*4.6. Quantitative Real-Time PCR Analysis*

In order to clarify the stress response variation tendency of Verbena at the transcriptional level on different stages of drought stress, we selected 10 DEGs to participate in different important biological processes for qRT-PCR with SsoFast™ EvaGreen® Supermix every 5 days. A 20 µL fluorescent quantitative reaction system contains 10 µL of stain, 2 µL of cDNA template and 300 nM of primers. The PCR settings are as follows:


Relative expression levels were calculated by the 2−∆∆*C*<sup>t</sup> method, and a β-actin gene of *Verbena bonariensis* (Forward primer: GAAAGATGGCTGGAAGAGGG, Reverse primer: GCTATGAA CTCCCTGATGGTC) was used as the reference for quantitative expression analysis. The expression pattern of DEGs was analyzed by melting furnace curve. The qRT-PCR primers are shown in Table SA6.
