Identification and Analysis of MADS-box, WRKY, NAC, and SBP-box Transcription Factor Families in Diospyros oleifera Cheng and Their Associations with Sex Differentiation

Abstract

Transcription factors are crucial for plant growth and development. MADS-box, WRKY, NAC, and SBP-box, some of the most vital transcription factors, are not well identified in Diospyros spp., and their floral development and sex differentiation related functions remain unknown. We identified and analysed 53 MADS-box, 66 WRKY, 83 NAC, and 17 SBP-box transcription factors using the chromosomal D. oleifera genome. There were six DolSBPs identified as miR156 and miR157 targets. According to the multiple sequence alignments of Arabidopsis and D. oleifera proteins and their conserved domains and motifs, DolMADSs were divided into 23 type I and 30 type II. The DolWRKYs, DolNACs, and DolSBPs were divided into 7, 16, and 6 subgroups, respectively. It was found that one DolMADS, five DolWRKYs, one DolNAC, and four DolSBPs may promote unisexual female flowers development, while three DolMADSs, four DolWRKYs, and one DolNAC may enhance unisexual male flowers development. The functions among the different members of the same family may, thus, vary widely. The results will help to elucidate the evolution of the MADS-box, WRKY, NAC, and SBP-box genes families in D. oleifera and to determine their functions in flower development for Diospyros spp.

1. Introduction

Persimmon (Diospyros kaki Thunb.) is a type of fruit tree that is widely distributed around the world [1]; however, the short shelf life of its fruits negatively affects consumption preference. The development of superior breeding varieties is, thus, crucial for the sustainability of the persimmon industry [2]. Crossbreeding is an important method of germplasm innovation and seed-breeding. However, most persimmon cultivars are gynoecious, and only a few are androgynomonoecious or monoecious. Non-bearing androecious fruit trees are randomly distributed in wild persimmon populations [3], and the lack of male flowers strongly impacts on persimmon pollination. Therefore, studies on sex differentiation in persimmon are beneficial for artificially controlling the sexuality of flowers and promoting persimmon breeding.

Diospyros spp. have various sex types. For example, diploid Diospyros lotus Linn (D. lotus) is dioecious, and this includes both androecious and gynoecious types. In contrast, diploid Diospyros oleifera Cheng (D. oleifera) and hexaploid Diospyros kaki Thunb. (D. kaki) both have a diverse array of sex types, and these include gynoecious, androecious, monoecious, polygamomonoecious, and andromonoecious. The mechanisms underlying the sexual diversity of Diospyros spp. are not yet understood. This is, in part, because research has been limited by the absence of a hexaploid D. kaki genome. Nevertheless, chloroplast genome analysis has suggested that D. kaki has a close relationship with D. oleifera [4]. Moreover, D. oleifera, whose chromosomal genome has been constructed [5] and sexual expression is similar to D. kaki, is ideal for investigating sexual diversity and differentiation.

Transcription factors are characterised as proteins with at least one domain corresponding to a specific DNA binding site, and they control the transcriptional regulatory schemes in plants. Floral development and morphological structures are complex projects involving many transcription factors and interactions [6]. To date, 58 different transcription factor families in a variety of plants, especially model plants, have been identified and are shown in the PlantTFDB database [7]. Among these transcription factor families, MADS-box, WRKY, NAC, and SBP-box have been identified as the most important and widely distributed in plants and they are actively involved in sex differentiation.

MADS-box genes are important for a diverse array of biological functions, especially the control of flowering time, flower architecture, pollen formation, and seed and fruit development [8]. Genome-wide studies have been conducted on the MADS-box family in different species such as Arabidopsis [9], Rhododendron simsii [10], Nelumbo nucifera [11], and Rhododendron hainanense [12]. Phylogenetic analysis classifies MADS-box genes into two lineages, type I (M-type) and type II (MIKC), based on their conserved domains, gene structures, and phylogenetic relationships with Arabidopsis proteins [9]. Moreover, some genes in the MIKC subclass belong to the ‘ABC/DE’ model. Different combinations of A [APETALA1 (AP1)], B [PISTILLATA (PI) and APETALA3 (AP3)], C [AGAMOUS (AG)], and E [SEPALLATA (SEP)] class genes determine the floral organ specification, such as the A + B + E, A + E, B + C + E, C + E, and D + E genes combinations that regulate, respectively, the formation of the petal, sepals, stamen, carpel, and ovule [13].

WRKY transcription factors make up one of the largest transcription regulator families in plants, and they are vital for the regulation of various growth and development processes, particularly in anther/pollen development. In 1994, the first member of the WRKY transcription factor family, SPF1 (sweet-potato-factor-1), was cloned from sweet potato (Ipomoea batatas) [14]. Since this point, numerous WRKY family members have been identified in dozens of plants, including 72 in Arabidopsis [15], 116 in apples [16], 69 in pomegranates [17], 104 in poplars [18], 62 in strawberry [19], and 97 in kiwifruit [20]. WRKY transcription factors are also divided into three distinct groups (I to III) based on the number of WRKY domains and the characteristics of the zinc-finger motif. Group I contains two WRKY domains and a C2H2 zinc-finger motif, group II includes one WRKY domain and a C2H2 zinc-finger motif, and group III has one WRKY domain and a C2HC zinc-finger motif [21].

The NAC (NAM, ATAF, and CUC) transcription factor is a specific transcription family in plants. In 1996, NAM, the first NAC transcription factor family member, was cloned from petunia, which affects apical meristem formation and differentiation [22]. Subsequently, the NAC transcription factors, the largest in plants, have been successively discovered in Arabidopsis [23], Populus [6], white pear [24], and other species. Furthermore, considerable evidence has been collected regarding the functions of NAC genes in plant growth and development. These include the regulation of floral morphogenesis [25], shoot apical meristem formation [26], lateral root development [27], and cell wall development [28].

SBP-box genes, known in Arabidopsis as SBP-Like (SPL) genes, encode a family of plant-specific transcription factors and play a significant role in regulating the growth and development of flowers, fruits, and other physiological processes [29]. Recently, members of this family have been discovered in different plant species such as Chinese Jujube [30], Arabidopsis [31], rice [32], and grape [33]. In addition, several genes in Arabidopsis SPL members have miR156/miR157 target sites. Members of the miR156/miR157 family regulate the transformation of vegetative and reproductive growth and flower development by negatively regulating SPL genes [32].

