Genome-Wide Identification and Expression Analysis of Eggplant Reveals the Key MYB Transcription Factor Involved in Anthocyanin Synthesis

Abstract

MYB transcription factors (TFs) represent one of the largest gene families in plants, and previous studies have demonstrated their involvement in regulating anthocyanin synthesis. Eggplant is an important anthocyanin-rich solanaceae vegetable crop. In this study, a total of 219 MYB gene-family members were identified using the complete and high-quality eggplant genome, comprising 105 1R-MYBs, 107 R2R3-MYBs, 5 3R-MYBs, and 2 4R-MYBs. Using phylogenetic analysis, we divided them into 33 subfamilies. Members of the SmeMYB gene family are unevenly distributed on 12 chromosomes, but are mainly concentrated at the upper and lower ends of the chromosomes. In addition, the expression levels of R2R3-MYBs in differently colored eggplant tissues (peel, calyx, stem, flower, and leaf) were investigated with RNA-seq (RNA sequencing). A total of 13 differentially expressed R2R3-MYB transcription factors related to the synthesis of anthocyanins in different eggplant tissues were screened out. SmeMYB110, SmeMYB175, and SmeMYB182 were also found to play significant roles in this process. Furthermore, 10 MYB TFs were identified as potential genes regulating anthocyanin synthesis in different tissues. The quantitative real-time PCR (qRT-PCR) analysis results showed that SmeMYB175 was regarded as the most likely key transcription factor regulating anthocyanin synthesis in calyx. These results not only deepen our understanding of the MYB gene family in eggplant but also lay a solid foundation for further research on the regulation of SmeR2R3-MYBs in anthocyanin synthesis across diverse eggplant tissues.

1. Introduction

Transcription factors play a crucial role in the regulation of gene expression. Typically, transcription factors have two main components: the DNA binding domain and the transcription activation/repression domain [1]. This implies that transcription factors generally control various biological processes through four regions: DNA binding motif, transcription activation motif, nuclear localization signal, and oligomerization site [2]. Based on the DNA domain, several transcription factors, such as MYC, MYB, bZIP, and BHLH, have been identified and categorized into different families and superfamilies. The v-MYB avian myeloblastosis viral oncogene homolog (MYB) gene family is among the largest members of the plant transcription-factor family. MYB transcription factors contain a variable number of conserved repeats (R) at the N-terminus which are primarily involved in DNA binding and protein–protein interaction. The variable C-terminal region regulates protein activity. Members of the MYB gene family are classified into four categories based on the number of repetitive domains in their sequence: 1R-MYB (MYB-related), 2R-MYB (R2R3-MYB), 3R-MYB (R1R2R3-MYB), and 4R-MYB [3]. Each repeat typically consists of approximately 52 amino acid residues forming three α-helices, and the second and third helices participate in forming a helix–helix (HTH) fold with a spacing of three tryptophan residues, creating a hydrophobic core in the three-dimensional HTH structure [4]. The MYB gene family is present in the majority of eukaryotes and plays a crucial role in regulating numerous physiological processes in plants, particularly those related to growth, development, secondary metabolism, and responses to external environmental stimuli [5].

MYB family proteins serve a variety of roles in regulation of a variety of plant-specific processes, including growth and development [6], stress responses [7,8], and secondary metabolic functions [9] like those involving anthocyanins, flavonols, and lignin. In plants, anthocyanins have been found to enhance drought resistance [10], cold tolerance [11], disease resistance [12], and photoprotection [13]. Previous research has demonstrated that MYB transcription factors play a crucial role in the biosynthesis of anthocyanins [14,15,16]. MdMYB10 has been shown to impact anthocyanin levels in apples [17], while VvMYBA1 and VvMYBA2 modulate peel color in grapes [18]. Wild-type lettuce was observed to accumulate anthocyanins, whereas overexpression of AtMYB60 inhibited the production and accumulation of anthocyanins in lettuce [19]. MYB transcription factors have also been identified in major Solanaceae plants. Quattrocchio et al. [20,21] discovered AN4, which regulates petunia blooms and anther coloration, and AN2, which controls anthocyanin synthesis in petals. Additionally, R2R3-MYB transcription factor PHZ regulates the flower buds and stalks of Petunia, as well as anthocyanin synthesis in vegetative tissue stems, the leaf margin, and the growth point under abundant light [22]. StAN2 is involved in the regulation of peel color in potato tubers [23], while StMYB44 acts as a negative regulator of anthocyanin synthesis in potatoes under high-temperature conditions [24]. Overexpression of NtAn2 in tobacco leads to the induction of anthocyanin synthesis throughout all parts of the plant [25]. LrAN2 and LrMYB113 play roles in anthocyanin synthesis in L. barbarum fruits [26], while SlANT1 and SlAN2, two highly homologous MYB transcription factors, are associated with anthocyanin synthesis and have been isolated from tomatoes [27,28]. The incomplete dominant gene A influences anthocyanin accumulation in pepper fruits [29].

