
==== Front
BMC Genomics
BMC Genomics
BMC Genomics
1471-2164
BioMed Central London

10681
10.1186/s12864-024-10681-9
Research
Genome-wide identification of CaWD40 proteins reveals the involvement of a novel complex (CaAN1-CaDYT1-CaWD40-91) in anthocyanin biosynthesis and genic male sterility in Capsicum annuum
Tang Peng 1
Huang Jingcai 1
Wang Jin 1
Wang Meiqi 1
Huang Qing 1
Pan Luzhao 12
Liu Feng jwszjx@hunau.edu.cn

1
1 https://ror.org/01dzed356 grid.257160.7 0000 0004 1761 0331 Engineering Research Center for Germplasm Innovation and New Varieties Breeding of Horticultural Crops, Key Laboratory for Vegetable Biology of Hunan Province, College of Horticulture, Hunan Agricultural University, Changsha, China
2 https://ror.org/04ejmmq75 grid.419073.8 0000 0004 0644 5721 Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, China
11 9 2024
11 9 2024
2024
25 85113 4 2024
1 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

The WD40 domain, one of the most abundant in eukaryotic genomes, is widely involved in plant growth and development, secondary metabolic biosynthesis, and mediating responses to biotic and abiotic stresses. WD40 repeat (WD40) protein has been systematically studied in several model plants but has not been reported in the Capsicum annuum (pepper) genome.

Results

Herein, 269, 237, and 257 CaWD40 genes were identified in the Zunla, CM334, and Zhangshugang genomes, respectively. CaWD40 sequences from the Zunla genome were selected for subsequent analysis, including chromosomal localization, phylogenetic relationships, sequence characteristics, motif compositions, and expression profiling. CaWD40 proteins were unevenly distributed on 12 chromosomes, encompassing 19 tandem duplicate gene pairs. The 269 CaWD40s were divided into six main branches (A to F) with 17 different types of domain distribution. The CaWD40 gene family exhibited diverse expression patterns, and several genes were specifically expressed in flowers and seeds. Yeast two-hybrid (Y2H) and dual-luciferase assay indicated that CaWD40-91 could interact with CaAN1 and CaDYT1, suggesting its involvement in anthocyanin biosynthesis and male sterility in pepper.

Conclusions

In summary, we systematically characterized the phylogeny, classification, structure, and expression of the CaWD40 gene family in pepper. Our findings provide a valuable foundation for further functional investigations on WD40 genes in pepper.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-024-10681-9.

Keywords

WD40 transcription factors
Pepper
MYB-bHLH-WD40 (MBW)
Functional diversification
Classification
Science and Technology Innovation Program of Hunan Province2021NK1006 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcBackground

The WD-repeat (WDR) proteins are commonly referred to as the WD40 repeat proteins or WD40 proteins owing to the highly conserved domain of approximately 40 amino acid stretches [1–4]. This conserved domain is characterized by a glycine histidine (Gly-His, GH) dipeptide at the N-terminal (start) and a tryptophan-aspartic acid (Trp-Asp, WD) dipeptide at the C-terminal (end) [1, 4]. Structurally, the WD40 proteins typically have multiple tandem repeated WD motifs, forming a series of four-stranded and antiparallel β sheets [2, 5, 6]. Functionally, the WD40 domain has no catalytic activity of its own and is considered an adaptor protein that recruits other factors to form protein-protein or protein-deoxyribonucleic acid (DNA) complexes [7–9]. WD40 proteins are involved in histone modification [10], DNA damage repair [11], signal transduction [9], and abiotic stress responses [12].

The MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat protein form an MYB-bHLH-WD40 (MBW) complex, which jointly regulates flavonoid biosynthesis, constituting among the most general functions of the WD40 genes. For example, the TTG1-bHLH-MYB complex promotes flavonoid accumulation and the formation of trichomes in Arabidopsis seedlings [13, 14]. Similarly, TT2 (AtMYB123), TT8 (AtbHLH42), and TTG1 (WD40) synergistically regulate the biosynthesis of the proanthocyanidins pigments in the Arabidopsis seed coat [15]. The expression of several structural genes is regulated by MBW complex, including dihydroflavonol-4-reductase (DFR), anthocyanin dioxygenase/ anthocyanin synthase (LDOX/ANS), BANYULS (BAN) [15, 16]. In pepper, CaTTG1 forms an MBW complex with CaAN1 and CaGL3 to control anthocyanin synthesis by activating the expression of structural genes in the synthesis pathway of anthocyanin [17].

Some studies have demonstrated the participation of the WD40 in various biological processes involved in reproductive development in Arabidopsis. For instance, the XPO1-interacting WD40 protein 1 (XIW1) can interact with ABA insensitive 5 (ABI5) in the nucleus and regulate seed germination and growth [18]. The JGB gene with the WD40 domain is a negative regulator of pollen germination [19]. WDR55 is essential for multiple plant developmental processes, including gametogenesis, seed, and endosperm development [20]. The SWA1 protein is necessary for the normal functioning of the mitotic division cycle by regulating cellular metabolism [21].

The WD40 repeats are widely found in all eukaryotes but are rare in prokaryotic organisms [3, 22–26]. In plants, the WD40 proteins form a superfamily, which are functionally diverse. With the completion of whole genome sequencing of different species, the WD40 protein family has been systematically identified in several [3, 25–29]. The 237 potential WDR proteins from Arabidopsis have been identified. These show conservation and divergence in structure and function [3]. In Triticum aestivum, 743 WD40 proteins have been grouped into 5 clusters and 11 subfamilies and may be involved in reproductive development and mediating responses to multiple stresses [25]. In Oryza sativa, 200 OsWD40 genes have been identified and are suggested to perform diverse functions through a complex network [26]. Li et al. [27] found 191 WDRs in cucumber (Cucumis sativus L.). In Arabidopsis, WD40s show strong conservation during the evolutionary process. Feng et al. [28] identified 220 WD40s in the peach genome, and corresponding proteins localized to different subcellular structures. Chen et al. [29] analyzed 164 barley HvWD40 proteins and revealed that the WD40 gene formed an MBW complex with MYB and bHLH and participated in anthocyanin synthesis. These results suggest that the WD40 gene family has diverse gene numbers, structures, and functions.

Pepper, a typical spicy vegetable, belongs to the genus Capsicum of the Solanaceae family. It originated in South and Central America [30]. Capsicum species have various morphological characteristics, and several pepper species lack anthocyanins [17, 31, 32]. In recent years, several genes controlling anthocyanin synthesis have been identified in pepper. Zhang et al. [32] reported that a splice acceptor site in CabHLH1 resulted in a frameshift mutation in a mutant of deficient anthocyanin and fertility reduction of anther (rpf1). Two genes, CaAN3 and Ca3GT, controlling anthocyanin biosynthesis in pepper fruit were successively reported [33, 34]. CaHY5 regulated anthocyanin accumulation in pepper hypocotyl by directly binding to the promoter of downstream structural genes in the anthocyanin pathway [35]. A structural gene of anthocyanin synthesis, flavonoid 3’,5’-hydroxylase (F3’5’H), controls pigment biosynthesis in stems and anthers in pepper [17].

In this study, 269 CaWD40 members were identified in the Zunla pepper genome. The physicochemical properties, sequence structural characteristics, phylogenetic construction, and tissue expression of the CaWD40 gene family were analyzed by bioinformatics methods. Based on yeast two-hybrid (Y2H) technology, we verified the potential involvement of a novel MBW complex (CaAN1-CaDYT1-CaWD40-91) in anthocyanin synthesis. These results are expected to facilitate the understanding of biological functions and molecular mechanisms of WD40 proteins and provide a basis for studying the anthocyanin regulatory network in pepper.

