B-box Proteins in Arachis duranensis: Genome-Wide Characterization and Expression Profiles Analysis

B-box (BBX) proteins are important factors involved in plant growth and developmental regulation, and they have been identified in many species. However, information on the characteristics and transcription patterns of BBX genes in wild peanut are limited. In this study, we identified and characterized 24 BBX genes from a wild peanut, Arachis duranensis. Many characteristics were analyzed, including chromosomal locations, phylogenetic relationships, and gene structures. Arachis duranensis B-box (AdBBX) proteins were grouped into five classes based on the diversity of their conserved domains: I (3 genes), II (4 genes), III (4 genes), IV (9 genes), and V (4 genes). Fifteen distinct motifs were found in the 24 AdBBX proteins. Duplication analysis revealed the presence of two interchromosomal duplicated gene pairs, from group II and IV. In addition, 95 kinds of cis-acting elements were found in the genes’ promoter regions, 53 of which received putative functional predictions. The numbers and types of cis-acting elements varied among different AdBBX promoters, and, as a result, AdBBX genes exhibited distinct expression patterns in different tissues. Transcriptional profiling combined with synteny analysis suggests that AdBBX8 may be a key factor involved in flowering time regulation. Our study will provide essential information for further functional investigation of AdBBX genes.


Introduction
Transcription factors are essential components of signal transduction pathways in plants. They often work as activators or suppressors, binding cis-acting elements in target promoter regions to regulate downstream gene expression [1,2]. Various transcription factor families are found in plants, which are involved in many different response pathways. Among these gene families, zinc-finger transcription factors, possess a conserved domain that can bind metal ions like zinc and interact with DNA, RNA or proteins, and members of that large transcription factor family play critical roles in plant growth and development [3][4][5][6]. Based on the diversification of their protein sequences and structures, zinc-finger genes are further classified into several distinct subfamilies [3]. A subgroup of zinc-finger proteins containing B-box (BBX) conserved domains, which are thought to be involved in protein-protein interactions, is designated as the BBX family and is highly conserved across all multicellular species [4,[7][8][9].
Two types of BBX domains, B-box1 and B-box2, have been identified based on their consensus sequences and the spacing of their zinc-binding residues [4,[10][11][12]. In Arabidopsis, 21 and 11 of the 32 BBX proteins contain two and one BBX domains, respectively [3,4]. In addition to the conserved BBX domain, some BBX family members contain additional specific domains, such as the CCT (for

Identification of BBX Members in A. duranensis
The amino acid sequences of the BBX conserved domain (PF00643) and Arabidopsis BBX proteins, downloaded from Arabidopsis genome database TAIR, were used as blast queries against the peanut genome database to search for A. duranensis, Arachis ipaensis, and Arachis hypogaea BBX genes (Peanut genome database version: Arachis hypogaea Tifrunner 1.0, Arachis duranensis 1.0 and Arachis ipaensis 1.0) [33]. All output candidate genes were assessed using the Pfam database (Pfam 32.0, September 2018, 17,929 entries) [38] and the InterPro program [39] to confirm the presence of BBX domains and remove genes without conserved BBX domains. The B-box1 and B-box2 domains were determined as described by Crocco et al. [12]. The first B-box that appeared in the N terminal position was called B-box1 and the second was termed B-box2. The positions of the BBX and CCT domains in each Arachis duranensis B-box (AdBBX) protein were analyzed with Pro Scan program. The genomic lengths, coding sequence (CDS) lengths, and amino acid numbers of the AdBBX genes were obtained from the peanut genome database. The gene GC contents were determined using MegAlign software