In this study, the MADS-box, WRKY, NAC, and SBP-box families of D. oleifera were identified and then the physicochemical properties of the proteins, as well as their phylogenetic relationships, protein structures, conserved domains, and conserved motifs were investigated to provide useful insights into the conserved regulatory modules of these transcription factors. The expression patterns of the DolMADS, DolWRKY, DolNAC, and DolSBP genes and their protein interaction networks under different flower bud types, such as female flowers of the gynoecious type, male flowers of the androecious type, and female and male flowers of the monoecious type were also investigated. Based on our results, we were able to predict the functions of the aforementioned genes and reveal the corresponding regulation mechanisms for female and male flower organ development in D. oleifera, providing a theoretical basis for the artificial control of sexuality in Diospyros spp.

2. Materials and Methods

2.1. Identification of Gene Family Members and Protein Physicochemical Property Analysis

An optimised version of the D. oleifera genome was generated using a previously published reference version of its genome [5] with BioNano optical mapping-assisted assembly [34]. The optimised version of the D. oleifera genome [34] was then used to identify gene family members. The profiles for the hidden Markov models (HMMs) profile of the MADS-box (PF00319), WRKY (PF03106), NAC (PF02365), and SBP-box (PF03110) DNA-binding domains were downloaded from the Pfam database (http://pfam.xfam.org/ (accessed on 20 September 2021)) and used to identify all potential MADS-box, WRKY, NAC, and SBP-box genes with TBtools v1.098661 ‘Simple HMM Search’ function [35], and genes with an e-value < 10⁻⁵ were selected as candidate genes. An NCBI Conserved Domains Database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 20 September 2021)) was utilised for domain analysis of the above genes, and sequences without conserved domains were removed, and subsequently the MADS-box, WRKY, NAC, and SBP-box gene family members were determined. The molecular weights (Mw) and isoelectric points (pI) for the MADS-box, WRKY, NAC, and SBP-box protein sequences were predicted using the ExPASy website (http://web.expasy.org/protparam/ (accessed on 25 September 2021)). Furthermore, the homologous MADS-box, WRKY, NAC, and SBP-box genes in Arabidopsis were identified using PlantTFDB (http://planttfdb.gao-lab.org/index.php (accessed on 20 October 2021)) [7].

In addition, different groups of WRKY and SBP-box proteins were used for multiple protein sequence alignment with DNAMAN software (Version 7.0, http://www.lynnon.com/index.html (accessed on 23 October 2021)). TAPIR (http://bioinformatics.psb.ugent.be/webtools/tapir/ (accessed on 14 November 2021)) predicted the miR156 and miR157 target sites for the 17 DolSBP cDNA sequences, for which the score was set to 5.0 and the free energy ratio was set to 0.7. A target point map was drawn using the DNAMAN software (Version 7.0, http://www.lynnon.com/index.html (accessed on 14 November 2021)). The miR156 and miR157 sequences of Arabidopsis were obtained from the small RNA database (https://www.mirbase.org/index.shtml#opennewwindow (accessed on 14 November 2021)).

2.2. Phylogenetic Analysis and Gene Duplication Analysis

The MADS-box, WRKY, NAC, and SBP-box protein sequences of Arabidopsis were obtained from the Arabidopsis Information Resource database (https://www.arabidopsis.org/ (accessed on 20 September 2021)). ClustalW and Muscle were used to perform multiple sequence alignments for the MADS-box, WRKY, NAC, and SBP-box proteins between Arabidopsis thaliana and D. oleifera using a full alignment setting [36]. Subsequently, phylogenetic trees were constructed using the Neighbour-Joining and p-distance methods with 1000 bootstrap replications using MEGA7.0 [37]. The MADS-box, WRKY, NAC, and SBP-box genes were then renamed in accordance with their subfamily.

The chromosome location distribution information and whole-genome duplication (WGD) or segmental, tandem, and dispersed duplicates of DolMADSs, DolWRKYs, DolNACs, and DolSBPs were analysed and drawn using TBtools v1.098661 [35]. The collinear relationships of the MADS, WRKY, NAC, and SBP-box transcription factors for D. oleifera and Arabidopsis were also analysed using a dual synteny plotter [35].

2.3. Protein Structure, Conserved Domains, and Conserved Motifs Analysis of Transcription Factor Family in D. oleifera

The conserved motifs of the MADS-box, WRKY, NAC, and SBP-box gene families in D. oleifera were predicted using the MEME website (http://meme-suite.org/tools/meme (accessed on 21 September 2021)) [38], with the motif discovery number set to 10 and the default setting was used for the other genes. Thereafter, the NCBI Conserved Domains Database was used to predict the conserved protein domains. Finally, the analysis results for the conserved motifs, conserved domains, and gene structures were visualised using TBtools v1.098661 [35].

2.4. Promoter cis-Regulatory Element Analysis

The upstream regions (2000 bp) of the initiation codon (ATG) of MADS-box, WRKY, NAC, and SBP-box genes were used as the gene promoter sequence and deprived from the D. oleifera genome, and its cis-acting elements were analysed using the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 25 August 2022)). The results were visualized using TBtools v1.098661 [35].

2.5. Expression Analysis of the Transcription Factor Families

Transcriptome sequencing of female flowers of gynoecious, male flowers of androecious, and female and male flowers of monoecious in the critical period of sex differentiation [39] has previously been performed by our research group (unpublished), and we described the transcriptome sequencing samples (Table S1) and procedure in detail in Methods S1–S4 [40,41,42,43,44,45,46,47]. Grouped female flowers of gynoecious and male flowers of androecious were named group 1, and grouped female and male flowers of monoecious were referred to as group 2. The Fragments per Kilobase of Transcript per million Fragments Mapped (FPKM) for the differential genes (Q < 0.05) was used to calculate the expression level of DolMADS, DolWRKY, DolNAC, and DolSBP genes, and TBtools v1.098661 [35] was used to draw the heat map.

The same plant materials used for transcriptome sequencing were used for real-time quantitative PCR (RT-qPCR) reactions. The total RNA from the female flowers of gynoecious, male flowers of androecious, and female and male flowers of monoecious of D. oleifera were extracted following the manufacturer’s instructions for the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China), and the concentrations were measured using a NanoPhotometer (P330, Implen, Munich, Germany). An equivalent amount of RNA was then reverse-transcribed into cDNA using HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme, Nanjing, China). Candidate genes were selected to confirm the RNA-seq data. RT-qPCR was performed using a Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The reaction conditions were 30 s at 95 °C followed by 39 cycles of 10 s at 95 °C and 10 s at 58 °C. The primer sequences for selected genes are listed in Table S2, and GAPDH was used as the internal reference gene. In group 1, male flower buds of androecious were used as the control group, while in group 2, male flower buds of monoecious were used. The relative expression of the selected genes was calculated using the 2^−ΔΔCT method, and the experiment was repeated three times.

