*3.1. Low Expression of Anthocyanin Genes Causing the Lack of Pigments in Non-Spot Areas*

In this study, we found anthocyanin accumulated in the upper epidermal cells in spot area, while no anthocyanin accumulated in cells in non-spot areas of 'Panda' (Figure 1). Transcriptome data and qPCR indicated 7 anthocyanin genes were low expression in the non-spot areas, especially for the very different expression levels of *4CL2, F3'H*, *F3H* and *F3H1* (Table 2, Figure 6). In *Phalaenopsis* 'Everspring Fairy', which also has purple spot on the white petal and sepal, DFR is the main gene which differently expressed between the purple and white part of the petals and sepals [12]. However, in *Phalaenopsis* 'Panda' expression of *DFR* in spot tissue was not significantly changed in comparison to non-spot area. These results showed that the same phenotype may be caused by different genes in the pigmentation in *Phalaenopsis.*

#### *3.2. PeMYB7 and PeMYB11 Are Important Genes in Spot Formation*

MYBs have been found to be very important in the floral pigmentation patterning in *Phalaenopsis* spp. In the sepals/petals, silencing of *PeMYB2*, *PeMYB11*, and *PeMYB12* resulted in the loss of the full-red pigmentation, red spots, and venation patterns, respectively; *PeMYB11* was responsive to the red spots in the callus of the lip, and *PeMYB12* participated in the full pigmentation in the central lobe of the lip [21]. In *P. amabilis* and *P. schilleriana*, anthocyanin-specific Myc and Wd were expressed, however, Myb specific for anthocyanin biosynthesis were undetectable in *P. amabilis*; in *Phalaenopsis* 'Everspring Fairy' petals and sepals, high levels of anthocyanin-specific Myb transcripts were present in the purple, but not in the white sectors [12]. Hsu, et al. [29] verified *PeMYB11* as the major regulatory R2R3-MYB transcription factor for regulating the production of the black color, and a retrotransposon, named Harlequin Orchid RetroTransposon 1 (HORT1), resulted in strong expression of *PeMYB11* and thus extremely high accumulation of anthocyanins in the harlequin flowers of the *P.* Yushan Little Pearl variety.

In our study, *PeMYB7* and *PeMYB11* expressed significantly different between the spot and non-spot areas, while *PeMYB2* and *PeMYB12* had not different expression levels (Table 2 and Figure 6). This result indicated that the gene of *PeMYB11* may also play an important role in spot pigmentation in *Phalaenopsis*'Panda', which was similar to these previous researches [12,21,29,30]. *PeMYB7* had different expression levels between the spot and non-spot areas of sepals in this research, which indicated this gene may also associate with purple spot patterning in *Phalaenopsis* 'Panda'. The function of *PeMYB7* is not very clear presently, however, in the phylogenetic tree inferred from MYB genes of *Phalaenopsis equestris* and *Oryza sativa* (Figure 8A), *PeMYB7* was in the same clad of *PeMYB2* and *PeMYB8*, suggesting *PeMYB7* may have similar function as *PeMYB2* which can activate anthocyanin synthesis [30].