Chromosomal Distribution of AdBBXs
To investigate the chromosomal locations of AdBBXs, we mapped the genes to the peanut genome to determine their physical positions. The AdBBX genes were named from AdBBX1 to AdBBX24 based on their chromosomal distribution ( Figure 1, Table 1). Among the 24 AdBBX genes, 13 were located on the plus strand and 11 were located on the minus strand ( Table 1). Nine of the 10 peanut chromosomes contained AdBBX genes, and only chromosome 2 contained none ( Figure 1 and Table 1). Chromosome 3 contained the largest number of AdBBX genes, with six AdBBX members, followed by chromosomes 6, 7, 9, and 10, with three AdBBXs on each. AdBBX2, AdBBX3, and AdBBX4 were located close together on chromosome 3 (the distance between AdBBX2 and AdBBX3 was 1,443,512 bp and the distance between AdBBX3 and AdBBX4 was 165,285 bp), as were AdBBX5 and AdBBX6 (the distance between AdBBX5 and AdBBX6 was 1,050,315 bp). AdBBX20 and AdBBX21 (the distance between them was 608,968 bp) were also located close together on chromosome 9. Most AdBBX genes were located on the chromosome arms, but three (AdBBX14, AdBBX17, and AdBBX18) were found on the middle portions of the chromosomes. members, followed by chromosomes 6, 7, 9, and 10, with three AdBBXs on each. AdBBX2, AdBBX3, and AdBBX4 were located close together on chromosome 3 (the distance between AdBBX2 and AdBBX3 was 1,443,512 bp and the distance between AdBBX3 and AdBBX4 was 165,285 bp), as were AdBBX5 and AdBBX6 (the distance between AdBBX5 and AdBBX6 was 1,050,315 bp). AdBBX20 and AdBBX21 (the distance between them was 608,968 bp) were also located close together on chromosome 9. Most AdBBX genes were located on the chromosome arms, but three (AdBBX14, AdBBX17, and AdBBX18) were found on the middle portions of the chromosomes.

Protein Sequence and Classification Analysis of AdBBX Genes
BBX proteins are classified into five subgroups based their conserved domains, including the types and numbers of BBX and CCT domains [3,4]. We analyzed conserved domains in the AdBBX

Protein Sequence and Classification Analysis of AdBBX Genes
BBX proteins are classified into five subgroups based their conserved domains, including the types and numbers of BBX and CCT domains [3,4]. We analyzed conserved domains in the AdBBX proteins and found two distinct BBX domains (B-box1 and B-box2) and one CCT domain. To further investigate the conserved amino acid sequences in these domains, logos of A. duranensis B-box1 (CX 2 CX 8 CX 2 DXAXLCX 2 CDX 2 VHX 2 NXLX 3 H, where X represents any amino acid), B-box2 (CX 2 CX 4 AX 3 CX 7 CX 2 CDX 3 HX 9 H), and CCT (RX 5 RX 3 KX 7 KX 2 RYX 2 RKX 2 AX 2 RXRXKGRFXK) were produced using Weblogo [45] (Figure 2). The amino acid sequences of the B-box1, B-box2, and CCT domains were also aligned to analyze corresponding positions of the conserved amino acid sequences ( Figure 3).     To further investigate the evolutionary relationships among the AdBBXs, we created a phylogenetic tree based on their amino acid sequences ( Figure 4a). Conserved domain analysis revealed that 16 of the 24 AdBBX proteins had two BBX domains (i.e., B-box1 and B-box2), 11 members contained a CCT domain, and seven had both BBX domains and a CCT domain (Figure 4b). The AdBBX proteins were also grouped into five subfamilies based on the diversity of their conserved domains ( Figure 4c). Group I and II, which differed in the details of their B-box2 domain, had a B-box1, B-box2, and CCT domain. There were three and four members of classes I and II, respectively, in A. duranensis. Class III had a B-box1 and a CCT domain and contained four members. Group IV had both a B-box1 and B-box2 domain and was the largest group in A. duranensis with nine members. Most AdBBX genes in the same class were clustered together, except for AdBBX5 (AraduJ5IAH), which had a distinct relationship with other class III genes and a closer relationship with class V subfamily members ( Figure 4a). However, the number of BBX genes from A. ipaensis and A. hypogaea in each group showed differences from A. duranensis. Group I, II, III, IV, and V contained 2, 4, 5, 9, and 5 members in A. ipaensis, and 1, 2, 10, Agronomy 2020, 10, 23 7 of 17 8, and 19 in A. hypogaea, respectively (Figures S1 and S2). To analyze the evolutional relationship of peanut BBX genes, a phylogenetic tree was constructed using BBX genes from A. duranensis, A. ipaensis, and A. hypogaea ( Figure S3). Most of the genes from A. duranensis and A. ipaensis in each group were clustered into the same clades, while many genes from A. hypogaea in the same group were not clustered together, indicating BBX genes in cultivated peanut might have changed a lot during evolution.
Group IV had both a B-box1 and B-box2 domain and was the largest group in A. duranensis with nine members. Most AdBBX genes in the same class were clustered together, except for AdBBX5 (AraduJ5IAH), which had a distinct relationship with other class III genes and a closer relationship with class V subfamily members ( Figure 4a). However, the number of BBX genes from A. ipaensis and A. hypogaea in each group showed differences from A. duranensis. Group I, II, III, IV, and V contained 2, 4, 5, 9, and 5 members in A. ipaensis, and 1, 2, 10, 8, and 19 in A. hypogaea, respectively ( Figure S1,2). To analyze the evolutional relationship of peanut BBX genes, a phylogenetic tree was constructed using BBX genes from A. duranensis, A. ipaensis, and A. hypogaea ( Figure S3). Most of the genes from A. duranensis and A. ipaensis in each group were clustered into the same clades, while many genes from A. hypogaea in the same group were not clustered together, indicating BBX genes in cultivated peanut might have changed a lot during evolution.  To obtain information from well-studied BBX genes in other species, we also analyzed the evolutionary relationships among BBX genes from Arabidopsis, rice, and A. duranensis ( Figure S4). Phylogenetic analysis revealed that AdBBX8 and AdBBX24 grouped with the well-studied flowering time genes At5g15840 (CO) and Os06g16370 (Hd1) from Arabidopsis and rice, respectively [4], indicating that one or both of these genes may play an important role in flowering time regulation in A. duranensis.