The eggplant (Solanum melongena) is a Solanum species within the Solanaceae family. According to FAO data (2020–2022), while the global eggplant cultivation area decreased to 1.89 million hectares, yield increased to 59.31 million tons. China produced 38.31 million tons in 2022, accounting for 64.6% of global production. China is the largest producer, consumer, and exporter of eggplant in the world, and eggplant plays an important role in China’s agricultural industrial structure. Eggplant has high anthocyanin content, which is distributed across multiple tissues. Due to the different levels of anthocyanin content, the eggplant peel primarily comes in four colors: purple-black, purple-red, green, and white; the potential flower colors include, roughly, dark purple, purple, and white; and the calyx, stem, and leaf contain three main color types: dark purple, purple-green, and green. Given the crucial role of MYB transcription factors in regulating anthocyanin synthesis in eggplant [30,31,32,33], we utilized the latest version of the eggplant genome (genome size 1.16 Gb, with 99.5% genes annotated) to identify all members of the MYB gene family on the whole genome. We then systematically analyzed the physicochemical properties, gene structure, motifs, conserved domains, and cis-acting response elements of SmeMYB gene-family members. Moreover, considering the significant role of R2R3-MYB transcription factors in plant anthocyanin synthesis, we examined the expression levels of SmeR2R3-MYB in various tissues (peel, calyx, stem, flower, and leaf) exhibiting different colors. The R2R3-MYB genes potentially involved in the regulation of anthocyanin content in different tissues were screened, which provided a reference for future studies on R2R3-MYB involvement in eggplant anthocyanin synthesis. The identification of the complete eggplant MYB gene family and thorough exploration of potential MYB TFs responsible for regulating different colors of eggplant across various tissues will significantly contribute to our understanding of eggplant varieties with diverse coloration.

2. Materials and Methods

2.1. Identification and Physicochemical Characteristics of MYB Gene-Family Members in Eggplant

The genome, gene annotation, and protein sequence files of the eggplant were obtained from the T2T Eggplant Genome database (unpublished) for analysis. A total of 197 Arabidopsis MYB protein sequences were downloaded from the Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/, accessed on 1 June 2023) database [34]. HMMER3.0 software was utilized to construct a hidden Markov model (HMM) based on the known Arabidopsis MYB protein family sequences, which was then used for domain alignment to identify potential MYB family sequences in eggplant protein sequences. Using Blastp (version: ncbi blast-v2.10.1+) [35], we obtained the candidate eggplant protein sequences, which were subsequently evaluated and annotated using the Pfamscan (version: v1.6) and Pfam A (version: v33.1) databases for domain information [36,37]. The candidate protein sequences that contain the complete MYB domain (PF00249, PF11831, PF13921, PF14379) were selected as the MYB gene-family sequence of eggplant (SmeMYB). Additionally, the ExPASY-Compute pI/Mw tool (https://web.expasy.org/compute_pi/, accessed on 1 June 2023) [38] was employed for further characterization of the identified eggplant MYB gene-family members’ basic physical and chemical properties.

2.2. Multiple Sequence Alignment and Phylogenetic Analysis of SmeMYBs

Utilizing the Interpro (https://www.ebi.ac.uk/interpro/result/InterProScan/#table; accessed on 1 June 2023) web platform for the classification of SmeMYB gene-family members (1R-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB), we investigated the evolutionary relationship between MYB gene-family members in eggplant and R2R3-MYB gene-family members in major Solanaceae crops (tomato, pepper, eggplant, and potato). The identified SmeMYB gene-family members were aligned to construct a phylogenetic tree of eggplant. Multiple sequence alignments of the R2R3-MYB family sequences of eggplant (SmeR2R3-MYB), tomato [39], pepper [40], and potato [41] were constructed to build the Solanaceae R2R3-MYB phylogenetic tree. The multiple sequence alignment was carried out using MAFFT (version: v7.427), and the phylogenetic tree was constructed using maximum likelihood (ML) with 1000 bootstraps through MEGA10 [42].

2.3. Gene Structure and Motif Analysis of SmeMYB Family Members

The SmeMYB gene structure (exon–intron) was determined using the Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/, accessed on 1 June 2023) online software [43]. The MEME (https://meme-suite.org, accessed on 1 June 2023) tool was utilized to identify conserved motifs included in the MYB sequence with a limit of 15 and default values for the remaining parameters [44]. TBtools software (version: v1.045) was employed to visualize the phylogenetic tree, gene structure, and conserved motifs of eggplant [45]. Additionally, the WebLogo platform (http://weblogo.berkeley.edu/, accessed on 1 June 2023) was used to visualize the conserved SmeR2R3-MYB domain [46]. Using TBtools, we extracted the 2000 bp sequence region upstream of SmeMYB gene-family members for prediction of transcription-factor binding sites using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 1 June 2023) [47]. The location and number of the top 12 cis-acting element species were displayed in the physical map of the gene promoter.

2.4. Chromosomal Localization and Collinearity Analysis of SmeMYBs

The physical location and chromosomal distribution information of the MYB gene-family members in eggplant were determined using MapInspect software (http://mapinspect.software.informer.com/, accessed on 1 June 2023) [48] and visualized with MG2C (http://mg2c.iask.in/mg2c_v2.1/, accessed on 1 June 2023). Collinearity analyses between the MYB gene-family members in eggplant was conducted using MCScanX software (version: v2) with default parameters [49]. The segmental and tandem duplicates resulting from gene duplication events in the data were also examined [50].

2.5. Analysis of SmeR2R3-MYB Gene Expression Levels

The natural population materials for RNA-seq were cultivated in a plastic greenhouse at the Qiaosi experimental field of the Zhejiang Academy of Agricultural Science in the spring of 2023. Eggplant fruits were harvested approximately 20 days after pollination. Peel (excluding pulp) was collected from 1825 (purple-black, B), HQ-1315 (purple-red, P), 1815 (green, G), and 1820 (white, W). Calyx (excluding pulp) was obtained from 1825 (dark purple, DPC), 1838 (purple-green, PGC), and 1818 (green, GC). Flower samples were obtained from HQ-1315 (dark purple, DPF), 1873 (purple, PF), and 1828 (white, WF). Only petals open on that day were selected. Stem samples were obtained from young stems of materials 1825 (dark purple, DPS), 1818 (purple-green, PGS), and 1828 (green, GS). Leaf samples were obtained from young leaves of materials 1897 (dark purple, DPL), 1838 (purple-green, PGL), and 1815 (green, GL). The term “dark purple” refers to shades ranging from purple to black. Detailed information as to the RNA-seq materials is listed in Table S1. The experimental samples were collected from the same plant population material and set up in three biological replicates. The experimental samples collected were immediately placed in liquid nitrogen and then stored in an ultralow-temperature refrigerator at −80 °C. We analyzed the expression level of the whole eggplant genome at the transcription level across five different tissues (peel, calyx, flower, stem, and leaf) of different colors. The total RNA was isolated from the tissues and subjected to RNA-seq analysis using the Illumina HiSeq sequencing platform (Metware, Wuhan, China).