Results

The CaWD40 gene family shows great variations in sequence length and physicochemical properties

The Hidden Markov Model (HMM) profile of the WD40 repeat domain (PF00400) was used as a query sequence to search WD40 proteins in the Zhangshugang, Zunla, and CM334 genome databases. Preliminarily, 267, 276, and 245 WD40s were retrieved from the Zhangshugang, Zunla, and CM334 reference genomes, respectively. All examined pepper WD40 members were verified, and partial WD40 genes with incomplete WD40 domains, considered pseudogenes, were further removed. Finally, 257, 269, and 237 WD40s were identified in the Zhangshugang, Zunla, and CM334, respectively. Since the knowledge of WD40 members in the Zunla genome is relatively complete, the information on this genome was subject to subsequent analyses. For convenience, pepper WD40 candidates were named CaWD40-1–CaWD40-269 according to their positions on the 12 chromosomes in the Zunla reference genome. Detailed information on gene name, chromosome location, protein length, molecular weight, isoelectric point (pI), and domain is listed in Table S1.

CaWD40 genes varied greatly in terms of protein length and physicochemical properties. The length of these CaWD40 fragments ranged from 100 aa (CaWD40-85, CaWD40-212) to 3,595 aa (CaWD40-87). The molecular weights (MWs) ranged from 10.75 kDa (CaWD40-85) to 399.13 kDa (CaWD40-87). The isoelectric point (pIs) ranged from 4.21 (CaWD40-267) to 9.96 (CaWD40-151) (Table S1). The prediction results for the subcellular localization of the 269 CaWD40s were as follows: 139 in the nucleus, 56 in the chloroplasts, 55 in the cytoplasm, six in the plasma membrane, five in the cytoskeleton, four in the mitochondria, two in the vacuole membrane, one in the endoplasmic reticulum, and one in the peroxisome.

Chromosome localization reveals multiple tandem duplication events

The 245 out of the 269 CaWD40 genes were randomly distributed across 12 chromosomes in the Zunla genome, and the remaining 24 CaWD40s were not anchored to chromosomes (Fig. 1A). The vast majority of CaWD40 members were concentrated on the distal end of each chromosome, with a few genes in the middle of the chromosome. The number of CaWD40s was highest on chromosome 3 (34), followed by chromosome 1 (33); the lowest was on chromosome 5 (8) (Fig. 1B). The distribution of CaWD40s indicated the accumulation of some genes on specific chromosomes.

We analyzed tandem duplications in the CaWD40 gene family in pepper. Nineteen pairs (I to XIX) of genes among 269 CaWD40s were identified as tandem duplication events (Table S2), crucial in gene functional differentiation. Tandem duplication events were unevenly distributed across the 10 chromosomes and were usually concentrated in regions with high gene density. Some tandem repeat genes belonged to the same phylogenetic branch, while others belonged to different branches, hinting at functional differentiation and conservation among the CaWD40 gene family (Fig. 2).

Fig. 1 Chromosome mapping and gene number of CaWD40 proteins in the Zunla genome. (A) Chromosome mapping and tandem duplication events of CaWD40s. Chromosome numbers are represented on the top, and the scale is shown on the left. Predicted tandem duplicate pairs are indicated in red font, and the categories are marked in parentheses using Roman numerals. (B) Statistical analysis for CaWD40s on 12 chromosomes

The combination of phylogenetic and structural analysis reveals the diversity of CaWD40s

In this study, the protein sequences of the 269 predicted CaWD40s were used to generate an unrooted tree to analyze the classification of the CaWD40 gene family in pepper (Fig. 2). Phylogenetic analysis showed that the CaWD40 gene family was categorized into six main branches (branch A to F) with 15, 22, 15, 42, 68, and 107 members, respectively. Among them, branch F was divided from F1 to F4 (Fig. 2). Branches A, B, and C were the earliest clusters in the phylogenetic tree, and genes in the same branch shared similar motif compositions (Fig. 2 and Fig S1). Similarly, most genes in branches D, E, and F followed this rule. Combined with chromosome localization analysis, gene pairs with tandem duplication were mostly distributed in the E and F branches, suggesting that the differentiation of gene function was gradual along with gene duplication (Figs. 1 and 2).

