**1. Introduction**

*Prunus mume* Sieb. et Zucc. (2*n* = 2*x* = 16), a well-known ornamental tree, is widely grown for its fruits and its abundant, colorful flowers with their unique fragrance [1]. Following the domestication of this species more than 3000 years ago in China, the cultivation of *P. mume* has spread widely to other countries in East Asia. Its petal color, which ranges from white to pale yellow, pink, red, and reddish-purple, determines the desirability and economic value of individual plants and is one of the central ornamental features attracting viewers and admirers. In the 1940s, varieties with a novel characteristic, flower color chimerism, were discovered and have since served as important materials for landscaping applications and genetic improvement [2]. Drawing on our breeding experience with these varieties, we recognize five types of flowers according to petal color patterns and arrangements on individual trees: (i) bicolored flowers, which are mostly white with some red-spotted or streaked petals; (ii) pure white flowers and (iii) pure red flowers, which are both found together in the same cluster; (iv) white flowers from branches bearing white flowers only; and (v) red flowers from branches bearing red flowers only. Although graft-propagated branches bearing chimeric flowers (i, ii, and iii) produce chimeric individuals, branches with only single-color flowers (iv and v) generate single-color clones. However, at present, little information is available on the genetic mechanisms of *P*. *mume* floral chimerism.

At the cytological level, the formation of plant chimeras is believed to be linked to genetic changes in primordial cells located in the apical meristem that then proliferate mechanically [3]. The resulting somatic cell lines contain pigments, including flavonoid, carotenoid, and betalain secondary metabolites, that can be directly visualized [4–7]. At the molecular level, chimeric variation was initially explained by the action of transposable elements (TEs, or transposons), or, more specifically, the *Activator*/*Dissociation* (*Ac*/*Ds*) system that regulates the mixture of purple and yellow pigments in maize kernels by activating or repressing a *C* group gene [8]. Much later, the insertions of TEs *Tpn1*, *Tpn2*, and *Tpn3* into structural genes *dihydroflavonolreductase-B* (*DFR-B*), *chalcone isomerase* (*CHI*), and *chalcone synthase-D* (*CHS-D*), respectively, were revealed to be the critical factors giving rise to the corresponding variegated color mutants *flecked*, *specked*, and *r-1* [9–11]. The integration of *Tpn4*, an *En*/*Spm*-related transposon, into the *purple-mutable* (*pr-m*) gene encoding a vacuolar Na+/H<sup>+</sup> exchanger was found to be responsible for a mutation giving rise to purple flowers with blue sectors [12,13]. A similar functional mechanism has been linked to tobacco flower color [14]. Other examples include *Gret1* activation regulating the expression of *VvmybA1* to produce colorless grape skin [15]; *Tam1* transposon insertion in *Glycine max* [16]; and the insertion of either *Ty1dic1* or *Retdic1* in *AA5GT*, whose disruption prevents glycosylation at the 5 position of anthocyanins in *Dianthus caryophyllus* [17].

Other studies have uncovered evidence supporting a relationship between chimeric petals and the expression of structural/biosynthetic genes encoding enzymes and other regulatory factors involved in floral pigment biosynthesis and metabolism. For instance, *CHS*, *cinnamate-*4*-hydroxylase* (*C4H*), *flavanone* 3*-hydroxylase* (*F3H*), *DFR*, *anthocyanidin synthase* (*ANS*), and *UDP-glucose*: *flavonoid* 3*-O-glucosyltransferase* (*UFGT*) genes have been found to exhibit differential expression patterns between red and white flower petal tissues of individual higher plants [7,18–21]. The alternative splicing of *ANS* results in red flower petals [22], while the sequence-specific silencing of *CHS* generates white sectors in *Petunia hybrida* "Red Star" flowers [23]. Flower color variegation was also observed when the *regulator involved in anthocyanin transport* (*Riant*) gene, encoding a GST protein, was expressed while harboring an insertion–deletion polymorphism in exon 3 [24], and a TT2-like R2R3 MYB has been shown to regulate anthocyanin biosynthesis in flowering *P*. *persica* "Genpei" [25]. At the same time, epigenetic modification, such as the use of hypomethylated promoters of *A1*, *DFR*-*B*, and *OgCHS* genes driving the brick-red pelargonidin pigmentation of flower tissue [11,26,27], has been introduced to reveal genetic variation in variegated flowers.