Raw data from 48 samples were obtained after sequencing. First, fastQC software (version: 0.12.0) was used for quality control, and the low-quality reads were filtered using fastp [51]. A total of 336.18 Gb of clean data was obtained, with the average percentage of Q30 bases being 92% or above. Using HISAT2 software (version: v2.2.1) [52] with the default parameters, the clean reads were mapped to the reference genome, with an average of 96% of the reads being mapped for the samples. FeatureCounts software (version: v2.0.3) [53] was employed to quantify gene expression, and the obtained FPKM (fragments per kilobase of transcript per million fragments mapped) was adopted as an indicator for the expression levels of transcripts or genes. DESeq2 [54,55] was employed for differential expression analysis among sample groups to acquire the set of DEGs (differentially expressed genes) between two biological conditions. Furthermore, the Benjamini–Hochberg method was utilized to correct the probability of the hypothesis test (p value) for multiple hypothesis tests, obtaining the false discovery rate (FDR). The selection criteria for DEGs are |log2Fold Change| ≥ 1 and FDR < 0.05. The FPKM values for SmeR2R3-MYB expression were obtained with RNA-seq. The average FPKM values of SmeR2R3-MYB in different tissue sites of different colors were determined, with a screening criterion set at an average FPKM mean value of less than 0.8. Subsequently, TBtools was utilized to generate a heat map illustrating the DEGs of SmeR2R3-MYB in different tissues of varying colors.

2.6. RNA Extraction and qRT-PCR Analysis

The RNA used in the qRT-PCR analysis was extracted from the three materials, 1825 (dark purple, DPC), 1838 (purple-green, PGC), and 1818 (green, GC), from the RNA-seq experiment. Each sample was analyzed with three technical replicates. The first-strand cDNA was synthesized, according to the instructions provided, with the Hifair^® III Reverse Transcriptase Kit (Yeasen Biotechnology Co., Ltd., Shanghai, China). Primers were designed based on the CDSs (coding sequences) of SmeMYB26, SmeMYB110, SmeMYB175, and SmeMYB186 (Table S2). The qRT-PCR analysis was performed in the 96 Real-Time PCR Detection System (Analytik Jena AG, Jena, Germany) using Hieff UNICON^® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotechnology Co., Ltd., Shanghai, China). The cassava β-actin gene (TRX) was used as a reference gene (Table S2). Data analysis was performed using the 2^−ΔΔCT method, and significance testing for differences was conducted using GraphPad Prism. The statistical significance between the mean values was assessed using a t test, with significance thresholds set at p < 0.05, p < 0.01, and p < 0.001. The primer pairs used for this study are provided in Table S2.

3. Results

3.1. Identification and Distribution of SmeMYBs

A total of 219 SmeMYB gene-family members were identified in the eggplant in this study. According to the distribution of chromosome location, they were renamed as SmeMYB1-SmeMYB219 (Table S3). According to the number of domains in each gene, the two hundred and nineteen identified genes were assigned to four subfamilies: one hundred and five 1R-MYB members (containing MYB-related, MYB-CC, CDC, and so on), one hundred and seven R2R3-MYBs, five 3R-MYBs, and two 4R-MYBs. The encoded SmeMYB family members ranged from 99 to 1572 amino acids (AA), with a CDS length of 300–4719 bp, molecular weight (MW) of 11.45–159.83 kDa, and isoelectric point (PI) of 4.42–10.41. A total of 127 MYB proteins had an isoelectric point below 7, while 92 MYB proteins had an isoelectric point above 7, with the majority of the proteins being acidic. A total of 15 members of the MYB family exhibit an instability index below 40, whereas the remaining 204 members have an instability index ranging from 40.57 to 73.35, indicating protein instability. The relatively high aliphatic index of all MYB members suggests their ability to adapt to environmental diversity to some extent. With the exception of SmeMYB130, the average total hydrophilies of the other MYB family members ranges from −0.23 to −1.178, indicating their hydrophilicity. These details as to SmeMYB are provided in Table S3. The SmeMYB gene-family members in eggplant are unevenly distributed on 12 chromosomes (Figure 1), with a concentration at the upper or lower ends of each chromosome. Chromosome 10 exhibits the highest abundance, with 28 mapped SmeMYB genes, followed by chromosome 6, with 27 mapped genes. Chromosomes 5 and 1 contain 22 and 20 SmeMYB genes, respectively. The remaining chromosomes have between 10 and 18 mapped SmeMYB genes, with the lowest number being found on chromosome12, where only 10 were identified. Additionally, distinct gene clusters are present on some chromosomes; these clusters consist of highly homologous members that likely possess relatively conserved functions from an evolutionary perspective.

The SmeMYB gene family contains 52 pairs showing collinear and conserved linkage relationships, and these genes are all subject to purifying selection (Figure 2). The SmeMYB gene family consists of 51 pairs of fragment duplication, with a higher number of segmental duplication genes located on chromosomes 2, 6, and 10. Within chromosome 12, up to three pairs were formed by one gene, such as SmeMYB23 with SmeMYB34, SmeMYB50, or SmeMYB107. Meanwhile, there was a tandem repeat gene pair, SmeMYB190 and SmeMYB191. These gene pairs were all situated at the same position on chromosome 10 and exhibited similar conserved motifs and gene structures. In the plant gene family, both tandem and segmental repeats contribute to their amplification.

