*2.5. Analysis of Differential Transcription between WT and RT*

To reveal expression differences between WT and RT, we compared the transcriptomes of the six petal tissue samples. RNA-seq generated 44,202,732–55,656,090 clean reads per sample, of which 38,737,776–49,062,666 (88%; 85% unique) were mapped to the reference genome. Approximately half of the unique reads were mapped to the positive-sense strand of chromosomes (Figure S13, Table S3), and 294 transcription factors were detected (Table S4). Gene expression levels were distributed similarly, and significantly positively correlated among the sample genomes (*R* <sup>2</sup> > 0.93) (Figure S14a,b). In the next step, the six samples were divided into two groups—WT (WT1, WT2, and WT3) and RT (RT1, RT2, and RT3)—for further analysis.

In this analysis, 16,383 expressed genes, 221 WT-specific and 765 RT-specific, were detected using a FPKM > 1 threshold (Figure S14c), with FPKM standing for fragments per kilobase of exon per million fragments mapped. Screening with DESeq yielded 958 upregulated and 1134 downregulated differentially expressed genes (DEGs) widely distributed across WT and RT genomes (Figure 5a,b). These DEGs belonged to 55 functional groups, including 36 biological process, 14 cellular components, and five molecular function categories. Many of the enriched genes take part in metabolic processes (Figure S14d). KEGG pathway analysis was used to further explore DEGs associated with anthocyanin biosynthesis and metabolic regulation (Figure 5c). We found that the genes were enriched mainly in pathways controlling plant hormone signal transduction (ko pmum04075), biosynthesis of secondary metabolites (ko pmum01110), metabolism (ko pmum01100), and flavonoid biosynthesis (ko pmum00941). Structural genes, including *Pm020453* (*YUCCA8*), *Pm004176* (*ANGLT*), *Pm031359* (*UGT79B6*), *Pm006139* (*GSTF1*), *Pm011195* (*GSTXC*), *Pm012985* (*GSTF7*), and *Pm025127* (*GSTX6*), were downregulated, but other critical genes, such as *Pm013782* (*DFRA*), *Pm018402* (*DFRA*), *Pm023202* (*DFRA*), *Pm017146* (*FLRT*), and *Pm008680* (*UGFGT*), were upregulated (Supplementary Data 1). In addition, 189 transcription factor genes, including *MYB*, *bHLH*, and *WD*, were detected using iTAK software (Supplementary Data 2), which suggests that transcription factors take part in the regulation of bicolored flowers.
