RNA extract, qRT-PCR and RNA-seq analysis

Harvested tomato tissues were immediately frozen in liquid nitrogen and stored at −80 °C. Total RNAs were isolated using Trizol (Invitrogen) according to the manufacturer’s instructions and used for PCR and RNA-seq analysis. For qRT-PCR analysis, the first-strand cDNA was synthesized using an M-MLV system. The qRT-PCR was performed using specific primers (Supplementary Table S2) as described by Liu et al.⁴⁰. Three technical replicates were performed for each sample every time and three biological repeats were performed. The relative quantitative values were calculated using the 2^−△△Ct method⁴¹. The specificity of the amplification was determined by performing a dissociation curve analysis.

For RNA-seq, total RNA samples were prepared from the fruits at the breaker stage (55 dpa) using Trizol (Invitrogen) and purified with the RNeasy Plus Kit (Qiagen). Then, the RNA was analyzed using a high-throughput parallel sequencing using an Illumina genome analyzer II according to the manufacturer’s instructions. The false discovery rate (FDR) was set at 1% to determine the threshold of the P-value in multiple tests and analyses by manipulating the FDR value. P < 0.001 and the absolute value of log2 ratio >1 were used as the threshold to judge the significance of the gene expression difference according to Audic and Claverie⁴².