Genome

We searched PepperHub (Pepper Information Hub, http://pepperhub.hzau.edu.cn/) and PGP (Pepper Genome Platform, http://passport.pepper.snu.ac.kr/?t=PGENOME) with the conserved B-box domain HMM profile (PF00643) to obtain global putative BBX genes in pepper. Then the putative encoding protein sequences of these genes were further confirmed their B-box domain by using SMART, Pfam and InterProScan searches, six putative genes without B-box domain were removed. In total, we eventually identified 24 BBX genes in pepper, which were named CaBBX1 to CaBBX24. Afterwards, the detailed information gene name, gene annotation ID, genomic position, gene length, theoretical isoelectric point, and molecular weight of their encoding protein were listed in Table 1.

These BBX genes showed diverse in length leads to the various length, theoretical isoelectric point, and molecular weight of their encoding protein. These BBX genes with sequence of 582 to 1476 bp encoded ranging from 193 (least, CaBBX7) to 491 (most, CaBBX22) amino acid residues. And the isoelectric points of 24 BBX proteins were ranging from 4.60 (lowest, CaBBX23) to 9.17 (highest, CaBBX24), with the molecular weights of 21.20 ~ 54.98 kDa (Table 1).

To identify the phylogenetic relationship and division of CaBBX proteins, we constructed the phylogenetic tree of BBX family proteins in pepper (Fig. 1). The phylogenetic analysis of the CaBBXs with AtBBXs, PtBBXs, OsBBXs and SlBBXs was also carried out to confirm the subfamily clustering of CaBBXs (Fig. S1). The division of 24 CaBBXs were not even on the phylogenetic tree (Fig. 1A). All the 24 CaBBXs were divided into five subfamilies with the similarity of the amino acid sequences based on previous studies in tomato [1]. In addition, the phylogenetic relationship of first B-box domain was constructed, as well as two B-box and one CCT domain (Fig. 1B and C). In total, there were eight CaBBXs classified into subclass I, whose contain two B-box domains, making up the largest subclass. The subclass II and III both contained six members, and only two members (CaBBX12 and 13) clustered together in subclass IV, and CaBBX23 and 24 aligned together in subclass V. Members from subclass II owed two B-box domains and one CCT domain, while only one BBX proteins (CaBBX13) possessed one B-box and one CCT domain belonging to subclass IV. Other CaBBX proteins only contained one or two B-box domains without CCT domain. Moreover, based on the phylogeny of BBXs in Arabidopsis, rice, tomato and Populus tomentosa, most of the BBXs with two B-box domains and one CCT domain were classified into subgroup II, and most of whom with two B-box domains and none CCT domain were classified into subgroup I. While, BBXs contain one single B-box domain were most together classified into subgroup V.

Phylogenetic analysis of BBX genes in pepper. A. Phylogenetic tree of BBX members from pepper. B. The trees shown were based on the alignments of the first B-box domain sequences. C. The trees shown were based on the alignments of the protein sequences with the two B-box plus the CCT domain. Ca: Capsicum annuum L. The protein sequences used to construct the tree were CaBBX1 (Capana02g003201), CaBBX2 (Capana02g003200), CaBBX3 (Capana02g003199), CaBBX4 (Capana01g004030), CaBBX5 (Capana12g000414), CaBBX6 (Capana07g000030), CaBBX7 (Capana00g004028), CaBBX8 (Capana07g001114), CaBBX9 (Capana00g004489), CaBBX10 (Capana03g003558), CaBBX11 (Capana00g001486), CaBBX12 (Capana11g002294), CaBBX13 (Capana03g000377), CaBBX14 (Capana02g002620), CaBBX15 (Capana08g002625), CaBBX16 (Capana12g000659), CaBBX17 (Capana04g000266), CaBBX18 (Capana07g002062), CaBBX19 (Capana09g000394), CaBBX20 (Capana06g000735), CaBBX21 (Capana08g002611), CaBBX22 (Capana05g001195), CaBBX23 (Capana07g001588), CaBBX24 (Capana00g004911). The phylogenetic tree was constructed based on peptide sequences using the Neighbor-Joining method

To determine the domains, motif structure and gene structure of CaBBXs, the conserved domain information were confirmed by CDD in NCBI, and motif and CDS were also plotted to identify structure analysis of CaBBXs (Fig. 2).