2.6. Go enrichment Analysis and Predicted Interaction Network of Transcription Factor Family

GO analysis was conducted to identify the roles of all the DolMADS, DolWRKY, DolNAC, and DolSBP proteins in sex differentiation using TBtools v1.098661 [35] with p-values < 0.01. For protein interaction networks, the homologs of the DolMADS, DolWRKY, DolNAC, and DolSBP proteins in Camellia sinensis were constructed using STRING (https://string-db.org/ (accessed on 12 August 2022)) with an option value > 0.7 [48]. The network was further visualised and analysed using Cytoscape version 3.7.1 (https://cytoscape.org/, Developer: Cytoscape Consortium, San Diego, CA, USA (accessed on 12 August 2022)) [49].

3. Results

3.1. Identification and Analysis of MADS-box Transcription Factor Family Members in D. oleifera

3.1.1. Identification of MADS-box Members of D. oleifera

In this study, 53 MADS-box transcription factor family members were identified, and named DolMADS1–DolMADS53, respectively (Text S1). The molecular characteristics of these proteins, including chromosomal distribution, amino acid sequence length, molecular weight, and isoelectric points were analysed (Table S3). The results showed that the DolMADSs ranged from 78 to 400 aa, and the predicted molecular weights varied from 8.9 KDa (DolMADS31) to 45.10 KDa (DolMADS22), and the isoelectric point values (pI) ranged from 4.35 (DolMADS16) to 11.11 (DolMADS5). To further facilitate our understanding of their functions, previously characterised orthologous genes in Arabidopsis were identified (Table S3).

3.1.2. Phylogenetic and Gene Duplication Analysis of the MADS-box Members

Phylogenetic analyses of 53 DolMADSs and 106 AtMADSs protein sequences from D. oleifera and Arabidopsis, respectively, revealed 23 type I (including 11 Mα, 4 Mβ, 4 Mγ, and 4 Mδ) (Figure 1a) and 30 type II (including 30 MIKCc) MADS-box proteins in D. oleifera (Figure 1b). For the ‘ABC/DE’ model, the A- (AP1/FUL), B- (AP3/PI), and C- (AG) classes all included three members, and the E-class (SEP) included two members.

These genes were distributed on 13 chromosomes. There were no DolMADS identified on Chr1 or Chr11, and Chr4 and Chr14 had the largest distributions (eight DolMADSs), while Chr7 and Chr10 had the lowest (one DolMADS) (Figure 2). To analyse DolMADSs duplication events, 14 paralogous gene pairs in the D. oleifera genome were identified (Table S4), and all the DolMADSs homologous gene pairs were located within the same group in the phylogenetic tree except for DolMADS37/DolMADS26 and DolMADS24/DolMADS29 (Figure 2). Moreover, comparative syntenic maps of D. oleifera versus Arabidopsis were constructed, and 35 pairs of orthologous MADS-box genes were identified in D. oleifera and Arabidopsis (Figure 3a).

3.1.3. Conserved Motif, Gene Structure, and Conserved Domain Analyses for the DolMADSs

Conserved motifs in the DolMADSs were searched using the MEME website [38]. Ultimately, a total of 10 motifs were identified and named motif 1–10, respectively (Figure S1, Table S5). MADS-box proteins pertaining to the same subfamily had similar conserved motif patterns. According to the Pfam domain search, motif 1 was the SRF-TF domain, and it was the most frequent, as it was present in 46 DolMADS proteins (Figure S1). Although motif 1 was not detected in DolMADS2, DolMADS12, DolMADS15–DolMADS17, DolMADS47, and DolMADS50, these DolMADSs were found to possess the SRF-TF domain using the NCBI Conserved Domains Database domain search (Figure S2). Motif 10 was only present in type II DolMADS, and the type I specific motifs were also identified. Motifs 6, 7, and 9 were specifically present in the Mα subfamily. Simultaneously, non-conserved motifs were present in the C-terminal of different MADS-box protein sequences with varied positions, lengths, and amino acid compositions (Figure S1). In addition, the K-box was found only in Type II DolMADSs (DolMADS24–DolMADS53) when assessing the conserved domain results (Figure S2).

The GFF genome data and TBtools v1.098661 [35] were used for gene structure analysis (Figure S1). The structures and number of exons and introns in the DolMADSs varied greatly among the different types. For example, the longest DolMADS49 was approximately 180,000 bp, while the shortest, DolMADS5, had no intron and was only 287 bp. Furthermore, the number of introns in Type I DolMADSs was considerably less than that in the Type II DolMADSs. Moreover, the number of exons in the DolMADSs varied from 1 to 10, of which DolMADS1, DolMADS2, DolMADS5-DolMADS11, and DolMADS13–DolMADS19 had only one exon, and DolMADS22 and DolMADS23 had 10 exons.

3.1.4. Analysis of Promoter cis-Acting Elements of the DolMADSs

The cis-acting regulatory elements of the promoter sequence among 53 DolMADSs genes were analysed. About 108 cis-acting elements were effectively expressed, and the top 20 cis-acting elements were shown in Figure S3. Among the 108 elements, there are some hormone response elements, such as the abscisic acid-responsive element (ABRE), gibberellin-responsiveness (TATC-box), salicylic acid responsiveness (TCA-element and SARE), MeJA-responsiveness (TGACG-motif and CGTCA-motif), and auxin-responsive element (TGA-element, AuxRE and TGA-box). These elements may be more important for sexual differentiation of flower buds.

