**1. Introduction**

Flower spots are heterochromatic dots or streaks with a specific texture and pattern appearing on the corollas of plants, which can affect the behavior of pollinators and the ornamental value of flowers [1–4]. Previous studies have confirmed the flower spot is caused by the accumulation of anthocyanins in a specific area of corollas. For example, peonidin-3-O-glucoside, malvidin-3-*O*-glucoside,

delphinidin-3-*O*-diglucoside, and cyanidin-3-*O*-glucoside are the main anthocyanins found in petal and sepal spots in *Oncidium* [5], and cyanidin and delphinidin are the main anthocyanidins in the spot of pansy (*Viola* × *wittrockiana* Gams.) petals [6].

Molecular mechanism of anthocyanins accumulation has been clearly studied in some plants, such as *Dianthus hybrida* [7], *Antirrhinum majus* [8], *Petunia hibrida* [9], *Platycodonis Radix* [10], *Gerbera jamesonii* [11], and *Phalaenopsis equestris* [12], and the anthocyanin pathway, which is a branch of the flavonoid pathway, has been elucidated as well [13,14]. Three kinds of transcription factor genes families—MYB transcription factor, bHLH transcription factor, and WD40 repeat protein family (MBW)—were also found to regulate the anthocyanin biosynthesis genes [15].

As for the accumulation of anthocyanin in specific regions of colloras, the direct cause attributes to the specific expression of the biosynthesis genes involved in the anthocyanin pathway. For example, high level expression of the genes of OgCHI and OgDFR results in anthocyanin accumulation and pigmented spot formation in yellow lip in *Oncidium* [5,16]. Upregulation of *LhCHSA, LhCHSB*, and *LhDFR* is detected within the spots located in the center of the petals, in comparison to the low expression levels in the margin in *Lilium* 'Sorbonne' [17]. In *Dendrobium moniliforme*, pigment accumulation in the base of the column has been caused by a consequence of preferential expression of *DmF3 5 H* [18]. In *Clarkia gracilis*, precise spatiotemporal regulation of the expression of the anthocyanin genes *F3 H*, *F3 5 H*, *DFR1*, and *DFR2* produces spotted petals [19]. In pansy, *VwDFR*, *VwF3 5 H* and *VwANS* have more significantly higher level expression in cyanic flower areas [6]. Moreover, the MYB genes also play an important role for the production of flower spot by regulating the anthocyanin biosynthesis genes. For example, the large purple spots in *Phalaenopsis* 'Everspring Fairy' was mainly caused by the expression of *MYB* [12], and the *LhMYB6* and *LhMYB12* positively regulate anthocyanin biosynthesis and determine organ- and tissue-specific accumulation of anthocyanin in Asiatic hybrid lily 'Montreux' [20]. Hsu also conducted a detailed study on three *MYBs* in *Phalaenopsis*, and found that the color patterning of flower sepals, petals and lips is regulated by different MYB genes combinations, and the pigmented veins and spots on the petals are also regulated by these three *MYB* genes [21].

RNA interference, regulating gene expression by post-transcriptional mechanisms, has also been recognized to play an important role in the color special patterning model of some plants [22]. Koseki et al. [23] found that the star-type color pattern of *Petunia hybrida* Red star' flowers is induced by sequence-specific degradation of chalcone synthase RNA. Their further study found that the formation of bicolor flower types of petunia was due to RNA interference in two *CHSA* alleles (*PhCHS-A1* and *PhCHS-A2*) [23]. In *Arabidopsis thaliana*, miRNAs may function as regulators in anthocyanin biosynthesis by targeting on related transcription factors and lead to the different accumulation of anthocyanin [24].

*Phalaenopsis* spp. have become important ornamental plants worldwide for their long-lasting and various colorful flowers [25]. There are varieties of flower colors and corolla pigmentation patterning styles of *Phalaenopsis* spp., so it is very important to find more details about the flower color patterning for its breeding and production. However, there was little research in this field because of the complex anthocyanin synthesis pathways and the gene expression networks in *Phalaenopsis* spp. RNA sequencing can effectively identify the subtle differences in gene expression and find the targeted genes of small RNA in transcription level, so it has been used to study flower color patterning recently, such as in *Lilium* 'Tiny Padhye' [26], monkeyflowers (*Mimulus*) [27], and tree peony [28]. In this study, the transcriptome sequencing and small RNA sequencing in spot and non-spot sepal of *Phalaenopsis* 'Panda' were carried out by genomics Illumina sequencing platform, and the expression levels of candidate genes, as well as microRNAs, were verified by qPCR. Meanwhile, the model of flower spot formation pattern was predicted by the interactions of the structural genes, regulatory genes together with small RNA. This study created a joint research of transcriptome sequencing and small RNA sequencing to explain the spot pigmentation in *Phalaenopsis.* spp., and the results will be helpful for the breeding of new colorful cultivars of *Phalaenopsis* spp.
