Quantitative Gene Expression Analysis in Peach and Arabidopsis

Total RNA was isolated from peach tissue samples and Arabidopsis by using a modified cetyltrimethylammonium bromide (CTAB) method (Liu et al., 2015 (link)) and the TRIzol Reagent Kit (Ambion, Hopkinton, MA, United States), respectively. First-strand cDNA was synthesized with HiScript^® II Q Select RT SuperMix (Vazyme) after removal of genomic DNA contamination by TURBO DNase (Ambion) following the manufacturer’s protocol. Reverse transcription quantitative PCR (RT-qPCR) was performed using the SsoFast EvaGreen Supermix kit (Bio-Rad, CA, United States) with the CFX96 instrument (Bio-Rad, CA, United States) according to the following program: 95°C for 3 min, followed by 45 cycles of 95°C for 10 s, 60°C for 30 s and then 95°C for 10 s followed by a continuous increase from 65°C to 95°C at the heating rate of 0.5°C/s for dissociation curve analysis (Zhao et al., 2017 (link)). The PpTEF2 (GenBank accession: No. JQ732180) and ATACT2 (GenBank accession: No. AT3G18780) genes were used as the internal reference genes to normalize expression values for peach and Arabidopsis, respectively (Tong et al., 2009 (link); Zhou et al., 2013 (link)). The primer sequences for RT-qPCR were designed with Primer Premier 5 and listed in Supplementary Table 1.