Primescript 4 1st strand cdna synthesis mix kit

The total RNA of the materials has been extracted using a TransZol Up reagent kit (Transgen, Beijing, China). The RNA quality was analyzed with a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The first-strand cDNA was synthesized with a PrimeScript™ IV 1st Strand cDNA Synthesis Mix Kit (TaKaRa, Beijing, China) for qRT-PCR. The DNAMAN software was used to design the primers of the qRT-PCR (Table 1), and the qRT-PCR (Roche Lightcyler 480, Roche, Basel, Switzerland) used was SYBR Green chemistry (2 × SYBR Green PCR Master Mix, Xiamen Biocontrast SciTech Co, Ltd., Xiamen, China). DlFeSOD (EU330204) was used as a reference gene for internal control [37 (link)]. The operating parameters of the qRT-PCR are as follows: 95 ℃ for 30 s, followed by 40 cycles of 95 ℃ for 10 s and 58 ℃ for 30 s. The relative expression of the genes involved in PA biosynthesis and metabolism were calculated via the 2^{− ΔΔCt} method [37 (link)].

The total RNA of grape branch was extracted using an OMEGA plant RNA kit (Omega Norcorss, GA, USA) according to the protocols. RNA samples (1 μg) were used for first-strand cDNA synthesized using PrimeScript™ IV 1st strand cDNA Synthesis Mix kit (TaKaRa, Japan) accordance with the manufacturer’s instructions. qPCR was performed using TB Green® Premix Ex Taq™ II (TaKaRa, Japan) and according to the manufacturer’s instruction. The reaction volume (20 μl) consist of 1 μl of cDNA (100 ng/μl), 10 μl of TB Green® Premix Ex Taq™ II, 2 μl of gene–specific primers (1 μl of forward and reverse primer, respectively), 7 μl of nuclease–free water. The qPCR program was initiated with a preliminary step of 1 min at 95 °C, followed by 40 cycles at 95 °C for 10 s, 55 °C for 30s and 72 °C for 20 s using Light Cycler®96 Real-Time PCR system (Roche, Switzerland). The primer was listed in Supplementary Table S1 that used for qPCR. The reference gene of grape was VvGAPDH (NCBI accession no. 973647) and data for each sample was calculated in relation to the reference gene using the 2^−ΔΔCT method [62 (link)].