Gene Structures and Conserved Motifs of AdBBX Genes
To investigate exon-intron organization, the genomic and CDS sequences of AdBBXs obtained from the peanut genome database were analyzed using the Gene Structure Display Server [43]. The exon numbers of AdBBX genes ranged from one (AdBBX19 and AdBBX22) to five (AdBBX4), and the intron numbers ranged from zero (AdBBX19 and AdBBX22) to six (AdBBX4 and AdBBX5). Nine AdBBX genes had both 5 and 3 untranslated regions (UTRs), four genes had only 3 UTRs, three genes had only 5 UTRs, and eight genes had no predicted UTRs (Figure 5a). To further investigate the conservation and diversity of AdBBX protein structures, the putative motifs of these genes were predicted using MEME tools. Fifteen distinct motifs were found across the AdBBX proteins (Figure 5b, Figure S5). Among these motifs, motifs 1 and 5 were found in all the AdBBX proteins. Conservation of AdBBX Agronomy 2020, 10, 23 8 of 17 protein structures was observed among genes that clustered into the same classes. For example, all class I members shared five motifs, including motifs 1, 2, 3, 4, and 5, and all class II members shared six motifs, including motifs 1, 2, 4, 5, 14, and 15. In addition, structural diversity was also found among the AdBBX proteins. The motif numbers in AdBBX proteins varied from two (AdBBX19) to seven (AdBBX7, AdBBX10, and AdBBX21), and some motifs were only found in specific AdBBX proteins. For example, motif 2 was specific to class I, II, and III proteins and was considered to be the CCT domain, and motif 15 was only found in class II proteins. To obtain information from well-studied BBX genes in other species, we also analyzed the evolutionary relationships among BBX genes from Arabidopsis, rice, and A. duranensis ( Figure S4). Phylogenetic analysis revealed that AdBBX8 and AdBBX24 grouped with the well-studied flowering time genes At5g15840 (CO) and Os06g16370 (Hd1) from Arabidopsis and rice, respectively [4], indicating that one or both of these genes may play an important role in flowering time regulation in A. duranensis.

