Abi 7500 fast real time detection system

To validate the transcriptome data, the relative expressions of 12 DEGs identified in transcriptome analysis were evaluated by qRT-PCR using three biological and three technical replicates in the analysis. RNA extraction and cDNA synthesis were conducted using the Tiangen total RNA extraction kit (Tiangen, Beijing, China) and PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan), respectively. The primers for qRT-PCR were designed using the Primer Premier 5 software (http://www.premierbiosoft.com/primerdesign/index.html) (Accessed 20 April 2021). qRT-PCR tests were carried out with the TaKaRa SYBR Green Mix kit (TaKaRa, Beijing, China) using the ABI 7500 Fast Real-Time Detection System. PCR amplification was performed in 20 µL containing 10 µL 2xSYBR Primix ExTaq, 0.8 µL upstream primer, 0.8 µL downstream primer, 0.4 µL Rox-Reference dye, 2 µL cDNA template and 6 µL ddH₂O under a standard PCR program of 95 °C for 30 s; 40 cycles at 95 °C for 5 s; 60 °C for 35 s; 95 °C for 15 s; and 60 °C for 1 min, followed by 95 °C for 15 s. The relative expression analysis of quantitative data was performed by using 2^−ΔΔCt with 18S-rRNA as the reference gene.

Nine genes were selected from the DEGs and detected using real-time quantitative techniques to verify the correctness of the transcriptome results. The RNA of each sample was extracted using a plant total RNA isolation kit (Tiangen, Beijing, China) based on the manufacturer’s instructions. The cDNA of all samples was obtained using the Prime Script RT reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan), and the specific operation steps followed the instructions of the manufacturer. In addition, qRT–PCR was performed on the ABI 7500 Fast Real-Time Detection System by using the TaKaRa SYBR Green Mix kit (TaKaRa, Kyoto, Japan). The PCR amplification experiment was performed with 10 μL 2 × SYBR Green premix ExTaq II, 0.4 μL Rox Reference Dye II, 0.8 μL primer-F/R, 2 μL cDNA and ddH₂O to 20 μL. Then, qRT–PCR was performed as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 35 s, 95 °C for 5 s, 60 °C for 1 min and 95 °C for 15 s. The reference gene was 18S-RNA, and all primers are listed in Supplementary Table S18. The relative expression analysis of quantitative data was performed using the 2^−ΔΔCT method [95 (link)].

To further verify the reliability and accuracy of the transcriptome data, we initially screened 12 DEGs associated with phenylpropanoid, flavonoid, and anthocyanidin biosynthesis to detect expression levels in GL, HRL and RL with qRT-PCR. The Tiangen total RNA extraction kit (Tiangen, Beijing, China) and PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan) were used for RNA extraction and cDNA synthesis, respectively. Primer 3 software (version 2.5.0, https://primer3.ut.ee/) was used to design specific primers for qRT-PCR analysis. qRT-PCR was carried out with the TaKaRa SYBR Green Mix kit (TaKaRa, Beijing, China) using an ABI 7500 Fast Real-Time Detection System. The PCR amplification experiment was performed with a total volume of 20 μl (10 μl 2xSYBR Primix ExTaq, 0.4 μl ROX-reference, 6 μl ddH₂O, 0.8 μl downstream primer, 0.8 μl upstream primer, and 2 μl cDNA template under a standard program (95 °C for 30 s, 40 cycles at 95 °C for 5 s, 60 °C for 35 s, and 95 °C for 15 s, and 60 °C for 1 min, followed by 95 °C for 15 s). The relative expression analysis of quantitative data was performed using the 2^−ΔΔCT method^{69 (link)} with reference gene 18S-RNA as a control.