3.2. Phylogenetic and Gene Structure Analysis of SmeMYBs

To elucidate the evolutionary relationship and function of MYB gene-family members in eggplant, the phylogenetic tree of the MYB gene family in eggplant was constructed using the neighbor-joining method with MEGA10 (Figure S1). The phylogenetic tree was classified in terms of branches, with at least 50% bootstrap support, into 33 subfamilies (Figure S1). However, 17 MYB gene-family members could not be assigned to these subfamilies, due to their bootstrap values being less than 50%. Among the identified subfamilies, subfamily 16 was found to be the largest, comprising 21 gene-family members, followed by subfamilies 18 and 14, which contained 18 and 17 gene-family members, respectively. The subfamilies labeled in the phylogenetic tree of SmeR2R3-MYB (Figure 3) correspond one by one to the subfamily names in the phylogeny tree of SmeMYB (Figure S1), and the genes in their subfamily classification are basically consistent. Differing from the phylogeny tree of the SmeMYB system (Figure S1), the phylogenetic tree of the SmeR2R3-MYB system (Figure 3) has one more gene, SmeMYB20, clustered in subfamily 1, and subfamily 7 is split into two parts.

The gene structures of the SmeMYB gene-family members exhibit similarity. An analysis of the gene structure characteristics was conducted to gain insight into the potential functions of additional gene-family members and identify subfamilies with similar functions (Figure 3c). The statistical analysis of the intron and exon of the SmeMYB members revealed that the 219 SmeMYB members displayed a range of introns from 0 to 17, and of exons, from 1 to 18. Among these, SmeMYB92 exhibited the highest number of exons (18) and introns (17). Furthermore, there were significant differences in the numbers of introns and exons among gene-family members in different eggplant MYB subfamilies, while members within the same subfamily shared similar gene structures. For instance, all gene-family members in subfamily 2 contained two introns and three exons.

3.3. Analysis of Protein Conserved Domains and Motifs

The conserved motifs of the MYB family in eggplant were analyzed using MEME, and the results are presented in Figure S1. A total of 15 amino acid conserved motifs, named motif1–15, were identified. Each eggplant MYB protein contained a minimum of one motif and a maximum of eight motifs (Figure S1). For example, SmeMYB34, SmeMYB131, SmeMYB166, SmeMYB75, SmeMYB123, and SmeMYB183 each consisted of one motif, while SmeMYB51 contained eight motifs. In addition, the composition and distribution of conserved motifs within the same subfamily were generally consistent when combining with phylogenetic analysis; however, differences were observed between different subfamilies. Meanwhile, almost all of the 107 R2R3-MYB family members contained highly conserved motifs such as motif1, motif2, motif3, and motif5, which corresponded to predicted MYB conserved domains in terms of location (Figure 3b and Figure 4). To further investigate the conserved domain of R2R3-MYB in eggplant, a total of 107 R2R3-MYB protein sequences were aligned and used to generate the R2 and R3 logos of MYB through the online website WebLogo (https://weblogo.berkeley.edu/logo.cgi, accessed on 1 June 2024) (Figure 4). The analysis revealed that the eggplant R2R3-MYB gene-family members exhibit typical characteristics of the MYB conserved domain, with each of the R2 and R3 repeats containing approximately 52 amino acid residues (Figure 4). Specifically, motif3, motif5, and motif2 constitute the sequence of the R2 repeat, while motif1 constitutes the sequence of the R3 repeat (Figure 3b and Figure 4). The structural domains for R2 and R3 are defined by specific amino acid sequences [-W-(X19)-W-(X19)-W-] and [-F/I/W-(X18)-W-(X18)-W-], respectively. W represents tryptophan (trp), and X is any amino acid. The results showed that the R2 repeat contains three highly conserved tryptophan residues (W), forming a hydrophobic core zinc HTH structure. The first tryptophan residue (W) of the R3 repeat is frequently replaced by phenylalanine (F), isoleucine (I), or leucine (L), whereas the second and third tryptophan residues are highly conserved, a result that is consistent with Arabidopsis [56]. We also observed that some amino acid residues are highly conserved, such as G-3, 9, E-D-10, 13, L-G-21, 34, L-R-36, K-39, 40, S-C-41, 42, R-L-43, 44, R-N-47, L-49, and P-51 in the R2 repeat and E-10, G-22, N-23, I-28, A-29, P-33, G-34, R-35, T-36, D-37, N-38, K-41, and N-47 in the R3 repeat. These conserved amino acids also possess the helix-turn-helix (HTH) structure that maintains MYB transcription factors.

3.4. Analysis of Cis-Acting Elements of SmeMYBs

The presence of a large number of cis-acting elements in the promoter region is an indispensable part of the gene. Genes control their expression by binding to transcription factors and responding to changes in different environmental conditions, providing valuable insights for predicting gene function and studying gene differentiation. In this study, the 2000 bp upstream region of the SmeMYB gene was analyzed using PlantCARE, revealing a total of 108 potential cis-acting elements, with 72 having clear functions (Figure S2). It was observed that the non-uniformly distributed cis-acting elements across all genes can be categorized into two main groups: those associated with light response and those associated with hormone response (Table S4). Light-responsive cis-acting elements (Box4, G-Box, GT1-motif, TCT-motif, ATCT-motif, etc.) accounted for approximately 48.6% of all types of elements and were identified as the primary cis-acting elements. Box4 and G-box elements were particularly prevalent and were found in the promoters of most SmeR2R3-MYB genes. Most SmeR2R3-MYBs contain at least one light-responsive cis-element in their promoter region. Hormone-responsive elements accounted for 16.67% of the total number of components, including methyl jasmonate (MeJA) response (CGTCA-motif and TGACG-motif), abscisic acid (ABA) response (ABRE), and gibberellin (GA) response (P-box, TATC-box, and GARE-motif). ABRE elements were the most numerous and common in the promoter of the eggplant R2R3-MYB gene. Additionally, low-temperature response elements (LTRs), anaerobically induced elements (ARE and GC-motif), defense and stress response elements (TC-rich repeats), circadian rhythm control elements (circadian), regulatory cell development elements (CAT-box, GCN4-motif, and CCAAT-box), and other cis-acting elements were involved in growth and development. The growth and development of eggplant may be continuously regulated by various cis-acting elements.