Gene Structures and Conserved Motifs of AdBBX Genes
To investigate exon-intron organization, the genomic and CDS sequences of AdBBXs obtained from the peanut genome database were analyzed using the Gene Structure Display Server [43]. The exon numbers of AdBBX genes ranged from one (AdBBX19 and AdBBX22) to five (AdBBX4), and the intron numbers ranged from zero (AdBBX19 and AdBBX22) to six (AdBBX4 and AdBBX5). Nine AdBBX genes had both 5′ and 3′ untranslated regions (UTRs), four genes had only 3′ UTRs, three genes had only 5′ UTRs, and eight genes had no predicted UTRs (Figure 5a). To further investigate the conservation and diversity of AdBBX protein structures, the putative motifs of these genes were predicted using MEME tools. Fifteen distinct motifs were found across the AdBBX proteins ( Figure  5b, Figure S5). Among these motifs, motifs 1 and 5 were found in all the AdBBX proteins.

Duplication Analysis of BBX Genes in A. duranensis
Polyploidy is a common feature of flowering plant evolution and produces many duplicated gene pairs. The wild peanut A. duranensis is thought to have experienced one round of whole genome duplication [33][34][35][36][37]. We investigated the duplication of AdBBX genes and found two interchromosomal duplicated gene pairs (AdBBX2/AdBBX17 and AdBBX8/AdBBX24), but no tandem duplicated gene pairs ( Figure 6). These duplicated genes were located on chromosomes 3, 4, 8, and 10, respectively. Moreover, the duplicated genes AdBBX2/AdBBX17 were found to belong to group IV and AdBBX8/AdBBX24 belonged to group II, and no duplicated gene pairs were found among the other groups. We also investigated duplication gene pairs in A. ipaensis and found two duplication events (AraipAS7FB/AraipP77MW, AraipWH6UQ/AraipS98FB), and all these genes were found to belong to group IV ( Figure S6). Moreover, the allotetraploid A. hypogaea contained 16 duplication events, and these genes, group II, III, IV, and V contained 1, 10, 4, and 17 members, respectively ( Figure S7).
IV and AdBBX8/AdBBX24 belonged to group II, and no duplicated gene pairs were found among the other groups. We also investigated duplication gene pairs in A. ipaensis and found two duplication events (AraipAS7FB/AraipP77MW, AraipWH6UQ/AraipS98FB), and all these genes were found to belong to group IV ( Figure S6). Moreover, the allotetraploid A. hypogaea contained 16 duplication events, and these genes, group II, III, IV, and V contained 1, 10, 4, and 17 members, respectively ( Figure S7).

Analysis of Cis-Acting Elements in AdBBX Promoter Regions
Cis-acting elements in promoter regions have critical roles in regulating plant gene expression. To further understand the expression responses of AdBBX genes, we identified cis-acting elements in AdBBX promoter regions 2 kb upstream of the initiation codon using PlantCARE [51]. Ninety-five kinds of cis-acting elements were identified, and 53 were predicted to have putative functions. These included seven development-related elements, five environmental stress-related elements, four site-binding-related elements, nine hormone-responsive elements, four promoter-related elements, and 24 light-responsive elements (Table S1). Binding sites in all 24 AdBBX genes related to development, including circadian control, metabolism regulation, stem expression, seed-specific regulation, cell differentiation, and cell cycle regulation (Figure 7a), environmental stress, including anaerobic conditions, drought, low temperature, and defense (Figure 7b), and hormones, including abscisic acid (ABA), gibberellic acid (GA), auxin, and jasmonic acid (MeJA) (Figure 7c), were identified in these promoters. The numbers and types of cis-acting elements varied among the AdBBX promoters, suggesting that the AdBBX genes have diverse roles in plant developmental regulation ( Table 2). All the AdBBX promoters contained light-responsive elements, which were represented to be the most abundant element type in each of the AdBBX promoters,