3.1.5. Expression Analysis of the DolMADSs

To identify DolMADSs related to reproductive growth, the transcriptome data for female flowers of gynoecious, male flowers of androecious, and female and male flowers of monoecious were obtained from previously Illumina RNA-Seq data that were generated by our research group (unpublished), and the statistics of transcript sequencing are shown in Table S6. The FPKM values of all DolMADSs are shown in Table S7 and we only screened the differentially expressed genes in the groups for study. As shown in Figure 4a, five of 53 genes were differentially expressed in groups 1 and 2, while seven were specifically expressed in group 1, and two in group 2. In addition, in group 1, except for DolMADS31 and DolMADS45, the expression levels of the remaining aforementioned genes in the male flowers were higher than those in the female flower buds. Furthermore, in group 2, except for DolMADS42 and DolMADS52, the expression levels of the remaining aforementioned genes in the male flowers were higher than those in the female flower buds (Figure 4a). We also identified genes with exceptionally high levels of expression (FPKM values > 100), such as DolMADS24 and DolMADS43 in female flowers of gynoecious and male flowers of androecious, DolMADS33 and DolMADS43 in male flowers of monoecious, and DolMADS42 and DolMADS43 in the female flowers of monoecious. Simultaneously, for the ‘ABC/DE’ model, the B-class gene DolMADS43, the C-class gene DolMADS33, and the E-class gene DolMADS24 were all found to be more highly expressed in the male flowers than in the female flowers.

3.2. Identification and Analysis of WRKY Transcription Factor Family Members

3.2.1. Identification of WRKY Members in D. oleifera

The WRKY transcription family plays a vital role in regulating the development of flower buds. In this study, 60 WRKY proteins were identified and designated as DolWRKY1–DolWRKY66 (Table S8 and Text S2). The homologous genes in Arabidopsis, gene localisation, amino acid number, isoelectric point, and molecular weight for each DolWRKY are listed in Table S8. The lengths of the DolWRKY proteins varied from 122 aa (DolWRKY23, DolWRKY25, and DolWRKY26) to 600 aa (DolWRKY8), and their molecular weights were between 13.36 kDa (DolWRKY26) and 65.42 kDa (DolWRKY8). Predicted isoelectric point values ranged from 4.66 (DolWRKY56) to 10.46 (DolWRKY2) (Table S8).

The WRKY and zinc finger domains are vital structures in WRKY proteins [50]. This study identified 54 DolWRKY with the highly conserved sequence ‘WRKYGQK’ in D. oleifera (Figure S4 and Table S9). In addition, DolWRKY57 and DolWRKY58 both had an incomplete zinc finger domain. DolWRKY1, DolWRKY2, and DolWRKY23–DolWRKY26 were deficient in the WRKY domain but had a complete zinc finger domain, whereas DolWRKY32 and DolWRKY62 were deficient in the zinc finger domain but had a complete WRKY domain. DolWRKY46 and DolWRKY47 were deficient in the zinc finger domain and the complete WRKY domain. Furthermore, three DolWRKYs, DolWRKY42–DolWRKY44, had the most common sequence variant, which was ‘WRKYGKK’, and DolWRKY9 had the uncommon sequence variant of ‘WIKYRQK’. Moreover, group III proteins except for DolWRKY57, DolWRKY58, DolWRKY62, and DolWRKY65 contained a C2HC (Cx7Cx23HxC) zinc finger motif (Figure S4 and Table S9).

3.2.2. Phylogenetic and Gene Duplication Analysis of the WRKY Members

Based on a previous study of WRKYs in Arabidopsis, the DolWRKYs were divided into seven subgroups, named subgroups I, IIa–IIe, and III (Figure 5). Based on the conservation of the sequence, the phylogenetic results demonstrated that DolWRKYs could be categorised into three clades: group I + IIc, IIa + IIb, and IId + IIe + III. Subgroup I contained 13 members, subgroup III contained 13 members, and subgroups IIa, IIb, IIc, IId, and IIe included 2, 7, 22, 3, and 6 members, respectively (Figure 5). Moreover, the phylogenetic analysis indicated that the classification of the DolWRKY proteins was similar to that of Arabidopsis, confirming the classification accuracy.

In addition, it was found that these genes were distributed on 15 chromosomes, with Chr1, Chr2, Chr4, Chr7, and Chr13 all having six DolWRKYs, while Chr6 only had one DolWRKY (Figure 6). To analyse the DolWRKYs duplication events, 31 paralogous gene pairs located in the genome of D. oleifera were identified (Table S10), and all the DolWRKYs homologous gene pairs were located within the same group in the phylogenetic tree (Figure 6). Moreover, comparative syntenic maps of D. oleifera versus Arabidopsis were constructed, and 74 pairs of orthologous WRKY genes were identified (Figure 3b).

3.2.3. Conserved Motif, Gene Structure, and Conserved Domain Analyses for the DolWRKYs

We used TBtools to demonstrate the genetic structure of DolWRKYs (Chen et al., 2020). As represented in Figure S5, 66 DolWRKYs contained between one and six exons (four with one exon, eight with two exons, 31 with three exons, 11 with four exons, seven with five exons, and five with six exons), indicating the appearance of exon gain and loss incidents during the development of the WRKY transcription family, which may result in functional multiplicity among the DolWRKYs. Concurrently, it was also observed that DolWRKYs in the same subgroup showed analogous exon and intron structures, such as subgroup IIa, IId, and IIe (Figure S5). In addition, by observing the conserved domains of the DolWRKYs, almost all of those in subgroup I contained two WRKY domains, while all the others contained one WRKY domain (Figure S6).

Conserved motifs of DolWRKYs were forecasted using the MEME website to further illustrate the comparability and multiplicity of the motif composition. In total, 10 conserved motifs were identified in the 66 DolWRKYs, each containing between 15 and 50 amino acids (Figure S5, Table S11). Additionally, motifs 1 and 2 were shared by almost all DolWRKYs, while motif 3 was identified only in subgroup I. Motif 4 existed in subgroups I, IIa, IIb, and IIc. Motifs 5 and 10 were found mainly in IIc, whereas motifs 6, 7, and 8 were identified in subgroups I, IIa, and IIb. Motif 9 was predominantly found in subgroups IId, IIe, and III (Figure S5). In general, nearly all DolWRKYs belonging to the same subfamily had very similar motif constitutions, indicating that these DolWRKYs had similar functions.

3.2.4. Analysis of Promoter cis-Acting Elements of the DolWRKYs

The cis-acting regulatory elements of the promoter sequence among 66 DolWRKYs genes were analysed. About 112 cis-acting elements were effectively expressed, and the top 20 cis-acting elements were shown in Figure S7. Among the 112 elements, there are some hormone response elements, such as the abscisic acid-responsive element (ABRE), gibberellin-responsiveness (TATC-box), salicylic acid responsiveness (TCA-element and SARE), MeJA-responsiveness (TGACG-motif and CGTCA-motif), and auxin-responsive element (TGA-element and TGA-box). These elements may be more important for sexual differentiation of flower buds.

