Quantitative Analysis of Grape VvHSP20 Genes

The RNAprep Pure Plant Kit (TIANGEN, Beijing China) was used to isolate total RNA. cDNAs were obtained by total RNA reverse transcription using HiScript® II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing China). Primers for the VvHSP20 genes were designed by Primer Premier 5.0 software and listed in Additional file 2: Table S1. The grape ubiquitin1 gene was used as the reference gene [57 (link), 58 (link)] and the expression level of K1 was used as the calibrator. Quantitative real-time PCR was conducted with a total volume of 10 μL of TransStart Top Green qPCR SuperMix kit (TRANSGEN, Beijing China) in CFX96 Real-Time PCR Detection System (Bio-Rad). The relative expression changes of VvHSP20s genes were calculated using the 2^-ΔΔCt method from three independent replicates [59 (link)]. SPSS version 21.0 was employed to analyze the statistical significant differences of the gene expression levels by ANOVA with Duncan’s multiple range test.
The FPKM values of VvHSP20 genes were from the RNA-Seq data (Accession codes, SRA: PRJNA541089). The average FPKM value of each repetition was converted to log10. Pheatmap (R package) was used to generate the heatmap.