*2.2. Di*ff*erentially Expressed Genes Between the Two Turnips and Analysis of Major Regulator Genes*

To identify the differentially expressed genes (DEG) related to turnip skin coloration, we compared the FPKM values of each gene in PT to GT at the different developmental stages and retained DEGs with fold change > 2 and a false discovery rate (FDR) correction set at *p* < 0.01 [44]. We detected 242, 194, 807, 459 and 199 DEGs at S1, S2, S3, S4 and S5, respectively (Figure 3A). The marked change in gene expression between the two turnips observed at the S3, implies that S3 may represent a key stage for turnip skin coloration. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 6 of 19

**Figure 3.** Transcription factors (TF) regulating the gene expression between the two turnips. (**A**) Number of up- and down-regulated genes between PT and GT at different developmental stages S1-S5. PT represents the purple turnip while GT represents the green turnip; (**B**) overview of the enriched TF family. The combined histograms showed the number of occurrence of genes belonging to each TF family or at each developmental stage; (**C**) Heatmap displaying the expression fold change (Log2 fold change) between PT and GT for the gene encoding transcription factors. **Figure 3.** Transcription factors (TF) regulating the gene expression between the two turnips. (**A**) Number of up- and down-regulated genes between PT and GT at different developmental stages S1-S5. PT represents the purple turnip while GT represents the green turnip; (**B**) overview of the enriched TF family. The combined histograms showed the number of occurrence of genes belonging to each TF family or at each developmental stage; (**C**) Heatmap displaying the expression fold change (Log2 fold change) between PT and GT for the gene encoding transcription factors.

*2.3. Detection of Anthocyanin Compounds in the Two Turnips*  Anthocyanins are the most important flavonoid colorants in plants [46]. We detected and determined 18, 18, 18 and 19 diverse anthocyanins at S1, S3, S4 and S5, respectively, resulting in 21 unique anthocyanin compounds in skin of the two turnips using the targeted-metabolomics approach (Table 4, Table S2). It is worth mentioning that the skin samples collected at the S2 from GT were deteriorated, so have not been used for this analysis. We did not detect procyanidin A1 and procyanidin A2 in PT, while GT did contain four anthocyanins: pelargonin and pelargonidin 3-*O*-beta-D-glucoside, pelargonidin *O*-acetylhexoside and cyanidin *O*-acetylhexoside (Table 4). With the determined quantities of all anthocyanins combined together, PT skin contained 20 times higher Transcription factors (TF) are the major regulators of gene expression profiles [45]. We therefore extended the study on the major transcription factor families differentially expressed between the two turnips. Our analysis showed that nine main TF families modulate the global gene expression levels among the two turnips (Figure 3B,C). In addition, the highest number of TF could be noticed at S3, which correlates well with the observed significant DEGs at this developmental stage (Figure 3A,B), showing that TFs are the main drivers of gene expression changes leading to the differential turnip skin coloration. Among the detected TF families, MYB, bHLH and WRKY families showed more active members involved in gene regulation (Figure 3B,C), therefore we deduce that these TF families may be crucial for the regulation of structural genes involved in turnip skin coloration. Distinctly, the genes

unique anthocyanin compounds differentially accumulated at least at one developmental stage between the two turnips. In addition, nine anthocyanins were constantly differentially accumulated in skin of the two turnips at all the four developmental stages (Figure 4F, Table S3). For most of these anthocyanin compounds, they showed presence/absence patterns among the two turnips skins,

indicating that they are the key components conferring the differential pigmentation.

level of anthocyanins as compared to GT (Table S2). Also, the highest difference in total anthocyanin content between PT and GT during the developmental stages was observed at S3 (Figure 4A), which *c42189.graph\_c1* (WRKY) was strongly down-regulated over most of the developmental stages in PT while the genes *c33188.graph\_c0* (MYB), *c44079.graph\_c0* (MYB) and *c37493.graph\_c0* (bHLH) exhibited the opposite trend. Given the role of WRKY, MYB and bHLH TFs in the regulation of structural genes involved in pigment (flavonoid-anthocyanin) biosynthesis in plants [33], it is tempting to speculate that these four key genes are the major regulators during turnip skin color formation.