3.2.5. Expression Analysis of the DolWRKYs

Based on the transcriptome data surveyed in previous studies (unpublished), the expression models for the WRKY genes were analysed. The FPKM values of all DolWRKYs are shown in Table S12 and we only screened the differentially expressed genes in the groups for study. The results showed that three of the 66 DolWRKYs were differentially expressed in groups 1 and 2, while 19 were specifically expressed in group 1, and three were specifically expressed in group 2 (Figure 4b). Additionally, in group 1, except for DolWRKY5, DolWRKY38, DolWRKY60, and DolWRKY63, the expression levels of the DolWRKYs in the female flower buds were higher than in the male flower buds. Similarly, in group 2, except for DolWRKY5, the expression levels of the DolWRKYs in the female flower buds were higher than in the male flower buds (Figure 4b). Concurrently, we identified genes with extraordinarily high levels of expression (FPKM values > 100), such as DolWRKY14 in female flowers of monoecious, DolWRKY55, and DolWRKY63 in female flowers of gynoecious (Figure 4b).

3.3. Identification and Analysis of NAC Transcription Factor Family Members in D. oleifera

3.3.1. Identification of NAC Members in D. oleifera

A total of 83 NAC proteins in D. oleifera were identified and confirmed using the HMM profile of the Pfam NAC domain (PF02365). These proteins were named DolNAC1 to DolNAC83, respectively (Table S13 and Text S3). Detailed information for these DolNACs is shown in Table S13, including the homologous genes in Arabidopsis, gene localisation, molecular weight (Mw), amino acids sequence lengths (aa), and isoelectric point (pI). The protein sizes of the DolNACs varied from 97 aa (DolNAC71) to 759 aa (DolNAC82), with an average length of 326 aa. The relative molecular weight (Mw) varied from 11.05 kDa (DolNAC71) to 86.11 kDa (DolNAC82). The isoelectric point values ranged from 4.55 (DolNAC9) to 10.22 (DolNAC45) (Table S13).

3.3.2. Phylogenetic and Gene Duplication Analysis of the NAC Members

An unrooted tree was established to illustrate the phylogenetic relationships amongst the NAC family of proteins in D. oleifera and Arabidopsis. The DolNAC proteins were diversified as Arabidopsis NAC proteins, and 83 DolNACs were inconsistently distributed in 16 subgroups (Figure 7). Each subgroup contained members ranging from three to nine. The majority of members were OsNAC7, which contained nine family members, and the least members were NAC1, AtNAC3, TIP, OsNAC8, and ANAC063, and each contained one family member. The proportions of the DolNACs in each subgroup were similar to those of the Arabidopsis NAC genes (Figure 7).

Furthermore, according to their positions in the D. oleifera genome, all the DolNACs were mapped on the chromosomes. As illustrated in Figure 8, 83 DolNACs were inconsistently distributed on all 15 chromosomes. Chr4 contained the largest number of DolNACs at 10, followed by nine on Chr2 and Chr13. In contrast, only two genes were discovered on Chr10 (Figure 8). To analyse the DolNACs duplication events, 31 paralogous gene pairs located in the genome of D. oleifera were identified (Table S14), and all the DolNAC homologous gene pairs were located within the same group in the phylogenetic tree except for DolNAC30/DolNAC31, DolNAC28/DolNAC34, DolNAC44/DolNAC70, and DolNAC22/DolNAC73 (Figure 8). Moreover, comparative syntenic maps of D. oleifera versus Arabidopsis were constructed, and 95 pairs of orthologous genes were identified between the NAC genes in D. oleifera and Arabidopsis (Figure 3c).

3.3.3. Protein Structure, Conserved Domains, and Conserved Motifs Analysis for the DolNACs

To identify the structural diversity of the DolNACs, their exon and intron content was analysed. As shown in Figure S8, among the 83 DolNAC, one had no introns and 82 possessed 2–17 exons (15 with two exons, 46 with three exons, seven with four exons, five with five exons, seven with six exons, one with 11 exons, and one with 17 exons). Furthermore, DolNACs within the same subgroup usually had similar exon and intron numbers and structures. For instance, all the NAC1 and TERN subgroup members contained two introns and three exons. However, the gene structures in subgroups ANAC011, ANAC2, AtNAC3, and ANAC001 were variegated and distinct (Figure S8). The results were in accordance with the NAC genes reported in Musa acuminata and Populus trichocarpa, which indicated that exon shuffling occurred during evolution [6,51].

The MEME program was used to investigate the structures of the DolNACs, and 10 motifs (motif 1–10, respectively), were detected. As expected, similar motif constitutions were found among the closely related DolNAC members (Figure S8; Table S15). Most of the conserved motifs were located in the N-terminal region of the NAC domain, indicating their importance for DolNAC function. In addition, similar motif compositions were also found to cluster in the same subgroup. For instance, the subgroup of ANAC063 primarily included five motifs (motif 2, 3, 7, 8, and 9), while those in ANAC2 contained five motifs (motif 1, 2, 3, 4, and 5), indicating that DolNACs in the same subgroup may have similar functions (Figure S8). In addition, by observing the conserved domains of the DolNACs, it was found that they all contained the NAM domain, and DolNAC30 and DolNAC59 contained two NAM domains (Figure S9).

3.3.4. Analysis of Promoter cis-Acting Elements of the DolNACs

The cis-acting regulatory elements of the promoter sequence among 83 DolNACs genes were analysed. About 117 cis-acting elements were effectively expressed, and the top 20 cis-acting elements are shown in Figure S10. Among the 117 elements, there are some hormone response elements, such as the abscisic acid-responsive element (ABRE), gibberellin-responsiveness (TATC-box), salicylic acid responsiveness (TCA-element and SARE), MeJA-responsiveness (TGACG-motif and CGTCA-motif), and auxin-responsive element (TGA-element and AuxRE). These elements may be more important for the sexual differentiation of flower buds.