Analysis of Cis-Acting Elements in AdBBX Promoter Regions
Cis-acting elements in promoter regions have critical roles in regulating plant gene expression. To further understand the expression responses of AdBBX genes, we identified cis-acting elements in AdBBX promoter regions 2 kb upstream of the initiation codon using PlantCARE [51]. Ninety-five kinds of cis-acting elements were identified, and 53 were predicted to have putative functions. These included seven development-related elements, five environmental stress-related elements, four site-binding-related elements, nine hormone-responsive elements, four promoter-related elements, and 24 light-responsive elements (Table S1). Binding sites in all 24 AdBBX genes related to development, including circadian control, metabolism regulation, stem expression, seed-specific regulation, cell differentiation, and cell cycle regulation (Figure 7a), environmental stress, including anaerobic conditions, drought, low temperature, and defense (Figure 7b), and hormones, including abscisic acid (ABA), gibberellic acid (GA), auxin, and jasmonic acid (MeJA) (Figure 7c), were identified in these promoters. The numbers and types of cis-acting elements varied among the AdBBX promoters, suggesting that the AdBBX genes have diverse roles in plant developmental regulation ( Table 2). All the AdBBX promoters contained light-responsive elements, which were represented to be the most abundant element type in each of the AdBBX promoters, hormone-responsive elements, and promoter-related elements ( Table 2, Supplementary Table S1), suggesting that the AdBBX genes share some common pathways in plant developmental regulation. The promoter-related elements CCAAT-box and TATA-box were found in all AdBBX promoter regions and likely constitute the basic components of the promoters. The light-responsive element, Box4, was identified in all AdBBX promoters except that of AdBBX13 (Table S1), indicating that AdBBX genes play important roles in Box4-mediated light response pathways.

Expression Patterns of AdBBX Genes in Different Tissues
To shed light on the potential functions of AdBBX genes during plant development, we investigated the expression levels of the 24 AdBBX genes in 22 different tissues (Figure 8). AdBBX genes showed distinct expression patterns among tissues, highlighting their functional diversity. For example, AdBBX1 and AdBBX23 were expressed at high levels in most tissues. In contrast, AdBBX3, AdBBX5, AdBBX15, AdBBX18, and AdBBX24 showed low expression in all tissues, suggesting that they may function only during specific stages of development in these tissues. AdBBX21 was expressed at high levels in root nodules but showed low expression in other tissues, including roots, indicating that it may be involved in the formation of A. duranensis root nodules. they may function only during specific stages of development in these tissues. AdBBX21 was expressed at high levels in root nodules but showed low expression in other tissues, including roots, indicating that it may be involved in the formation of A. duranensis root nodules.
CO homologs are key factors in flowering time regulation in many species, and we therefore investigated the expression of A. duranensis orthologs of Arabidopsis CO and rice Hd1 (i.e., AdBBX8 and AdBBX24). AdBBX8 was highly expressed in leaves, flowers, pistils, and Aerial Gyn Ti, while AdBBX24 was expressed at low levels in all tissues. Some duplicated gene pairs, such as AdBBX3/AdBBX15, were expressed at similar levels in all tissues (Figure 8), suggesting functional conservation in the duplicated genes. In contrast, other duplicated pairs differed in their expression patterns. For example, AdBBX9 was highly expressed in leaves and roots, while its duplicate AdBBX4 exhibited low expression in the same tissues ( Figure 8).

Discussion
In the past decades, the characterization of BBX genes, such as Arabidopsis CO and rice Hd1, has greatly increased our understanding of the molecular mechanisms involved in plant development. Peanut is an important crop around the world and provides essential oil for daily life, thus the CO homologs are key factors in flowering time regulation in many species, and we therefore investigated the expression of A. duranensis orthologs of Arabidopsis CO and rice Hd1 (i.e., AdBBX8 and AdBBX24). AdBBX8 was highly expressed in leaves, flowers, pistils, and Aerial Gyn Ti, while AdBBX24 was expressed at low levels in all tissues. Some duplicated gene pairs, such as AdBBX3/AdBBX15, were expressed at similar levels in all tissues (Figure 8), suggesting functional conservation in the duplicated genes. In contrast, other duplicated pairs differed in their expression patterns. For example, AdBBX9 was highly expressed in leaves and roots, while its duplicate AdBBX4 exhibited low expression in the same tissues ( Figure 8).