3.5. Phylogenetic Tree of R2R3-MYB in Main Solanaceae Crops

To further study the evolutionary relationships among the solanaceae vegetables eggplant, tomato, pepper, and potato, the phylogenetic tree of R2R3-MYB in Solanaceae was constructed based on the amino acid sequences of 107, 122, 116, and 124 R2R3-MYBs in eggplant, tomato, pepper, and potato, respectively (Figure 5). According to the bootstrap values, the Solanaceae R2R3-MYB phylogenetic tree could be divided into 39 subfamilies named E1-E39. The analysis revealed that 34 subfamilies in the Solanaceae R2R3-MYB phylogenetic tree are shared by eggplant, tomato, pepper, and potato. Among the 39 divided R2R3-MYB subfamilies, the E36 subfamily has the most members, with a total of 30 R2R3-MYB gene-family members, followed by the E6 subfamily, which has 27 R2R3-MYB gene-family members. The subfamilies with the fewest members were E16 and E17, both composed of three gene families, and neither including the potato MYB gene. These results suggest the existence of a common ancestor of R2R3-MYB genes among solanaceae vegetables which also underwent specific amplification and divergence during evolution after separation.

3.6. Analysis of Expression of SmeR2R3-MYBs in Different Tissues of Different Colors

During the course of long-term evolution, gene families often undergo functional divergence or new functionalization. The analysis of gene expression patterns is crucial for understanding the function of R2R3-MYB transcription factors. A total of 29, 46, 48, 53, and 7 SmeR2R3-MYB DEGs were identified in peel, calyx, stem, flower, and leaf, respectively. TBtools was utilized to visualize the gene expression levels of these SmeR2R3-MYB DEGs across different tissues of different colors (Figure 6). A total of 26 SmeR2R3-MYB DEGs exhibited high expression in dark-purple stem (DPS), while four were highly expressed in purple-green stem (PGS), and 18 in green stem (Figure 6a). SmeMYB7 was utilized as the threshold to categorize the DEGs regulating the different colors of the calyx into two groups. A total of 28 DEGs, including SmeMYB209, were found to be expressed in at least two colors in the upper portion, suggesting their potential role in regulating the formation of two calyx colors. In contrast, the lower portion exhibited a total of 18 DEGs, including SmeMYB7, which showed high expression specifically in dark-purple calyx (DPC), while displaying low or no expression in purple-green calyx (PGC) and green calyx (GC) (Figure 6b). Figure 6c can be divided into three parts. The first part shows high expression of four DEGs, including SmeMYB125, which showed high expression in white flower (WF) and low expression in purple flower (PF). In the second part, 26 DEGs, including SmeMYB154, are mainly highly expressed in deep-purple flower (DPF), together with a few DEGs such as SmeMYB10, SmeMYB110, and SmeMYB143 that are also expressed in WF, indicating that these DEGs may regulate the formation of multiple flower colors simultaneously. The third part consists of a total of 23 DEGs, including SmeMYB186, that were mainly expressed in PF, and some of these DEGs were also expressed in DPF. SmeR2R3-MYB DEGs regulating anthocyanin synthesis in eggplant peel and leaf could be clearly divided into two parts. In the peel (Figure 6d), a total of eight DEGs were identified, namely, SmeMYB42, SmeMYB154, SmeMYB11, SmeMYB174, SmeMYB189, SmeMYB218, SmeMYB136, and SmeMYB148. These DEGs showed high expression levels in peel without anthocyanins. The remaining 21 DEGs exhibited high expression in anthocyanin-containing peel, particularly in purple-black peel (B), and low or no expression in the peel without anthocyanin. In the leaf, only seven R2R3-MYB DEGs were identified, due to potential environmental influences (Figure 6e). These R2R3-MYB DEGs regulate anthocyanin synthesis in leaves and contribute to the formation of dark-purple and purple-green leaves. In conclusion, there appears to be functional diversity among members of the SmeR2R3-MYB gene family during eggplant growth and development.

Further analysis revealed that some R2R3-MYB DEGs positively regulated anthocyanin synthesis in multiple tissues. SmeMYB110 and SmeMYB182 were found to be expressed in five different tissues. The expression trend of SmeMYB110 in calyx was correlated with anthocyanin accumulation, with the highest expression level being observed in DPC, followed by PGC, and no expression detected in GC. However, there was no discernible pattern of expression in other tissues. SmeMYB182 exhibited high expression in various anthocyanin-containing tissues and low or negligible expression in tissues lacking anthocyanins. These results suggest that this gene is not tissue-specific and is the gene that most likely positively regulates anthocyanin synthesis across different tissues of eggplant. Thirteen DEGs were co-expressed in four different tissues. Both SmeMYB1 and SmeMYB26 showed high expression levels in four anthocyanin-containing tissues, and the expression trend was consistent with anthocyanin accumulation. There were 23 and 20 DEGs involved in the regulation of anthocyanin content in three and two tissues, respectively. The results indicated that among the forty-three DEGs, only four (SmeMYB7, SmeMYB105, SmeMYB175, and SmeMYB186) exhibited higher expression levels in anthocyanin-rich tissues compared to those with little or no anthocyanins. Twelve DEGs were identified in one tissue apiece; these may have tissue specificity. Notably, SmeMYB84 and SmeMYB87 had the highest expression in DPF. SmeMYB44 exhibited high expression in the DPS, while SmeMYB61 demonstrated the highest expression in purple leaf (PL). Among the screened DEGs, only SmeMYB125 showed high expression in both stem and flower without anthocyanins, with an expression trend opposite to anthocyanin accumulation, suggesting a potential negative regulation of anthocyanin synthesis. Therefore, we identified the above 13 DEGs (SmeMYB1, SmeMYB7, SmeMYB26, SmeMYB44, SmeMYB61, SmeMYB84, SmeMYB87, SmeMYB105, SmeMYB110, SmeMYB175, SmeMYB182, SmeMYB186, and SmeMYB125) as candidate genes for regulating anthocyanin synthesis in eggplant (Table S5).