Fig. 2 Phylogenetic relationships among CaWD40s in the Zunla genome. Different branches are marked by shading with diverse colors. The gray dots represent the bootstrap, and those with a bootstrap greater than 0.5 are shown. Predicted tandem duplicate pairs are marked with fonts of diverse colors

The structural domain of the predicted 269 CaWD40s was counted using SMART (Table 1, Table S1) to analyze the relationship between domain composition and phylogenetic classification. The CaWD40 gene family was categorized into 17 distinct classes based on domain composition (Table 1). Among them, 191 members contained only a single WD40 domain, and the remaining had at least one or more WD40 domains and other domains.

Table 1 Domain composition of the 17 classes of 269 CaWD40s

Classes	Domain composition	Gene number	
Class 1	OnlyWD40	191	
Class 2	WD40 + LISH + CTLH	8	
Class 3	WD40 + Ubox or Fbox	6	
Class 4	WD40 + Utp12 or Utp13	5	
Class 5	WD40 + NLE	3	
Class 6	WD40 + PH_BEACH + Beach or Beach or DUF4704 + PH_BEACH + Beach	3	
Class 7	WD40 + RING + TYKc or RING or CLH + RING	3	
Class 8	WD40 + Transmembrane region	3	
Class 9	WD40 + Coatomer_WDAD + COPI_C or Coatomer _WDAD	3	
Class 10	WD40 + Katanin_con80	3	
Class 11	WD40 + PFU	2	
Class 12	WD40 + STYKc	2	
Class 13	WD40 + BCAS3	2	
Class 14	WD40 + CAF1C_H4-bd	2	
Class 15	WD40 + LRRcap	2	
Class 16	WD40 + ZnF_C3H1 or ZnF_C2HC	2	
Class 17	Other	29	

Gene structural characteristics can elucidate the relationship between gene function and evolution. The number of exons and introns in the 269 CaWD40s varied greatly (Fig. S1A and B). Twenty-three CaWD40 genes (8.55%) had only one exon and were intronless, including CaWD40-80 and CaWD40-91 (Fig. S1B). A total of 146 CaWD40s (54.28%) contained 1–10 exons; 83 CaWD40s (30.86%) contained 11–20 exons, and the remaining 17 CaWD40s (6.32%) contained more than 20 exons. Specifically, CaWD40-218 and CaWD40-181 had two exons; the former belonged to branch A and the latter to branch F (Fig. 2, Fig. S1B). All three genes (CaWD40-62, CaWD40-124, CaWD40-160) belonged to branch A. CaWD40-124 had four exons. CaWD40-160 and CaWD40-62 had nine and 29 exons, respectively. These results suggest that the different exon-intron compositions of 269 CaWD40s are randomly distributed across different branches of the phylogenetic tree, implying a high diversity of the CaWD40 gene family in the process of evolution.

Using the Multiple Em for Motif Elicitation (MEME) tool, we identified 10 putative conserved motifs (motif 1 to motif 10) in the CaWD40 gene family (Fig. S1C). No common motifs were observed in 269 CaWD40s. Among the 10 motifs, motif 1 was the most widely distributed, found in 264 CaWD40 genes. Some motifs were present in only a few genes, and motif 9 and motif 10 were present in only 4 (CaWD40-57/117/233/256) and 6 (CaWD40-86/89/98/147/195/222) CaWD40 genes, respectively. Some CaWD40s with the same exon–intron structure exhibited a similar motif composition, such as CaWD40-69 and CaWD40-266 (Fig. S1C).