Discussion
In the past decades, the characterization of BBX genes, such as Arabidopsis CO and rice Hd1, has greatly increased our understanding of the molecular mechanisms involved in plant development. Peanut is an important crop around the world and provides essential oil for daily life, thus the investigation of peanut BBX genes is therefore useful for understanding and improving peanut development. In this study, we identified and characterized 24 BBX proteins from the wild peanut A. duranensis and carried out a comprehensive analysis of these genes.
BBX genes have changed during plant evolution, and the numbers and types of BBXs vary among different species [3,4,7,8,54]. For example, A. duranensis, Arabidopsis, rice, and pear contain 24, 32, 30, and 25 BBX members, respectively, and class IV contains the largest number of BBX genes in each species (Table S2). The genome sizes of diploid A. duranensis [33,55], Arabidopsis [56], rice [57], and pear [58] are 1.25 GB, 125 Mb, 466 Mb, and 512 Mb, respectively. Thus, genome size has no direct relationship to the number of BBX genes in these plants. Genes containing both BBX and CCT domains encode CO or CO-like (COL) proteins, and many Arabidopsis CO-like genes (COL) are thought to be involved in circadian clock or flowering time regulation [4]. Approximately half of the BBX proteins are identified as CO or COL members (classes I, II, and III) ( Figure 4) in Arabidopsis (53.13%), rice (56.67%), pear (44%), and A. duranensis (45.83%), suggesting that the evolution of CO and COL genes may be broadly conserved in these plants. In addition, the genome size of A. hypogaea (40 BBX members) is close to the sum of A. duranensis and A. ipaensis genomes, but the number of BBX genes in A. hypogaea is less than the sum of those in the A. duranensis (24 BBX members) and A. ipaensis (25 BBX members) genomes, indicating that BBX genes might be lost during evolution.
The cis-acting elements in promoter regions influence gene transcription, and differences in the type and number of cis-acting elements are responsible for differences in gene expression. AtBBX genes participate in the regulation of many pathways, including flowering time, the circadian clock, abiotic stress response, and photomorphogenesis [3,4]. Different numbers and types of cis-acting elements were found in the AdBBX promoter regions, underscoring the functional diversity of these genes. Many BBX genes in Arabidopsis are involved in light input signal pathways [4]. Light responsive elements were the most abundant elements in each of the AdBBX promoters, suggesting that AdBBXs may also be involved in light-dependent regulation pathways. Moreover, many cis-acting elements were also identified in the promoter regions of low expressed genes, including AdBBX3, AdBBX5, AdBBX15, AdBBX18, and AdBBX24 ( Figure 8). Many factors in addition to cis-acting elements affect gene expression in plants. For example, epigenetic modification and somatic genome variation also influence gene expression in many organisms [59]. Whether low-expressed AdBBX genes were influenced by these factors requires further investigation.
CO is an important factor involved in the regulation of flowering time in Arabidopsis, and it is highly expressed at the apex of seedlings and young leaves [60]. CO accelerates flowering time by activating the transcription of the Rafkinase inhibitor protein (RAF)-kinase-inhibitor-like protein, FT. AdBBX8 and AdBBX24 are close A. duranensis homologs of CO ( Figure S4). Soybean CO orthologs, GmCOL1a, GmCOL1b, GmCOL2a, and GmCOL2b, are also involved in flowering time regulation [61]. Genes derived from the same common ancestor may have similar functions, and we therefore investigated synteny relationships among CO orthologs/homologs from soybean (GmCOL1a, GmCOL1b, GmCOL2a, and GmCOL2b) and A. duranensis (AdBBX8 and AdBBX24) ( Figure S8). Synteny analysis revealed that AdBBX8 had a closer relationship to soybean GmCOL1a and GmCOL1b than did AdBBX24. In contrast, AdBBX8 and AdBBX24 showed similar close relationships to soybean GmCOL2a and GmCOL2b. AdBBX8 was expressed highly in leaves, flowers, pistils, and aerial gyn Ti, but AdBBX24 exhibited extremely low expression in all tissues ( Figure 8). These results suggest that AdBBX8 may play a similar role to CO in flowering time regulation, and that AdBBX24 may be a redundant gene that has lost its functions during evolution. In addition, CO is regulated by the circadian clock and its expression changes during the day [62]. AdBBX24 may therefore be expressed at a different time of day than the one at which the plants were sampled. Much work remains to be done to fully understand the functions of AdBBX8 and AdBBX24 in flowering time regulation.
Gene duplication produces new genes during evolution in many species. Some duplicated genes lose function, and some evolve new functions, compared to the original gene [63,64]. As a result, the allotetraploid Arachis hypogaea produces much more duplicated events than the sum of that in wild species Arachis duranensis and Arachis ipaensis (Figures S6 and S7). Two duplicated gene pairs were found in A. duranensis, and these duplication events occurred in class II and IV subfamilies, respectively, whose members contains two BBX domains ( Figure 6). In addition, duplicated gene pairs had different exon-intron structures (Figure 5a), and their cis-acting promoter elements differed (Table 2), indicating functional differentiation of these gene pairs during evolution. The duplicated gene pairs contained similar motifs (Figure 5b), and some duplicated genes showed similar expression in specific tissues, such as AdBBX2/AdBBX17 (Figure 8), indicating that these duplicated genes may have retained some original functions and may participate in common pathways.

