*1.1. Bud Sport*

Many of the fruits we eat every day are extremely heterozygous in nature [1]. The genomes of fruit trees or vines are highly heterozygous, and in order to adapt to the natural environment, some fruit trees gradually develop inbred incompatibility; as a result, some of the excellent characteristics of fruit trees are lost. Most varieties of fruit trees, such as peach, grape, and citrus, are self-compatible. Most varieties of apple, pear, sweet cherry, and other fruit trees are self-incompatible, while male sterility sometimes occurs in grapes. The VviINP1 gene was identified as related to male sterility in grapes [2]. In order to maintain the excellent properties of fruit during production, asexual propagation (cuttings, strips, and grafting) is used to maintain the exceptional characteristics of fruit [3]. Among cultivation processes, some different mutative traits are observed in similar plants [4], and some mutations are stable to inherit and are called bud sport [5].

Plant bud sport is related to somatic cell mutation that occurs in the cells of the meristem of plant buds, usually expressed on branches, leaves, flowers, and fruits. The

**Citation:** Wen, W.; Fang, H.; Yue, L.; Khalil-Ur-Rehman, M.; Huang, Y.; Du, Z.; Yang, G.; Xu, Y. RNA-Seq Based Transcriptomic Analysis of Bud Sport Skin Color in Grape Berries. *Horticulturae* **2023**, *9*, 260. https://doi.org/10.3390/ horticulturae9020260

Academic Editor: Rossano Massai

Received: 5 November 2022 Revised: 5 February 2023 Accepted: 8 February 2023 Published: 15 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

phenotype displayed by the bud sport is significantly different from that of the rest of the plant [6]. In general, bud sport is produced by cell division in the apical meristem of plants, which is triggered by mutations in the stable somatic cells of the first single cell and then fills the cell layer and forms a stable chimera [7,8]. Mutation in this cell gradually fill some or all of the meristem tissue during later stages of growth, and the mutation can be transferred to offspring and can enable mutants to reproduce asexually [9]. Bud sport brings certain types of new traits in the plant itself, while the original qualities of the plant parents are retained, which shape a new mechanism of genetic mutation [10]. Different quantitative genetic studies have located the SDI 119 quantitative trait locus (QTL) on linkage group (LG) 18, explaining up to 70% of phenotypic variance in the 120 seed content parameters. Looking into the potentials of grape varieties for table purposes, mutation-breeding programs have started for other characteristics using chemical and physical mutagens. This is very important for plants because not only the quality of plants can be improved but also more economic value can be generated [11–13].

At present, researchers and growers have selected bud sport varieties that are related to the early ripening, peel color, fruit size, and disease resistance of fruit trees according to different needs [14]. For example, through natural selection, radiation, or colchicine treatment, bud sports varieties related to early fruit ripening and peel color have been found in apples and grapevines [15,16]. Bud sport varieties with enhanced disease resistance have been found in peaches, plums, strawberries, and citrus [17–20], and varieties with enlarged fruit and doubled chromosomes have been found in bananas and kiwifruit [21,22].

#### *1.2. Fruit Color*

In fruit trees or vines, especially in apples and grapevines, peel color acts as one of the criteria for judging the ripeness of fruit, which is an important indicator and quality parameter of fruits. Numerous examples of fruit berry skin and flesh types of bud sports were reported [23]; the most common type of bud sport changes the color of the flesh or berry skin. The color change in fruit is mainly related to the change in anthocyanin content. Anthocyanins are secondary metabolites of flavonoids. In plants, flavonoids are believed to have a variety of functions, including defense against light coercion. Anthocyanin compounds play an important reproductive role as attractants in plant–animal interactions [24]. Changes in the contents of anthocyanins and synthetic pathways have been fully studied through many plant experiments [25,26].

According to multifaceted verification, some key regulatory genes in the anthocyanin synthesis pathway were analyzed [27]. In the early stages of the flavonoid biosynthesis process, CHS generates chalcone from the 4-coumarinyl-CoA and malonyl-CoA substrates. Chalcone isomerase catalyzes the formation of naringenin, which is the main metabolite of other synthetic branches of this pathway. Downstream of the flavonoid biosynthetic pathway, anthocyanins and leucine are common key substrates for the synthesis of anthocyanins and proanthocyanidins (PAs). Leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS) can convert leucoanthocyanins to anthocyanidins, and anthocyanidins can be further glycosylated by uridine diphosphate (UDP)-glucose to forming flavonoid-O-glycosyltransferase (UFGT). O-methyltransferases (OMTs) catalyze the formation of O-methylated anthocyanins, such as petunidin, peonidin, and malvidin [28,29].