The domain, conserved motifs and gene structures of the BBX family members in pepper. A. The domain of BBX family members in pepper. B. The distribution of conserved motifs of BBX family members in pepper. C. The gene structures of BBX family members in pepper. The boxes and lines denote exons and introns, respectively. Eight conserved motifs of each subfamily were displayed in different colors. The scale on the bottom is in base pair (bp)

Eight motifs were identified in these CaBBXs, members from same subclass shared similar motifs according to the phylogenetic relationship (Fig. 2A). For example, all the members from subclass I contained motif 1, 3 and 7, only CaBBX20 also owed another motif 6. While, except CaBBX4 only owed two motif 1 and one motif 2, CaBBXs from subclass II possessed maximum motifs, containing motif 1, 2, 3, 5 and 7; moreover, three of them (CaBBX1, 2 and 3) contained all the 8 identified motifs. Other members carried one or two motif 1, several of them contained motif 2; in addition, CaBBX11 and 13 also had a motif 4 and 8 located in middle of their amino acid sequence, respectively. The detail sequence information of these eight motifs were shown in Fig. S2.

Furthermore, the gene structures of CaBBXs were constructed with TBtools by gff file from pepper genome 2.0 [49]. Among 24 CaBBXs, only CaBBX24 had no intron, others had one to five exons. To make clear the domains arrangement, we also plotted domains on the CDS directly. Nine BBX proteins were identified containing two B-box and a CCT domain, five of them share two same B-box, and four possessed two different B-box domains. Only one BBX proteins (CaBBX13) possessed one B-box and one CCT domain, while three and eleven CaBBX proteins contained one and two B-box without CCT domain, respectively (Fig. 2B). These results were consistent with the phylogenetic divergence analysis. Except for subclass I members, the B-box domains of members from other subclasses were located in the beginning of first exon. The B-box domains of subclass I members were on the first three exon. CCT domain were situated in the terminal of last two exon. Moreover, the two B-box (B-box1 and B-box2 domains) share similar conserved sequences and Zinc finger domain.

We have plotted the CaBBX genes to the chromosomes of pepper genome to confirm their genomic distribution (Fig. 3). Except for four CaBBX genes (CaBBX7, CaBBX9, CaBBX11 and CaBBX24), 20 CaBBX genes were distributed unevenly on 11 of 12 pepper chromosomes, no gene was on chromosome 10. Both chromosome 02 and 07 possessed four CaBBX genes, making up the maximum number of genes among all these 12 chromosomes. In addition, only one CaBBX gene was located on chromosome 01, 04, 05, 06, 09 and 11, respectively; and two on chromosome 03, 08 and 12, respectively.

Chromosome distribution and segmental duplication of pepper BBX genes. Chromosomal mapping was based on the physical position in 12 pepper chromosomes. The scale on the left is in megabases (Mb). Different color represented the classification of CaBBX genes, green: subclass I; purple: subclass II; orange: subclass III; blue: subclass IV; red: subclass V. The chromosome numbers are indicated at the side of each bar. The segmental duplicated genes are connected by lines

Potential duplication within pepper were marked on the 12 chromosomes by using TBtools [49]. Expect CaBBX7 (duplicated with CaBBX8) was not located on pepper chromosomes, the other three duplications only occurred on the 3 of 12 chromosomes, and these duplicated genes (CaBBX14, CaBBX15, CaBBX17 and CaBBX21) were all belonged to subgroup I (Fig. 3). And all the duplication events occurred between two different chromosomes, not within the same chromosome. In addition, we constructed a collinearity relationship analysis to identify the duplication events of BBX genes between pepper and the model Solanaceae plant tomato (Fig. 4). Twenty-six pairs of BBX genes were identified duplicate between pepper genome and tomato genome. All the subgroups of BBX genes involved in duplication. Among them, 13 pairs of subgroup I members play part in the replication events, account for half of the total duplication events. And we found 4 pairs of subgroup II and III members, 3 pairs of subgroup IV members and 2 pairs of subgroup V members were homologous in the pepper and tomato genome (Fig. 4).

