*2.6. Reverse Transcription and Quantitative Reverse-Transcription Polymerase Chain Reaction Analysis*

RNA was first treated with DNase I and then reverse-transcribed to cDNA using a HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), according to the manufacturer's instructions (Vazyme, Nanjing, China). Then, the concentration of cDNA was diluted to 100 ng/μL, and the internal reference gene *CaUBI-3* forward and reverse primers were used for PCR to detect whether the reverse transcription was successful [45]. For the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay, all primers were designed using Primer3 (http://primer3.ut.ee/) (accessed on 10 March 2021), and the specificity of the designed fragment was tested using SGN (https://solgenomics.net/) (accessed on 10 March 2021) (Table S8). qRT-PCR was performed using SYBR® Premix Ex Taq™ (Vazyme), with three technical replicates in 10 μL volumes containing 5 μL Fast SYBR™ Green Master Mix (2×), 2 μL cDNA template (100 ng/μL), 0.2 μL of each primer (10 μM), and 2.6 μL ddH2O. The PCR cycling conditions were as follows: 95 ◦C for 30 s, followed by 40 cycles of 95 ◦C for 5 s, and finally 60 ◦C for 20 s. The relative expression level of each gene was calculated using the 2−ΔΔCT method [46].