Most of the fruit and skin colors of different fruits, especially grape berries, are associated with the *MYB* gene regulation of anthocyanins [30–32]. The biosynthesis of fruit anthocyanins is controlled by a unique branching of R2R3 *MYB* transcription factors. Normally, the *MYB* gene interacts with the bHLH transcription factor and the WD40 complex protein to regulate the synthesis pathway of anthocyanins [33]. Studies related to grapes and apples have shown that the change in fruit color is due to the insertion of a reverse transcriptional transposon in the promoter region of *MYB* or is a deletion of the *MYB* gene and its upstream alleles that causes the fruit peel or flesh color change. When the *MYB* gene does not show expression or its related sequence alleles are missing, fruit color cannot change to red, blue, or purple [34].

#### *1.3. Grape Bud Sport*

Grapes (*Vitis vinifera* L.) are one of the most popular fruits in the world and are usually consumed fresh, as well as in the form of several value-added products. The varieties of grape are diverse, including color, fruit size, fruit type, aroma, and other characteristics that show difference in quality. Among them, color is one of the most important quality attributes for consumers. From the beginning, people have used fresh grapes and wine as a source of transmission to spread grapes all over the world. However, with the development of breeding technology, grape breeding started, and many somatic mutations associated with the quality of grapes have been discovered. Many new grape varieties have been developed through bud sport selection.

In the following figure, the color of line under a variety represents grape peel color: green represents green varieties, red represents red varieties, and black represents black and purple varieties.

The white grape 'Italia' could sport into red grapes of the 'Ruby Okuyama' and 'Benitaka' varieties. The red grape 'Okuyama Ruby' and the white grape 'Rosario Bianco' were crossed to produce the red grape 'Rosario Rosso'. The white grape 'Muscat of Alexandria' and the black-purple grape 'Schiava Grossa' were crossed to produce the black-purple grape 'Muscat Hamburg'. The hybridization of 'Bicane' white grapes and 'Muscat Hamburg' black-purple grapes produced the white grape 'Italia' (Figure 1).

**Figure 1.** 'Italia' is associated with several grape bud sports and related relationship maps.

After thousands of years of natural hybridization and human selection, the color of the berry skins of grapes has become very diverse [35]. According to the presence or absence of anthocyanins in grape berry skin, which is divided into red and black or white varieties, this phenotype is controlled by a single gene locus [29]. There are four *MYBs* at this chromosome with two locations; at least two of these *MYBs* are mutated in white grapes. Either *VvMYBA1* or *VvMYBA2* (or both) can regulate berry peel color. For white grape, two mutations in the coding region of the *VvMYBA2* allele cause its inactivation, while it is not transcribed in white grapes due to the presence of retrotransposons in the promoter region of *VvMYBA1* [36,37]. This results in no accumulation of anthocyanins or very minute accumulation, and the berry skins and flesh color change from dark to

light eventually. However, in some grape bud sport varieties, the deletion of the *Gret1* retransposon restores the function of *VvMYBA1*, and this deletion makes the color of grape berry skins and flesh white to black or purple [38]. However, some studies have shown that, in yellow-green or white bud sports of 'Cabernet Sauvignon' [39], with the exception of *VvMYBA1*, its homologous genes of *VvMYBA2r*, *VlMYBA1-1*, *VlMYBA1-2*, and *VlMYBA2* also regulate the synthesis of anthocyanins. In addition, there are functional and nonfunctional genes among these homologous genes and alleles [26]. Researchers found that, in white grapes, the allele of *VvMYBA1* is homozygous, while the alleles of *VvMYBA1* in red or black grapes are heterozygous [40]. It can be seen in many *MYB*-related genes in berries that play an important role in anthocyanin biosynthesis that the content of anthocyanins and the color of berry flesh and peels might be regulated by these genes.

#### *1.4. Transcriptome Sequencing*

Bud sport has been studied in many fruits; however, the mechanism of bud sport in grapes remains unclear. In order to understand the mechanism of berry peel color in relation bud sport, we utilize RNA-Seq technology to compare the 'Italia', 'Benitaka', 'Muscat of Alexandria', 'Flame Muscat', 'Rosario Bianco', and 'Rosario Rosso' varieties by selecting samples at 10 wpf (weeks post-flowering) and 11 wpf (12 samples in total). We conclude that, in addition to *UFGT*, the expression of the *LDOX* gene may also correlate with the expression of *VvMYBA1/A2*, and a new gene (gene ID: Vitvi19g01871) that exhibits the highest expression of all the detected genes in white varieties might play an important role at the véraison stage in 'green-red' bud sport berries.
