*2.4. The Expression Patterns of ABP Genes*

Genes involved in the flavonoid biosynthesis and ABPs that are related to color formation were isolated, including structural and regulatory genes, in order to identify those involved in the regulation of flower polymorphism in *P. limprichtii*. There were 21 ABP-related structural unigenes, which encoded 10 enzymes, expressed both in pigmented flowers and white flowers, with a total of 14 R2R3-MYB, 32 bHLH, and 23 WD40 regulatory unigenes. With regard to structural unigenes, six of the 10 belong to multigene families, with the exception of *F3H, F3 5 H, ANR*, and *FLS*, which were single copy genes (Table 4). All of these unigenes were used to analyze the expression pattern of the flower color polymorphism of *P. limprichtii*.


**Table 4.** Tentative anthocyanin biosynthesis pathway-related genes in *P. limprichtii*.

Since petal anthocyanins are detectable in different color petals, we inferred that floral color differences were caused by different expression patterns of ABP-related genes. The results show that the genes encoding flavonol synthese (*FLS*, *PlFLS*), anthocyanin synthese (*ANS*, *PlANS1*, *PlANS2*), and UDP-glucose anthocyanidin 3-O-glucosyltransferase (*UFGT*, *PlUFGT1*, *PlUFGT2*) were expressed at a higher level in pigmented flowers (rose-purple and pink) than in white flowers, and were more up-regulated in the rose-purple flowers than in the pink flowers (Figures 5a and 6). These genes were correlated with flower color intensity and color phenotypes. Besides these directly affected genes, other genes that have different expression patterns between pigmented flowers and white flowers also influence color formation, such as the flavanone 3 -hydroxylase gene (*F3 H*; *Pl F3 H3*) and dihydroflavonol 4- reductase (*DFR*, *PlDFR3*). These were both up-regulated in rose-purple flowers and contribute to red color formation. According to the ABP-related gene expression patterns and metabolites detected in the three distinct flower groups, we drew a putative ABP of *P. limprichtii*.

**Figure 5.** Expression pattern analysis base on RNA sequencing of anthocyanin biosynthesis pathway-related genes and transcription factors in *P. limprichtii*. (**a**) Anthocyanin biosynthesis pathway-related unigenes; (**b**) *bHLH* unigenes; (**c**) *R2R3-MYB* unigenes; (**d**) *WD40* unigenes. W-pe (petal of white flower), P-petal (petal of pink flower), R-petal (petal of rose -purple flower).

**Figure 6.** Real time quantitative reverse transcription-PCR of several genes in *P. limprichtii*. Each value is shown as average ± standard deviation from three biological replicate sampling.

The expression patterns of anthocyanin regulatory genes, including R2R3-MYB, bHLH, and WD40 were also investigated (Figure 5b–d). Phylogenetic analysis (Figure 7) shown that PlMYB13 (unigene0062421) and PlMYB4 (unigene0039181) were clustered with *AtMYB75*, *AtMYB90*, and *AtMYB113*, which belong to subgroup 6 of *A. thaliana* [27], and have been demonstrated to activate anthocyanin accumulation, while *PlMYB1*0 (Unigene0058559) was homologous to *AtMYB11*, *AtMYB12*, and *AtMYB111*, which belong to subgroup 7 in A. thaliana, and have been suggested to control flavonol biosynthesis [28]. The expression pattern of PlMYB13 was consistent with anthocyanin accumulation, while *PlMYB10* exhibited an inverse relationship between its expression and flower color intensity.

**Figure 7.** Phylogenetic analysis of R2R3-MYB DNA binding domains for *P. limprichtii* and Arabidopsis thaliana. (**a**) Circular phylogenetic tree; (**b**) amplification of S4, S6, and S7 branches. The R2R3 domains of the 14 MYBs identified in *P. limprichtii* petal transcriptome were aligned and analyzed using neighbor-joining phylogenetic methods.