With reference to the previous example, DNA methylation indeed appears to be one of the best-studied epigenetic modifications regulating eukaryotic growth and development [28–32] that also leads to morphological abnormalities in plants [33,34]. For instance, the extensive methylation and transcriptional silencing of a *Lcyc* gene leads to a fundamental change in floral symmetry, from bilateral to radial flowers [35], while methylated genes encoding MYB transcription factors are inversely associated with red and green-skinned fruits of apple and pear cultivars [36–38]. Importantly, DNA methylation modification is related to the silencing or reactivation of TEs that generally remain inactive [39–42]. However, little research has focused on the relationship between methylated TEs and chimeric traits. inversely associated with red and green-skinned fruits of apple and pear cultivars [36–38]. Importantly, DNA methylation modification is related to the silencing or reactivation of TEs that generally remain inactive [39–42]. However, little research has focused on the relationship between methylated TEs and chimeric traits.

bilateral to radial flowers [35], while methylated genes encoding MYB transcription factors are

In this study, we first assumed that the flower color chimerism of *P. mume* is associated with DNA methylation modification of structural genes or regulators, as well as methylated TEs, through the color regulation pathway. We performed transcriptome sequencing (RNA-seq) and advanced single base resolution methylome detection, which is a technique that has been used to elucidate fruit ripening in tomato [43], dynamic changes during seed development in soybean [44], photoperiodic sensitivity in cotton [45], and drought stress in cotton, apple, and rice [46–48], to examine three issues: (i) the methylome landscape of *P. mume*; (ii) differentially methylated region (DMR)-related genes contributing to pigment variation; and (iii) the question of whether TEs with DNA methylation modification contribute to bicolored flower formation. In this study, we first assumed that the flower color chimerism of *P. mume* is associated with DNA methylation modification of structural genes or regulators, as well as methylated TEs, through the color regulation pathway. We performed transcriptome sequencing (RNA-seq) and advanced single base resolution methylome detection, which is a technique that has been used to elucidate fruit ripening in tomato [43], dynamic changes during seed development in soybean [44], photoperiodic sensitivity in cotton [45], and drought stress in cotton, apple, and rice [46–48], to examine three issues: (i) the methylome landscape of *P. mume*; (ii) differentially methylated region (DMR)-related genes contributing to pigment variation; and (iii) the question of whether TEs with DNA methylation modification contribute to bicolored flower formation.

#### **2. Results 2. Results**

#### *2.1. Variation in Pigmentation in White (WT) and Red (RT) Petal Tissues 2.1. Variation in Pigmentation in White (WT) and Red (RT) Petal Tissues*

Three types of anthocyanins, namely, cyanidin 3,5-*O*-diglucoside (Cy3,5G; 0.077 mg/g fresh weight), cyanidin 3-*O*-glucoside (Cy3G; 0.103 mg/g fresh weight), and peonidin 3-*O*-glucoside (Pn3G; 0.124 mg/g fresh weight), were detected in red petal tissue (RT) samples by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). In contrast, no compounds similar to these secondary metabolite products were detected at an absorption wavelength of 520 nm in white petal tissue (WT) samples (Figure 1a,b and Figure S1). Three types of anthocyanins, namely, cyanidin 3,5-*O*-diglucoside (Cy3,5G; 0.077 mg/g fresh weight), cyanidin 3-*O*-glucoside (Cy3G; 0.103 mg/g fresh weight), and peonidin 3-*O*-glucoside (Pn3G; 0.124 mg/g fresh weight), were detected in red petal tissue (RT) samples by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). In contrast, no compounds similar to these secondary metabolite products were detected at an absorption wavelength of 520 nm in white petal tissue (WT) samples (Figure 1a,b and Figure S1).

**Figure 1.** Color phenotypes and petal-tissue anthocyanin contents of flowers of *Prunus mume* "Danban Tiaozhi" collected in this study. (**a**) Examples of the six types of sampled petals. Samples WT1 and RT1 were respectively collected from white and red petals of bicolored flowers; WT2 and RT2 were collected from flowers with only white or red petals, respectively; WT3 and RT3 were collected from flowers on branches with only white flowers and only red flowers, respectively. (**b**) Anthocyanin content (mg/g fresh weight) of white petal tissue (WT) and red petal tissue (RT) samples. Cy3,5G, cyanidin 3,5-*O*-diglucoside; Cy3G, cyanidin 3-*O*-diglucoside; Pn3G, peonidin 3-*O*-glucoside. **Figure 1.** Color phenotypes and petal-tissue anthocyanin contents of flowers of *Prunus mume* "Danban Tiaozhi" collected in this study. (**a**) Examples of the six types of sampled petals. Samples WT1 and RT1 were respectively collected from white and red petals of bicolored flowers; WT2 and RT2 were collected from flowers with only white or red petals, respectively; WT3 and RT3 were collected from flowers on branches with only white flowers and only red flowers, respectively. (**b**) Anthocyanin content (mg/g fresh weight) of white petal tissue (WT) and red petal tissue (RT) samples. Cy3,5G, cyanidin 3,5-*O*-diglucoside; Cy3G, cyanidin 3-*O*-diglucoside; Pn3G, peonidin 3-*O*-glucoside.

We used the BS-seq method, the "gold standard" of DNA methylation detection, to reveal the methylomes of six petal tissue groups from a single ornamental tree. In total, 40.0–50.9 million clean

*2.2. Genome Methylation Landscape of Prunus mume* 

sequenced genomes.

of any chromosome (Figure S7).