3.7. Relative Expression Levels of the Likely Candidate in Calyx via qRT-PCR Analysis

Further screening of the 13 candidate genes revealed that the expression trends of SmeMYB26, SmeMYB110, SmeMYB175, and SmeMYB186 were more consistent with the synthesis of calyx anthocyanin. Analysis using qRT–PCR was used to verify the expression of the four predicted genes related to anthocyanin synthesis in the eggplant calyx. The results of one-way analysis of variance indicated that there was an extremely significant difference among groups for SmeMYB26. Tukey’s multiple comparison analysis revealed that there were highly significant differences (p < 0.001) between the DPC and PGC groups, as well as between the DPC and GC groups, while no significant difference was observed between the PGC and GC groups. The expression levels of SmeMYB26 in DPC were higher than PGC and GC (Figure 7a). SmeMYB26 is not expressed in the PGC and GC of eggplant. However, DGC also contains anthocyanins, so the trend of SmeMYB26 expression does not correspond to the synthesis of anthocyanins in calyx. The results of one-way ANOVA show that both SmeMYB110 and SmeMYB175 exhibit significant differences between groups. Tukey’s multiple comparison analysis revealed that for SmeMYB110, the expression levels showed extremely significant differences between the DPC and PGC groups and the DPC and GC groups (p < 0.01), while no significant difference was found between the PGC and GC groups. Regarding SmeMYB175, the expression levels presented a significant difference between the DPC and PGC groups (p < 0.05), and an extremely significant difference between the DPC and GC groups (p < 0.01), while no significant difference was observed between the PGC and GC groups. The expression levels of SmeMYB110 and SmeMYB175 in DPC are higher than in PGC and GC, and both are expressed in PGC. There is no anthocyanin synthesis in the GC. Thus, compared with SmeMYB110, the overall expression level trend of SmeMYB175 is more in line with the trend of anthocyanin synthesis. The multiple-comparisons analysis showed significant differences between each pairing of the groups for SmeMYB186. The expression level of SmeMYB186 in PGC was higher than those in DPC and GC, and its expression trend at the transcriptional level was inconsistent with the synthesis of anthocyanin. In conclusion, SmeMYB110 and SmeMYB175 are considered to be transcription factors that may regulate the synthesis of calyx anthocyanin, and SmeMYB175 is the most likely candidate gene for regulating calyx anthocyanin synthesis.

4. Discussion

As the most significant category among vegetable crops, the economic value of solanaceae plants ranks only after those of Gramineae and Fabaceae. There are approximately 3000 species of solanaceous plants, including eggplant, tomato, and potato. With the continuous advancement of technology and multi-omics, the research on various aspects of its study, such as the identification, cloning, and functions of MYB transcription factors, has become a focus of attention. At present, MYB genome-wide identification studies have been conducted in Arabidopsis, tomato, pepper, and potato. The majority of identified MYB TFs in these species are R2R3-MYBs, with fewer 3R-MYBs and 4R-MYBs. A total of 196 MYB gene-family members were identified in Arabidopsis, including 126 R2R3-MYBs [56]. In solanaceous fruit vegetables, 127 MYB gene-family members were identified in tomato, including 122 R2R3-MYBs [39]. The 235 MYB gene-family members in pepper include 116 R2R3-MYBs [40], and the 217 MYB gene-family members in potato include 124 R2R3-MYBs [41]. With the advances in sequencing technology, an increasing number of near-complete genomes have been obtained for various plants, along with improved genome-wide annotations. The 73 eggplant R2R3-MYBs [57] identified from the draft genome sequence of eggplant (http://eggplant.kazusa.or.jp/, accessed on 1 June 2023) [58] represent an earlier version of the eggplant R2R3-MYB gene family. Additionally, relevant research on identifying the MYB gene family of eggplants using high-quality complete genomes has not been observable thus far. In this study, a total of 107 R2R3-MYBs were identified using the near-complete version of the genome, which is 33 more than the number of previously identified R2R3-MYBs. This demonstrates that higher-quality complete genomes enable identification of completer and more comprehensive MYB gene-family members. The identification of the complete MYB gene family is anticipated to enable more groundbreaking progress in the research of MYB transcription factors in the domain of solanaceous plants and expedite the application of transcription factors from theory to practice.

