2.8. RT-qPCR expression level analysis of the key DEGs

Total RNA was extracted from the roots using the RNAprep Pure Plant Plus Kit (Tiangen, Beijing, China) and SteadyPure plant RNA Extraction Kit (Accurate Biotechnology (Hunan, China) Co., Ltd.), and residual genomic DNA was removed during the extraction process. The RNA concentration and quality were assayed with Thermo Scientific ND One, and the RNA integrity was assayed by 1.0% agarose gel electrophoresis. Reverse transcription of 1,000 ng of RNA into 20 μL of system cDNA was performed using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2 (Accurate Biotechnology (Hunan, China) Co., Ltd.). ddH₂O was used to dilute the cDNA to 30 μL for qPCR.

To verify the reliability of the RNA-seq data, RT-qPCR validation analysis was performed on randomly selected candidate genes that were contained in Unigene0000019, Unigene0012859, Unigene002300, Unigene0028761, Unigene0030931, Unigene0030968, and Unigene0039043. The RT-qPCR expression level analysis was used to identify candidate genes of interest on the KEGG synthesis pathway, including Unigene0015669 (PAL3), Unigene0002103 (HMGR), Unigene0033249 (4CL1), Unigene0031514 (FPS2), Unigene0033266 (F26G), Unigene0003471 (IPP2), and Unigene0035269 (CHS2). Primer 5.0 was used for the primer design, and ACTIN was used as the internal reference gene. All primers were obtained from Biotechnology Co., Ltd. (Shanghai, China, https://www.sangon.com), and the primer information is shown in Supplementary Table S1 .

For RT-qPCR, the SYBR Green Pro Taq HS qPCR Kit (Accurate Biotechnology (Hunan) Co., Ltd.) was used. The 10-µL reaction system contained 5 µL of 2X SYBR Green Pro Taq HS Premix, 0.2 µL of forward primer (10 µmol/L), 0.2 µL of reverse primer (10 µmol/L), 1 µL of cDNA, and 3.6 µL of sterile water. Based on the study of Xu et al. (2018), we optimized the qPCR procedure as follows: 95°C, 3 min; 95°C, 15 s; 60°C, 30 s; the above steps were cycled 40 times, 65°C, 15 s; 95°C, 5 s. The Bio-Rad CFX96™ detection system was used. All reactions were repeated three times. The amplification products were detected by agarose gel electrophoresis and RT-qPCR solubility curves to verify the specificity of the primers. The relative expression levels of DEGs were calculated using the 2^−ΔΔCT method.