Conclusions
In the present study, we identified and characterized 24 BBX genes from wild peanut, A. duranensis. We investigated their conserved domains, gene structures, phylogenetic relationships, chromosomal distributions, gene duplications, synteny relationships, cis-acting elements, and gene expression. Our results will not only be useful for understanding AdBBX genes but will also provide essential information for further functional analysis of these genes in A. duranensis.
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/2073-4395/10/1/23/s1. Figure S1. Phylogenetic analysis of AiBBX proteins. A phylogenetic tree was generated from protein sequences in MEGA 7 using the neighbor-joining method. The yellow, red, blue, purple, and green gene names indicate class I, II, III, IV, and V members, respectively. Figure S2. Phylogenetic analysis of AhBBX proteins. A phylogenetic tree was generated from protein sequences in MEGA 7 using the neighbor-joining method. The yellow, red, blue, purple, and green gene names indicate class I, II, III, IV, and V members, respectively. Figure S3. Phylogenetic analysis of AdBBX, AiBBX, and AhBBX proteins. A phylogenetic tree was generated from protein sequences in MEGA 7 using the neighbor-joining method. The yellow, red, blue, purple, and green gene names indicate class I, II, III, IV, and V members, respectively. Figure S4. Evolutionary relationship analysis of AdBBX proteins. Amino acid sequences of BBX proteins from A. duranensis, rice, and Arabidopsis were used to generate a phylogenetic tree with MEGA 7.0 using the neighbor-joining method. Figure S5. Sequence logos of 15 distinct motifs in 24 AdBBX proteins. Figure S6. Duplication analysis of AiBBX genes. The chromosomes are indicated by different colors, and the duplicated gene pairs are marked with lines. Figure S7. Duplication analysis of AhBBX genes. The chromosomes are indicated by different colors, and the duplicated gene pairs are marked with lines. Figure S8. Synteny analysis of CO orthologs/homologs in soybean and A. duranensis. The putative orthologous genes surrounding CO orthologs/homologs from soybean (GmCOL1a, GmCOL1b, GmCOL2a, and GmCOL2b) and A. duranensis (AdBBX8 and AdBBX24) were identified by BLASTP search. Synteny between AdBBX8, AdBBX24, and (a) GmCOL1a, (b) GmCOL1b, (c) GmCOL2a, and (d) GmCOL2b are shown. The red boxes indicate our target genes, and the green boxes indicate genes surrounding the CO orthologs/homologs. Gm, Glycine max. Table S1. Function analysis of the cis-acting elements in AdBBX promoter regions. The classifications, names, and putative functions of related cis-acting elements are predicted and listed. Table S2. The numbers and types of BBX proteins in Arabidopsis, rice, pear, and Arachis duranensis.

Acknowledgments:
The authors thank the reviewers for their valuable suggestions during the revision of the manuscripts.

Conflicts of Interest:
The authors declare no conflicts of interest.