Forward specific primer

In each treatment group, strawberry fruits (n = 6) growing at the same rate were acquired using the random sampling method. For analysis of FaTPK1 transcripts by SqRT–PCR in RNAi and OE fruits, six fruits were randomly selected for mixed grinding. First‐strand cDNA was used as the template for 28 cycles of PCR amplification of FaTPK1 using 20 μL PCR mixture. The reactions contained 0.2 μL LATaq (5U/μL), 2 μL 10 × LA PCR buffer II (Mg²⁺ plus), 3.2 μL dNTP Mixture (2.5 mm), 1 μL forward specific primer (10 μm; Sangon, Shanghai, China), 1 μL reverse specific primer (10 μm; Sangon), 1 μL cDNA template and 11.6 μL ddH₂O. The experiment above was repeated three times. A 418‐bp FaTPK1 was amplified by primers (sense, 5′‐TGCTTCCAGGGGTCATAG‐3′; and antisense, 5′‐AGTCTGCTGCTTGG CTCA‐3′). These conditions were selected for the comparison of the relative accumulation of FaTPK1 and Actin. After silence and overexpression of FaTPK1 in strawberry, the ripening‐regulated genes GAL6, XYL2, PG1, CHS, TPK1, CHI and SUT1 were investigated by SqRT–PCR, primers as shown in Table S2. The primers used for SYBR real‐time PCR are showed in Table S3 and made according to the description by Chai et al. (2011). The experiment was performed with three replications.

Total RNA from leaves of about 40-day transgenic and wild-type plants were extracted using an Omega RNA extraction kit (Omega Bio-tek, USA) according to the manufacturer’s protocols. To generate first-strand cDNA, 400 ng of total RNA was reverse-transcribed using the Trans kit (Transgen, China) according to the manufacturer’s protocols. The first-strand cDNA was used as a template for PCR amplification for real-time PCR on a LightCycler 96 real-time PCR system (Roche Diagnostics GmbH, Mannheim City, Germany) using TransStart Top Green qPCR SuperMix (Transgen, China). The reactions of 10 µl contained 5 µl qPCR SuperMix, 0.25 µl forward specific primer (10 µM; Sangon, China), 0.25 µl reverse specific primer (10 µM; Sangon, China), 2 µl cDNA template, and 2.5µl ddH₂O. Actin was used as a reference gene. Relative gene expression was analyzed by LightCycler^® 96 SW 1.1.The primers used for real-time PCR are shown in Supplemental Table S3. The experiment was repeated three times.