*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 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 further supports the premise that S3 is the key for stage for turnip skin coloration. We investigated the differential accumulated metabolites (DAM) between the two turnips based on the variable importance in projection (VIP) ≥ 1 and fold change ≥ 2 or fold change ≤ 0.5 [47]). A total of 14, 14, 17 and 17 DAMs were recorded at S1, S3, S4 and S5, respectively (Figure 4A–E), resulting in all the 21 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.


**Table 4.** Anthocyanins detected in two turnip varieties.

**Table 4.** Anthocyanins detected in two turnip varieties.

**Index KEGG ID Compounds Class**  Bra18 - Peonidin *O*-hexoside Anthocyanins Bra23 - Rosinidin *O*-hexoside Anthocyanins Bra1 - Delphinidin *O*-malonylhexoside Anthocyanins Bra8 C08604 Cyanidin 3-*O*-glucoside Anthocyanins Bra22 - Delphinidin *O*-malonyl-malonylhexoside Anthocyanins Bra28 C08726 Peonidin Anthocyanins Bra32 - Cyanidin *O*-acetylhexoside Anthocyanins Bra35 - Pelargonidin *O*-acetylhexoside Anthocyanins Bra26 - Procyanidin A1 Proanthocyanidins Bra29 C10237 Procyanidin A2 Proanthocyanidins Bra20 - Procyanidin B2 Proanthocyanidins Bra34 - Procyanidin B3 Proanthocyanidins Bra17 C05908 Delphinidin Anthocyanins Bra27 C05904 Pelargonidin Anthocyanins Bra5 C12138 Delphinidin 3-*O*-glucoside Anthocyanins Bra12 C08620 Cyanidin 3-*O*-rutinoside Anthocyanins Bra9 C08718 Malvidin 3,5-diglucoside Anthocyanins Bra10 C08725 Pelargonin Anthocyanins Bra7 C12139 Petunidin 3-*O*-glucoside Anthocyanins

**Figure 4.** Temporal changes of anthocyanin content in two turnips. (**A**) Number of increased and decreased anthocyanins between PT and GT at different developmental stages S1–S5. The yellow line shows the difference in total anthocyanin content between PT and GT. PT represents the purple turnip while GT represents the green turnip; (**B–E**) differentially accumulated anthocyanins at S1, S3, S4, and S5 and their log2 fold change values between PT and GT. The asterisks mark the metabolites constantly differentially accumulated at the 4 developmental stages between PT and GT. (**F**) Venn diagram depicting the number of shared and unique differential accumulated anthocyanins between PT and GT at the 4 developmental stages. **Figure 4.** Temporal changes of anthocyanin content in two turnips. (**A**) Number of increased and decreased anthocyanins between PT and GT at different developmental stages S1–S5. The yellow line shows the difference in total anthocyanin content between PT and GT. PT represents the purple turnip while GT represents the green turnip; (**B**–**E**) differentially accumulated anthocyanins at S1, S3, S4, and S5 and their log2 fold change values between PT and GT. The asterisks mark the metabolites constantly differentially accumulated at the 4 developmental stages between PT and GT. (**F**) Venn diagram depicting the number of shared and unique differential accumulated anthocyanins between PT and GT at the 4 developmental stages.