3.3.5. Expression Analysis of DolNACs

The previously generated Illumina RNA-Seq data (unpublished) for group 1 and group 2 (Figure 4c) were examined to investigate the expression patterns of the DolNACs in the flower buds of the different sex types. The FPKM values of all DolNACs were shown in Table S16 and we only screened the differentially expressed genes in the groups for study. Among the 83 DolNACs, 23 DolNACs were differentially expressed in at least one group, among which eight DolNACs were differentially expressed in both groups 1 and 2, 10 were specifically expressed in group 1, and five were specifically expressed in group 2. In addition, in group 1, except for DolNAC12, DolNAC20, DolNAC30–DolNAC32, DolNAC56, DolNAC73, and DolNAC82, the expression levels of DolNACs in the female flower buds were higher than those in the male flower buds. In contrast, in group 2, except for DolNAC17, DolNAC29, DolNAC40, and DolNAC78, the expression levels of the DolNACs in the male flower buds were higher than those in the female flower buds. We also identified DolNACs with exceptionally high levels of expression (FPKM values > 100), such as DolNAC31 in the male flowers of androecious and the male flowers of monoecious and DolNAC57 in the female flowers of gynoecious.

3.4. Identification and Analysis of SBP-box Transcription Factor Family Members in D. oleifera

3.4.1. Identification of the SBP-box Members in D. oleifera

To identify SBP-box family members, the entire D. oleifera genome sequence and 26,164 protein sequences were used. After an HMM search, 17 SBP-box proteins were isolated (Text S4). These proteins were named DolSBP1–DolSBP17, respectively (Table S17). The deduced length of the DolSBPs ranged from 112 (DolSBP15) to 1220 (DolSBP16) aa, and the relative molecular weight ranged from 11.78 kDa (DolSBP12) to 88.37 kDa (DolSBP17), while the isoelectric point values ranged from 6.31 (DolSBP3) to 9.80 (DolSBP15) (Table S17).

DNAMAN software was used for multiple sequence alignments for the full-length protein sequences and confirmed the structures of the DolSBPs (Figure S11). There was a conserved SBP domain in all members. These SBP domains were highly conserved in certain positions, such as the CQQC sequences. Interestingly, nearly all members had two zinc finger-like structures (Zn-1 and Zn-2) and a highly conserved bipartite nuclear localisation signal (NLS). The NLS is partly overlapped with the Zn-2 [30]. Nearly all DolSBPs had the same zinc finger-like structure CysCysCysHis, while another zinc finger-like structure, CysCysHisCys, was present in all members except for DolSBP5 and DolSBP15–DolSBP17 (Figure S11). Furthermore, the miR156 and miR157 target sites in DolSBPs were predicted, and the results showed that DolSBP1–DolSBP5, DolSBP9, and DolSBP10 were all miR156 and miR157 target genes (Figure S12 and Text S5).

3.4.2. Phylogenetic and Gene Duplication Analysis of the SBP-box Members

To further comprehend the evolutionary relationship of the DolSBPs and uncover the functions of the DolSBPs in D. oleifera development, we used 33 SBP sequences from two species, D. oleifera and Arabidopsis, to construct a phylogenetic tree using MEGA 7.0 (Figure 9). These 33 sequences were classified into six groups (group 1–6, with members 1–5 in each group). The largest number of members was five and this was identified in group 1, and the smallest was one and this was identified in groups 2 and 3 (Figure 9).

In addition, the DolSBPs were mapped to the chromosomes, and this provided visual insights into their distribution (Figure 10). Based on the genomic data of D. oleifera, 17 DolSBPs were found to be unevenly distributed in more than nine chromosomes (excluding chromosomes 1, 3, 5, 10, 11, and 15) with one on Chr2, Chr9, and Chr13, two on Chr6, Chr7, Chr12, and Chr14, and three on Chr4 and Chr8 (Figure 10). To analyse the DolSBPs duplication events, five paralogous gene pairs in the D. oleifera genome were identified (Table S18), and all the DolSBPs homologous gene pairs were within the same group in the phylogenetic tree, except for DolSBP1/DolSBP11 (Figure 10). Moreover, comparative syntenic maps of D. oleifera versus Arabidopsis were constructed, and 12 pairs of orthologous genes were identified for the SBP-box genes in D. oleifera and Arabidopsis (Figure 3d).

3.4.3. Conserved Motifs, Gene Structure, and Conserved Domains Analysis for DolSBPs

To improve our understanding of the evolutionary relationships within the D. oleifera SBP transcription family, the exon–intron organisations was inspected for all of the DolSBPs. As previously reported, all DolSBPs were found to contain intron(s) at highly conserved positions [52]. Furthermore, with few exceptions, DolSBPs had a similar exon–intron structure within the same group, whereas the number of exons changed among groups. For instance, DolSBP6 in group 2 had two exons, while DolSBP7 in group 3 had three exons (Figure S13).

Ten conserved motifs were detected from the DolSBPs using the MEME website (Figure S13, Table S19). Based on the frequency of occurrence, motifs 1, 2, and 3 were identified as the most common, occupying nearly all subgroups from group 1 to group 6. In contrast, motifs 5 and 9 were specific to group 1 only. Furthermore, motifs 6, 7, 8, and 10 were only found in group 6 but were shared by all the members within the subgroup (Figure S13). The result, which predicted conserved protein domains, indicated that all the DolSBPs contained the SBP domain (Figure S14).

3.4.4. Analysis of Promoter cis-Acting Elements of the DolSBPs

The cis-acting regulatory elements of the promoter sequence among 17 DolSBPs genes were analysed. About 86 cis-acting elements were effectively expressed, and the top 20 cis-acting elements were shown in Figure S15. Among the 86 elements, there are some hormone response elements, such as the abscisic acid-responsive element (ABRE), gibberellin-responsiveness (TATC-box), salicylic acid responsiveness (TCA-element), MeJA-responsiveness (TGACG-motif and CGTCA-motif), auxin-responsive element (TGA-element). These elements may be more important for the sexual differentiation of flower buds.

3.4.5. Expression Analysis of DolSBPs

Transcriptomic data of the DolSBPs were analysed in the different flower buds sex types to determine correlations with flower organ development (Figure 4d). The FPKM values of all DolSBPs are shown in Table S20 and we only screened the differentially expressed genes in the groups for study. The results showed that among the 17 DolSBPs, five DolSBPs were differentially expressed in at least one group, four were differentially expressed in group 1 and group 2, and one was specifically expressed in group 1. Furthermore, in group 1, the expression levels of all the DolSBPs in the female flower buds were higher than those in the male flowers buds, and group 2 had similar results (Figure 4d).