MYB transcription factors participate in every aspect of the growth and development of solanaceous plants and have significant roles in the process of their growth and development. Recent studies on MYB transcription factors in eggplant have primarily focused on their involvement in anthocyanin synthesis and stress response. Zhang et al. utilized tomato SlANT1 and CaMYB gene sequences to clone SmMYB1 from purple fruit in eggplant [30]. Subsequently, Docimo et al. [59] successfully cloned the gene SmMYB1 using RACE technology, which had previously been reported by Zhang et al. [30]. SmMYB6 served as a crucial transcription factor in regulating anthocyanin synthesis in eggplant [57]. Li et al. [60] identified five MYB transcription factors associated with anthocyanin synthesis, with SmMYB113 being a positive regulator and SmMYB35, SmMYB44, SmMYB86, and SmMYB108 acting as negative regulators. The overexpression of SmMYB113 led to an increase in anthocyanin content in the peel and pulp of the offspring of the transformed plants, indicating that SmMYB113 plays an important positive regulatory role in anthocyanin synthesis in eggplant fruit [61]. Additionally, it was found that SmMYB35 is a light-responsive R2R3-MYB transcription factor [33]. Overexpression of SmMYB35 enhances anthocyanin synthesis in the stem and corolla of transgenic eggplant. Meanwhile, SmMYB86 inhibited anthocyanin synthesis in eggplant by directly binding to the promoters of SmCHS, SmF3H, and SmANS [62]. You et al. [63] identified SmMYB1 as an epiallelic gene regulating anthocyanin synthesis in eggplant peel through gene mapping, gene annotation, and functional analysis. Multiple sequence alignments of the identified MYB protein sequences are described above. We found that SmMYB1, SmMYB6, and SmMYB113 are identical to gene SmeMYB182 (Q > 60%, I > 100%). Shao et al. [64] utilized homologous cloning in combination with RACE technology to isolate the eggplant anthocyanin synthesis gene SmMYB. Two differentially expressed MYB transcription factors, SmMYB18 and SmMYB19, were identified using RNA-seq [65]. The qRT-PCR and VIGS analyses further confirmed that SmMYB18 and SmMYB19 were involved in anthocyanin synthesis in eggplant. A comparison of the protein sequences of SmMYB and SmMYB18 revealed a high similarity, suggesting that they may represent the same gene, SmeMYB175 (Q > 100%, I > 99%). Hirakawa et al. [58] identified two candidate MYB genes, Sme2.5_02513.1_g00003.1 and Sme2.5_05212.1_g00003.1, associated with anthocyanin synthesis in the eggplant corolla on chromosome 12. Two MYB genes, ouSmMYB and dongSmMYB, were previously cloned from the green calyx and purple calyx of eggplant [66]. However, it was concluded that ouSmMYB and dongSmMYB may actually be the same gene, Sme02G0995 (Q > 62%, I > 99%). He et al. [67] demonstrated that the MYB transcription factors SmMYB94 and SmMYB19 directly interact with the structural gene CHS and participate in the anthocyanin synthesis of eggplant through transcription-factor target gene prediction and yeast monoheterization analysis. Additionally, the R3-MYB gene SmelMYBL1 is suggested to inhibit the MBW complex by competing with MYB activators for the bHLH binding site, ultimately leading to inhibition of anthocyanin synthesis [31]. The R2R3-MYB gene SmMYB75 was induced by light and was specifically expressed in petals [32]. Overexpression of SmMYB75 led to a significant increase in anthocyanin accumulation, causing a color change in callus from green to purple. Li et al. [68] identified a positive anthocyanin regulator of jasmonic acid (JA) response factor SmMYB5 using RNA-sequencing and also investigated a light-dependent JA-SmMYB5 signaling pathway that promotes anthocyanin synthesis in eggplant peel. The reverse genetics of anthocyanin synthesis in eggplant primarily focused on MYB transcription factors. Furthermore, MYB transcription factors have been reported to regulate stress resistance in eggplant [15,69]. With the advancement of technology and the deepening of research, an increasing number of studies are being conducted on MYB related to eggplant. However, due to the updated version of the eggplant genome, the complete identification of the MYB gene family has not been achieved, leading to duplication in the naming of previously identified MYB genes. Therefore, based on the identified MYB transcription factors, these genes will be uniformly named after the MYB gene-family members identified in this study (

Table 1. Uniform table of research names of existing MYB transcription factors in eggplant.

Original Name	Main Expression Tissues	Query Cover	Ident	Chromosome	SmeMYB	MYB Type	Reference
SmMYB	peel	100%	100%	Chr10	SmeMYB175	R2R3	[64]
SmMYB1	peel	100%	100%	Chr10	SmeMYB182	R2R3	[59]
Sme2.5_02513.1_g00003.1	corolla	100%	100%	Chr12	SmeMYB216	R2R3	[58]
Sme2.5_05212.1_g00003.1	corolla	77%	94%	Chr12	SmeMYB218	R2R3	[58]
SmMYB1	peel	100%	100%	Chr10	SmeMYB182	R2R3	[59]
SmMYB6	peel	100%	100%	Chr10	SmeMYB182	R2R3	[57]
ouSmMYB	calyx	62%	99%	Chr02	SmeMYB24	R2R3	[66]
dongSmMYB	calyx	72%	99%	Chr02	SmeMYB24	R2R3	[66]
SmMYB18	peel	100%	99%	Chr10	SmeMYB175	R2R3	[65]
SmMYB19	peel	100%	80%	Chr12	SmeMYB217	R2R3	[65]
SmMYB19	peel	100%	100%	Chr09	SmeMYB162	R2R3	[67]
SmMYB94	peel	100%	100%	Chr03	SmeMYB50	R2R3	[67]
SmMYB44	-	100%	100%	Chr04	SmeMYB65	R2R3	[69]
SmelMYBL1	peel	45%	100%	Chr10	SmeMYB165	R1	[31]
SmMYB35	stem, corolla	100%	100%	Chr01	SmeMYB3	R2R3	[33]
SmMYB86	peel	100%	100%	Chr06	SmeMYB110	R2R3	[62]
SmMYB75	petal	69%	98%	Chr10	SmeMYB183	R1	[32]
SmMYB113	peel	67%	100%	Chr10	SmeMYB182	R2R3	[61]
SmMYB1	peel	100%	100%	Chr10	SmeMYB182	R2R3	[63]
SmMYB39	-	100%	100%	Chr07	SmeMYB132	R2R3	[15]
SmMYB5	peel	83%	99%	Chr08	SmeMYB135	R1	[68]

).

