Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA). Conditions for the reaction were as follows: 95 ◦C for 30 s, followed by 40 cycles of 95 ◦C for 5 s and 60 ◦C for 30 s. The specific primers for *SWEET* genes were designed using the Real-time PCR (TaqMan) Primer and Probes Design Tool (https://www.genscript.com/tools/real-time-pcr-taqman-primer-design-tooland, accessed on 18 December 2022) and have been listed in Table S1. *Rba-Tubulin* was used as the reference gene. All reactions were performed in three biological replicates, and the 2−ΔΔCt method was applied to calculate the relative expression. GraphPad Prism 8.4.0 (https://www.graphpad.com, accessed on 18 December 2022) software was used to generate graphs and to perform statistical analyses. A *p*-value of <0.05 was considered to determine the significance level of the data.