3.5. RT-qPCR Analysis of the DolMADSs, DolWRKYs, DolNACs, and DolSBPs

Three DolMADSs, 1 DolWRKYs, 2 DolNACs, and 3 DolSBPs were selected to validate the transcriptome data accuracy using RT-qPCR (Figure 11). The expression patterns for the genes in groups 1 and 2 were in accordance with the RNA-seq results. Thus, our sequencing data were determined to be reliable.

3.6. Go Enrichment Analysis and Protein Interaction Networks

A total of 219 proteins in the DolMADSs, DolWRKYs, DolNACs, and DolSBPs were annotated using the GO terms for molecular functions and were categorised as DNA-binding transcription factor activity, transcription regulator activity, DNA binding, nucleic acid binding, heterocyclic compound binding, organic cyclic compound binding, and binding (Figure S16), which was consistent with the functions of the transcription factors.

The homologous MADS-box, WRKY, NAC, and SBP-box proteins in Camellia sinensis, which was closely related to D. oleifera [5], were used to predict the protein–protein interaction networks of DolMADSs, DolWRKYs, DolNACs, and DolSBPs. The results showed that, 18 DolMADSs, eight DolWRKYs, 14 DolNACs, and one DolSBPs exhibited co-expression correlations with 41 nodes and 70 edges (Figure 12). Co-expressed genes and their correlation coefficients were provided in Table S21. Based on the connections between the nodes, 41 nodes were distinctly classified into eight groups. In the first group, 17 of the 41 nodes were connected, suggesting that these genes might be involved in the same regulatory pathway. In the other groups, two, three, five, or six nodes were jointed, implying that these genes might be involved in a relatively simple signalling network. In addition, proteins within the same family or those between different families interacted with each other. For example, DolMADS52 could interact with DolMADS33 and DolSBP7 (Table S21), which represent the possibility of the interplay between different families.

4. Discussion

The floral organs found in plants are diverse and crucial for reproduction in angiosperms. Plants mature from leaf bud to flower bud and subsequently develop into flower organ primordia via a process called flower bud differentiation [53]. The development of flower organs begins when plants receive the flowering signal, and the stem apical meristem transforms into an inflorescence meristem [13]. Due to their different growing environments and reproductive strategies, the floral organs of various plants have evolved to be diverse to improve their reproductive success. Most flower organs have a typical four-wheeled floral structure, which is organized from the outside to the inside as the sepal, petal, stamen, and pistil (carpel), respectively. In most unisexual plants, the stamen and pistil primordia are developed into a floral bud at an early developmental stage. Subsequently, due to complex genetic, epigenetic, physiological, and environmental factors, either the stamen or pistil primordium can be arrested at a certain stage, leading to a unisexual flower [54].

MADS-box transcription factors can reportedly arrest the development of stamens and promote the formation of unisexual female flowers. For example, the overexpression of BcAP3 in Arabidopsis was reported to result in abnormal anther wall development and low pollen viability [55]. The ectopic expression of WAG1 and WAG2 can lead to pistil-like stamens of alloplasmic wheat [56]. HAM59, a C-class gene of the sunflower, was expressed ectopically in chrysanthemum, and transgenic lines showed male sterility and flower structure transformation. Moreover, floret stamens in the disk can transform into petal-like structures, forming a double flower phenotype [57]. In rice, the C-class protein, OsMADS58, can combine with numerous photosynthetic genes, to inhibit their expression, which in turn affects chloroplast differentiation in the stamens [58]. In this study, the expression level of B-class gene DolMADS42 was found to be higher in female floral buds than in male buds, indicating its contribution to the formation of unisexual female flowers.

In contrast, some MADS-box genes facilitate the formation of unisexual male flowers. For example, OsTGA10, which encodes a bZIP transcription factor, was preferentially expressed during stamen development as a target protein of the E-class protein OsMADS8, and its mutation led to male sterility in rice [59]. The homeotic transformation of stamens into pistil-like structures (called pistillody) was observed in alloplasmic bread wheat lines with missing WPI1 [60]. Furthermore, some studies have confirmed that the loss of C-class gene function may cause stamen petaloidy. For example, the loss of CpAG1 expression in Cyclamen persicum led to stamen petaloidy with an increase of five petals, while the simultaneous loss of CpAG1 and CpAG2 expression led to the formation of multiple petals, resulting in rose-like, multi-petal flowers [61]. When the two C-class genes of Petunia hybrida, MADS3 and FBP6, were silenced, the stamens transformed into petals [62]. In this study, the expression of the B-class gene DolMADS43, the C-class gene DolMADS33, and the E-class gene DolMADS24 were all higher in male floral buds when compared with female buds. Moreover, the protein interactions between DolMADS33 and DolMADS43 were predicted. These results suggest that DolMADS24, DolMADS33, and DolMADS43 might have facilitated the formation of unisexual male flowers.

WRKY transcription factors are reported to be important for the regulation of floral development [63]. In Arabidopsis, several WRKY members were found to influence pollen development. For instance, AtWRKY34 was specifically expressed in the male gametophyte and participated in the regulation of early pollen development. The WRKY34 mutant exhibited higher levels of pollen vigour, pollen germination, and pollen tube growth [64], while the overexpression of AtWRKY27 led to abnormal plant growth and male sterility defects [65]. Transgenic Arabidopsis plants with GhWRKY22 from a cotton gene showed male sterility, and the number of pollen grains decreased when compared with the wild type [63]. After overexpressing OsWRKY51, rice plant height and pollen fertility decreased significantly [66]. In this study, the phylogenetic tree results demonstrated that DolWRKY48, DolWRKY49, and AtWRKY27 (AT5G52830) were closely related. DolWRKY55, DolWRKY54, and DolWRKY64 possessed protein interactions, were all differentially expressed only in group 1, and their expression levels were higher in female flower buds than in male flower buds. It was, thus, speculated that DolWRKY48, DolWRKY49, DolWRKY54, DolWRKY55, and DolWRKY64 may play significant roles in the formation of unisexual female flowers in D. oleifera.

