Transcriptome Data Analysis

To investigate the mechanisms corresponding with the anthocyanin accumulation and petal coloration, six cDNA libraries were constructed with petals of ZYR1 and HYR3 and subjected to RNA-seq analysis based on an Illumina HiSeq 2000 platform by Personal Biotechnology Co., Ltd. (Shanghai, China). The raw data were deposited in the National Center for Biotechnology Information (NCBI) with the accession number PRJNA549842. After the low-quality reads and the reads containing adapter and ploy-N sequence were removed from the raw reads, clean reads were obtained and were further aligned to radish reference genome sequences released by the Radish Genome Database⁴ using TopHat 2.0.12 (Trapnell et al., 2009). The mapped reads count was normalized with FPKM (Fragments-per-kilobase of transcript per-million-fragments mapped) (| log₂^FoldChange| > 1, P_value < 0.05) to provide a gene expression level estimation.

Expression analysis among samples was calculated using the DESeq R package. The significant P-value between samples was determined using the Benjamini and Hochberg method (Benjamini and Hochberg, 2000). DEGs (differentially expressed genes) were obtained using a DESeq2 program with an adjusted P value less than 0.05 and | log2FoldChange| > 1 based on the FPKM values (Love et al., 2014). A Gene Ontology (GO) enrichment analysis of the DEGs was implemented using the GOseq R package with a corrected P < 0.05 (Young et al., 2012). KOBAS (KEGG Orthology Based Annotation System) software was employed to identify the enriched pathways of DEGs based on the KEGG database (Kanehisa and Goto, 2000).

Real-time quantitative PCR (RT-qPCR) was used to verify the data from the transcriptome. RT-qPCR was carried out with ABI SYBR green on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems) following the manufacturer’s instructions. β-actin gene was used as the reference gene. The reaction parameters were carried out following a previous research paper (Liu et al., 2020) and the relative expression levels were evaluated using the 2 ^{–Δ Δ Ct} method (Livak and Schmittgen, 2001). All reactions were performed using three technical replicates. Sequences of primers for RT-qPCR are listed in Supplementary Table 1.