Whole-genome analysis is a crucial approach for investigating the biological function of the MYB gene family in plants. In this study, 219 MYB gene-family members were identified from high-quality eggplant genomes, of which 107 were R2R3-MYBs, accounting for approximately 48.85% of the total number of eggplant MYB gene-family members. The number of MYB gene-family members in eggplant is less than that in pepper and more than those in tomato and potato. The size of the genome and the presence of repetitive elements are factors potentially contributing to the variation in the number of MYB gene-family members [70]. This further illustrates the complexity and diversity of plant MYB gene-family members during the evolutionary process. The phylogenetic tree analysis revealed that at least 50% of the phylogenetic tree branches supported by bootstrap values were divided into 33 subclasses. Additionally, 17 members of the MYB gene family could not be classified into these 33 subfamilies due to bootstrap values lower than 50%. The Solanaceae R2R3-MYB phylogenetic tree is instrumental in investigating the evolutionary relationships among eggplant, pepper, tomato, and potato. The structural analysis of exons and introns is valuable for exploring the evolutionary relationship within gene families. The results of the motif analysis indicate that the differentiation of gene function has led to common conserved motifs among the MYB gene-family members within the same subfamily, as well as unique conserved motifs among different subfamilies. Furthermore, the visualization of cis-acting elements in the promoter region concurrently facilitates the identification of crucial recognition sites where transcription factors exert regulatory control. Moreover, the presence of abundant and diverse cis-acting regulatory elements in the promoter region also indicates the multifaceted nature of the functions they serve during transcription. The colors of different tissues of eggplant are primarily attributed to the syntheses of anthocyanins. According to the expression levels of R2R3-MYB transcription factors in different tissues with different anthocyanin contents in eggplant, a total of 12 R2R3-MYB DEGs, namely, SmeMYB1, SmeMYB7, SmeMYB26, SmeMYB44, SmeMYB61, SmeMYB84, SmeMYB87, SmeMYB105, SmeMYB110, SmeMYB175, SmeMYB182, and SmeMYB186, were identified as candidate genes for positively regulating anthocyanin accumulation. In contrast to the observed expression trend of these genes, it was found that the transcription factor known as SmeMYB125 negatively regulates anthocyanin accumulation. As shown in Table 1, the functions of the candidate genes SmeMYB110, SmeMYB175, and SmeMYB182 are relatively well defined. Additionally, both SmeMYB175 and SmeMYB182 belong to the same subfamily (E14, S6), with Table 1 indicating that SmeMYB175 also possesses the ability to regulate anthocyanin synthesis. The highly homologous genes of SmeMYB175 and SmeMYB182 in tomato (SlMYB113 and SlMYB75) [27,71] and potato (CaA) [72] perform the same function. Li et al. [62] proposed that SmeMYB110 inhibits the synthesis of anthocyanins in the eggplant peel, but our findings suggest that SmeMYB110 may actually promote anthocyanin synthesis in the eggplant calyx. Genes with the same subfamily may have similar functions. Previous studies indicate that the S4, S5, S6, S7, and S9 subfamilies of Arabidopsis thaliana are all associated with anthocyanin synthesis [73,74,75,76]. SmeMYB1 and SmeMYB105 were classified into subfamily E19, while SmeMYB87 belonged to subfamily E18, and SmeMYB84 was categorized under subfamily E20; they were assigned to the S4, S5, and S7 subfamilies of Arabidopsis thaliana, respectively. In conclusion, we posit that SmeMYB110, SmeMYB175, and SmeMYB182 serve as crucial transcription factors in regulating anthocyanin synthesis in different tissues of eggplant, with the remaining 10 transcription factors being the most probable potential candidate genes. Analysis using qRT-PCR was further used to validate the four related transcription factors that regulate anthocyanin synthesis in calyx, namely, SmeMYB26, SmeMYB110, SmeMYB175, and SmeMYB186. SmeMYB110 and SmeMYB175 are transcription factors that may regulate anthocyanin synthesis in calyx, with SmeMYB175 being the most likely candidate gene for regulation of anthocyanin synthesis.

5. Conclusions

In this study, the MYB gene family was identified and analyzed based on the whole genome. A total of two hundred and nineteen MYB gene-family members were identified, comprising one hundred and five 1R-MYBs, one hundred and seven R2R3-MYBs, five 3R-MYBs and two 4R-MYBs. Using phylogenetic analysis, we divided them into 33 subfamilies. We further investigated the expression levels of R2R3-MYB transcription factors in different tissues with varying anthocyanin contents and successfully screened 13 MYB transcription factors that might relate to anthocyanin synthesis. Among these candidates, SmeMYB110, SmeMYB175, and SmeMYB182 were identified as important transcription factors regulating anthocyanin synthesis in different eggplant tissues. Additionally, SmeMYB1, SmeMYB7, SmeMYB26, SmeMYB44, SmeMYB61, SmeMYB84, SmeMYB87, SmeMYB105, SmeMYB186, and SmeMYB125 are potential candidate genes for regulating anthocyanin synthesis in various eggplant tissues. Analysis using qRT-PCR was employed to further verify the key transcription factors for anthocyanin synthesis in calyx. SmeMYB175 was regarded as the most likely key transcription factor regulating anthocyanin synthesis in calyx. This study not only contributes to the production of eggplants but also provides a basis for understanding the function of the MYB gene family in eggplant, particularly through an in-depth analysis of different tissues of varying colors.