WRKY genes were also found to have a large role in the formation of unisexual male flowers [67]. In Arabidopsis, WRKY2, WRKY34, and VQ20 were related to the regulation of pollen development and tube elongation, and the simultaneous mutation of WRKY2, WRKY34, and VQ20 could lead to pollen abortion [68]. Loss of WRKY2/WRKY34 function led to a decrease in GPT1 expression, and a decrease in liposome accumulation in mature pollen, which led to impaired pollen vigour, germination, pollen tube growth, and male passage in Arabidopsis [67]. Furthermore, the loss of AtWRKY18 resulted in an abnormal pollen shape and reduced the pollen germination rate [69]. In this study, the phylogenetic tree results demonstrated that DolWRKY5, DolWRKY6, and AtWRKY2 (AT5G56270) were closely related, and the expression level of DolWRKY5 in male flower buds was higher than in female flower buds, in groups 1 and 2. Moreover, there was protein interaction between DolWRKY5, DolWRKY4, and DolWRKY1. It was, thus, indicated that DolWRKY1, DolWRKY4, DolWRKY5, and DolWRKY6 may impact the formation of unisexual male flowers.

NAC gene family members have primarily regulated flower bud development [70]. For example, when the VvDRL1 gene of grape was overexpressed in tobacco, pollen viability and germination rates were significantly reduced when compared with the wild type, and the expression level of the VvDRL1 increased as the flower development time was prolonged [71]. Compared with the wild type, ZmNAC84 overexpression in maize resulted in abnormal pollen phenotype, which included an abnormal pollen shape and shrunken cell nucleus [70]. When the GhFSN gene of grape was overexpressed in Arabidopsis, the pollen was found to be shrunken, collapsed, and the anthers did not disperse properly [72]. In this study, the expression level of DolNAC57 in the female flower buds was notably higher than in the male flower buds in group 1, indicating that DolNAC57 may accelerate the formation of unisexual female flowers.

In addition, some NAC genes also played an enormous role in the formation of unisexual male flowers [73]. In Arabidopsis, nst1 and nst2 mutants showed a lack of secondary walls in the anther endodermis and abnormal anther dehiscence [74]. The FveCUC2 mutant in strawberries showed leaves with smooth leaf margins and fewer stamens [73]. MtNST1 was a homolog of NST1/2/3 in Arabidopsis, and the Tnt1 retrotransposon insertion mutant population of MtNST1 found that the phloem fibre lignin was lost, the vascular element lignin was reduced, and the anther dehiscence failed [75]. In addition, studies have also shown that jasmonic acid has a notable role in the regulation of anther dehiscence [76]. Further to this, ectopic expression of the Arabidopsis NAC-like gene AIF-C was found to regulate genes in the jasmonic acid biosynthesis pathway, leading to an anther male sterility phenotype with anthers present throughout flower development [77]. In this study, the expression level of DolNAC31 in male flower buds was significantly higher than in female flower buds, in groups 1 and 2; it was, thus, speculated that DolNAC31 may have considerable effects in promoting the formation of unisexual male flowers in D. oleifera.

In plants, SBP-box transcription factors also play an enormous role in sex differentiation [78]. SPL8 was the first functional SPL gene reported in Arabidopsis and was not regulated by miR156/157 [79]. In this study, the phylogenetic tree results showed that DolSBP7 and AtSPL8 were closely related, and the expression level of DolSBP7 in female flower buds was significantly higher than that of male flower buds in group 1, indicating that DolSBP7 may be a key gene in the formation of unisexual female flowers. However, an opposite function was also reported in Arabidopsis; the AtSPL8 mutation, under normal growth conditions, caused a few male flowers to form in the early stages of development, which were sterile. This phenotype was exacerbated under stress conditions such as intense light. Through dissection and section observations, it was found that the sterility caused by the AtSPL8 mutation was mainly due to the reduction of anther size, anther sac, and pollen number. This was due to the failure of sporogenesis cells and anther parietal cells in some parts of the anther in AtSPL8 mutants, which was consistent with AtSPL8 expression in these cell parts [66,78,80].

In addition, other SPL genes may also be involved in the formation of the unisexual male flowers of Arabidopsis. For example, in spl8 spl2 spl9, spl8 spl9 spl15, spl8 spl2 spl9 spl15, and other multi-mutants, the fertility of plants was further reduced, especially in the tetrad spl8 spl2 spl9 spl15, as most flowers produced no pollen or only a small amount [78]. In this study, the phylogenetic tree results showed that DolSBP1, AtSPL2, DolSBP3, DolSBP4, DolSBP5, AtSPL9, and AtSPL15 were closely related, but the expression levels of DolSBP3, DolSBP4, and DolSBP5 in the female flower buds were higher than in the male flower buds for groups 1 and 2, which suggests that DolSBP3, DolSBP4, and DolSBP5 may play vital roles in the development of female flowers in D. oleifera.

Moreover, many studies have shown that members of the SBP-box gene family in Arabidopsis are involved in the promotion of the floral transition, and this is dependent on the miR156 [81]. A study reported that miR156/miR157 regulated flower development by negatively regulating SPL genes [32]. For example, the high levels of miR156b expression in spl8 mutants resulted in the complete sterility of plants, with no anthers and no pollen production [78,80]. The overexpression of GmmiR156 in soybean significantly reduced the transcriptional expression level of GmSPL3/9, resulting in a delayed flowering time and longer growth period [82]. MiR156 regulated the expression of AtSPL3 at the vegetative development stage; simultaneously, when the miR156 content was reduced, AtSPL3 expression was be enhanced, and the plants blossomed earlier [83]. In this study, DolSBP1-DolSBP5, DolSBP9, and DolSBP10 had miR156 and miR157 recognition sites, suggesting that they may also have an important role in the development of floral organs.

5. Conclusions

In this study, we identified 53 MADS-box, 66 WRKY, 83 NAC, and 17 SBP-box transcription factors in D. oleifera, and further classified them based on their phylogenetic relationships. Their gene structures and conserved domains and motifs were also analysed. Their expression patterns in the different flower bud sex types were assessed and used to predict their interaction networks. The final results showed that DolMADS42, DolWRKY48, DolWRKY49, DolWRKY54, DolWRKY55, DolWRKY64, DolNAC57, DolSBP3, DolSBP4, DolSBP5, and DolSBP7 may promote the formation of unisexual female flowers, while DolMADS24, DolMADS33, DolMADS43, DolWRKY1, DolWRKY4, DolWRKY5, DolWRKY6, and DolNAC31 may have a significant role in the formation of unisexual male flowers. This study represents the first whole genome analysis of the MADS-box, WRKY, NAC, and SBP-box gene families in D. oleifera, and it provides a firm foundation from which to further explore their roles in flower organ development and sex differentiation, as well as uncovering their evolutionary history in Diospyros spp.