**Comparative Transcriptomics Provides Insight into Floral Color Polymorphism in a** *Pleione limprichtii* **Orchid Population**

**Yiyi Zhang <sup>1</sup> , Tinghong Zhou 2, Zhongwu Dai 1, Xiaoyu Dai 1, Wei Li 1, Mengxia Cao 1, Chengru Li 1, Wen-Chieh Tsai 1,3, Xiaoqian Wu 1, Junwen Zhai 1, Zhongjian Liu 1,\* and Shasha Wu 1,\***


Received: 4 December 2019; Accepted: 26 December 2019; Published: 30 December 2019 -

**Abstract:** Floral color polymorphism can provide great insight into species evolution from a genetic and ecological standpoint. Color variations between species are often mediated by pollinators and are fixed characteristics, indicating their relevance to adaptive evolution, especially between plants within a single population or between similar species. The orchid genus *Pleione* has a wide variety of flower colors, from violet, rose-purple, pink, to white, but their color formation and its evolutionary mechanism are unclear. Here, we selected the *P. limprichtii* population in Huanglong, Sichuan Province, China, which displayed three color variations: Rose-purple, pink, and white, providing ideal material for exploring color variations with regard to species evolution. We investigated the distribution pattern of the different color morphs. The ratio of rose-purple:pink:white-flowered individuals was close to 6:3:1. We inferred that the distribution pattern may serve as a reproductive strategy to maintain the population size. Metabolome analysis was used to reveal that cyanindin derivatives and delphidin are the main color pigments involved. RNA sequencing was used to characterize anthocyanin biosynthetic pathway-related genes and reveal different color formation pathways and transcription factors in order to identify differentially-expressed genes and explore their relationship with color formation. In addition, qRT-PCR was used to validate the expression patterns of some of the genes. The results show that *PlFLS* serves as a crucial gene that contributes to white color formation and that *PlANS* and *PlUFGT* are related to the accumulation of anthocyanin which is responsible for color intensity, especially in pigmented flowers. Phylogenetic and co-expression analyses also identified a R2R3-MYB gene *PlMYB10*, which is predicted to combine with *PlbHLH20* or *PlbHLH26* along with *PlWD40-1* to form an MBW protein complex (MYB, bHLH, and WDR) that regulates *PlFLS* expression and may serve as a repressor of anthocyanin accumulation-controlled color variations. Our results not only explain the molecular mechanism of color variation in *P. limprichtii*, but also contribute to the exploration of a flower color evolutionary model in *Pleione*, as well as other flowering plants.

**Keywords:** *Pleione limprichtii*; flower color polymorphism; variation within populations; metabolome analysis; anthocyanin biosynthetic pathway; RNA sequencing; transcription factor

#### **1. Introduction**

Flower color is one of the most attractive characteristics of plants in nature. With such massive variation, flower color is regarded as an evolutionarily labile trait and has been shown to contribute to plant evolution [1–3]. In particular, flower color adaptive mutations mediated through pollinators are directly relevant to phenotypic evolution [4]. Color variation is considered a fixed difference between species and promotes the formation of population polymorphism [5,6]. Among angiosperms, sister species always display differences in flower hue and intensity, This pattern of flower color polymorphism is used as a model trait in the study of ecology, evolution, and gene regulation [7]. Color changes are related to flower pigment content. To date, the molecular mechanism of flower color transition has been investigated in several species, owing to the main floral pigments having been well characterized in many plants [8–13] providing sufficient information for studying floral color formation in non-model species and the opportunity to explore the relationship between phenotypic evolution and color variations.

Although flower color is influenced by many factors, flavonoids, especially anthocyanins which are produced by the anthocyanin biosynthesis pathway (ABP), are the primary components that contribute to floral pigments and they are produced by highly conservative structural and regulatory components [14]. The ABP involves multi-metabolic processes which mainly consist of seven core structural genes: *CHS*, *CHI*, *F3H*, *F3 H*, *F3 5 H*, *DFR*, and *ANS*, and several branch-enzyme genes [15]. Due to the instability of anthocyanidins, they exist mainly as anthocyanins, which are formed by anthocyanidins and various glycosides [16]. They play an irreplaceable role in the color development of plants and are primarily derived from three main anthocyanidins: Pelargonidin (brick red to scarlet), cyaniding (red to magenta), and delphinidin (purple to violet) [16]. Studies have shown that blocking the ABP can lead directly to variations in pigment production and affect flower color [17]. In addition to the structural genes in the ABP, transcription factors also contribute to flower color transition by regulating the spatial and temporal expression of the structural genes [18,19]. The ABP is regulated by three complex, interacting transcription factors: R2R3-MYB, basic helix–loop–helix (bHLH), and WD40-repeat (WDR) [20]. These transcription factors activate or suppress the transcription and expression of target genes, thereby regulating anthocyanin synthesis [21]. Generally, the structural and regulatory genes involved in the ABP have provided a number of targets to reveal the diversity of mutations that could block the ABP [22]. For flower polymorphism within populations, locating the blockage could elucidate the cause of flower color transition at the biochemical and molecular scales [8,10,11]. In addition, understanding their specific ABP is of benefit for predicting evolutionary influences from a genetic perspective.

The genus *Pleione* (Orchidaceae) comprises nearly 30 species of terrestrial, lithophytic, and epiphytic plants with high ornamental value [23]. There are 27 species in China, while Yunnan is the world biodiversity distribution center of this genus [24,25]. *Pleione* possesses different flower colors ranging between white, pink, lavender, magenta, light purple, and yellow [26]. In particular, populations of Huanglong, Sichuan Province, there remain a color polymorphism population, consisting of pink flowers of different intensities along with white mutant individuals, which can be considered an ideal situation to study the polymorphism formation mechanisms of *Pleione*, as well as benefit to explore potential correlation between color pattern and the species evolution.

The focus of our study was to understand the molecular mechanism of color polymorphism, including how the white individuals formed and the main reason caused pink flowers intensities, as well as summarize the general rules of the color distribution pattern. We aimed to investigate distribution of color monomorphic in the Huanglong *P. limprichtii* population and examine the transcriptome and biochemistry of their color polymorphic petals. RNA sequencing (RNA-seq) and ultra-performance liquid chromatography (UPLC) were used to identify the variation of related genes and the differences in flavonoid intermediates in the ABP that cause color transition, respectively.