Chromosome distribution and duplication events of pepper and tomato BBX genes. The segmental duplicated genes are indicated in red and connected by lines. (Orange pillar represent Capsicum annuum L chromosome, from left to right: chr11, chr00, chr10, chr02, chr01, chr12, chr04, chr03, chr06, chr05, chr08, chr07, chr09; Green pillar represent Lycopersicon esculentum chromosome,from left to right: chr09, chr08, chr07,chr06, chr05, chr04, chr03, chr02, chr01, chr12, chr00, chr11, chr10)

To investigate the tissue-specific and developmental expression pattern of all the CaBBX genes, we performed the heatmap by using TBtools based on the transcript data from Pepper Information Hub (http://pepperhub.hzau.edu.cn/) [49]. Several CaBBX genes showed organ-specific expression pattern, such as CaBBX19, expressed specifically in seed, respectively, and expressed arise as the tissues’ development (Fig. 5). This result indicated that CaBBX19 may play an important role in seed morphogenesis development, respectively. CaBBX7, CaBBX12, CaBBX13 and CaBBX22 mainly expressed in leaf, may showed their regulatory function in pepper leaf (Fig. 5). Additionally, CaBBX5 and CaBBX6 showed high expression levels in leaf and flower, CaBBX3 and CaBBX14 specifically expressed in the early developmental stage of flower, CaBBX4 and CaBBX20 expressed in almost all the detected tissues, and expressed most highly in fruit development, especially in the pericarp, however, expect in the seed (Fig. 4). This result may indicate that CaBBX4 and CaBBX20 involved in the pericarp development (such as pigmentation, enlargement, and so on).

Expression profiles of the pepper CaBBX genes in different organs, tissues and developmental stages. Data were normalized based on the mean transcript levels (log2scale) of each gene in all tissues analyzed. Genes were hierarchically clustered based on average Pearson’s distance metric and ‘average linkage’ method. Red and green circular indicate high and low expression levels, respectively, for each gene. 2 d (L1), 5 d (L2), 10 d, 15 d (L4), 20 d (L5), 25 D (L6), 30 d (L7), 40 d (L8), 50d (L9), 60 d (L10) after the emergence of new leaves. Leaf (AL), root (AR), stem (AS). According to the size of flowers, the flowers of different development stages can be divided into nine stages (F1, F2, F3, F4, F5, F6, F7, F8, F9) such as young bud stage and white stage, and the flowers that open on the same day (F10). Ovary (O10) and anther (STA10). The fruit pollination three days (FST0), seven days (FST1), from the third stage (10d after pollination), the fruit was divided into three tissues: pulp (G), placenta (T) and seed (S). 10 days (G1), 15 days (G2), 20 days (G3), 25 days (G4), 30 days (G5), 35 days (G6), 40 days (G7), 45 days (G8), 50 days (G9), 55 days (G10), 60 days (G11) after pollination

Furthermore, we also investigated the expression levels of 24 CaBBX genes by qRT-PCR. In particularly, CaBBX19 showed the highest expression level in seed, and CaBBX24 was expressed more highly in flower than that in the other tissues, indicating that CaBBX19 and CaBBX24 play essential roles in controlling pepper seed development and flowering (Fig. 6). CaBBX13 and 22 showed highly expression in leaf, while CaBBX6 expressed highly in both leaf and flower. These results were consisting with that of RNA-seq data transcript levels.

The expression of CaBBXs in different pepper tissues. Different tissues were arranged as ‘root, stem, leaf, flower, fruit and seed’. Three independent biological experiments were performed (P