Synteny analysis of CaWD40s among three genomes

A genome-wide synteny analysis was performed among the CaWD40s in the Zunla, Zhangshugang, and CM334 genomes. Zunla and Zhangshugang had 225 gene pairs co-linked and Zunla and CM334 had 178 gene pairs co-linked (Fig. 3, Table S3). Zunla and Zhangshugang showed a close synteny for the CaWD40 gene, suggesting that these two species may share more similar functions. The relationship of species origin indicates that Capsicum annuum cv. Zhangshugang and Zunla are derived from China [31, 56]; the CM334 pepper is from South Korea [57].

Fig. 3 Synteny analysis for WD40s among different pepper genomes. The identified collinear genes are shown with blue lines

CaWD40s exhibit distinct expression profiles in various tissues and stages

By RNA-seq analysis, expression profiles of the putative CaWD40 genes at various tissues and developmental stages of Capsicum line 6421 were analyzed (Fig. 4). CaWD40s showed varied expression patterns, which could be divided into six types (type i–vi) based on a hierarchical clustering of their expressional characteristics (Fig. 4, Table S4). Specifically, type i contained 48 genes, most relatively highly expressed in different tissues and periods. Type ii comprised 101 genes with medium or low expression in various tissues or stages. Type iii included 21 CaWD40s, highly expressed in almost all tissues. Type iv included 49 genes showing low-to-no expression in most tissues. Type v had 16 members with tissue-specific high expression in specific tissues or stages. For example, four genes (CaWD40-51, CaWD40-135, CaWD40-199, and CaWD40-223) were highly expressed in flower and stamen tissues and showed almost no expression in other tissues. CaWD40-93 and CaWD40-125 showed high expression levels in fruit or placenta. Group vi contained 34 genes with low expression levels in all tissues.

Fig. 4 Expression analysis of CaWD40 genes in various tissues and stages based on RNA-seq data. Cluster hierarchy based on expression levels. Heat map; blue/yellow/red colors represent low/medium/high expressions, respectively

Verification of expression in the eight CaWD40s

We randomly selected eight CaWD40s from each hierarchical cluster in the heatmap to determine their expression in different tissues by RT-qPCR to verify the expression of the CaWD40 gene family. The expression trend identified by RT-qPCR was similar to that of RNA-seq (Figs. 4 and 5). For example, the expression of CaWD40-26 was not detected in diverse tissues; CaWD40-69 and CaWD40-266 were highly expressed in almost all examined tissues and stages (Fig. 5), and CaWD40-135 and CaWD40-223 tended to be expressed later during flower development.

Fig. 5 Relative expressions of CaWD40-26, CaWD40-57, CaWD40-69, CaWD40-91, CaWD40-127, CaWD40-135, CaWD40-223, and CaWD40-266. F1, F4, F5, F7, F8, and F9 represent different stages of buds in Capsicum line 6421

A novel MBW complex may be involved in anthocyanin biosynthesis and genic male sterility

Previous reports have shown that WD40 typically acts as part of the MBW complex to regulate anthocyanin biosynthesis [13–15, 17]. A recent report has demonstrated that the MBW complex of CaAN1-CaGL3-CaTTG1 affects pigment accumulation and male gametophyte development in the Cha1 mutant of pepper [17]. In this study, CaWD40-91 (Capana04g000080) protein showed high phylogenetic and structural similarity with CaTTG1 (CaWD40-80/Capana03g001813) (Figs. 4 and 5, Fig S2, 3). Subcellular localization results also demonstrated CaWD40-91 protein localization in the nucleus and cytoplasm (Fig. 6A), consistent with the localization results for CaTTG1 [17]. Therefore, we reasonably speculate that CaWD40-91 might function similarly to the CaTTG1 gene.

To confirm whether CaWD40-91 can bind to the CaGL3 and CaAN1 proteins, a Y2H experiment was performed. CaWD40-91 could interact with CaAN1 but not with CaGL3 (Fig. 6B). Considering that CaWD40-91 could indeed bind to CaAN1, we replaced other bHLH proteins to verify the protein interaction. A recent study from our laboratory showed that a candidate bHLH (CaDYT1) activated CaDFR expression to control anthocyanin synthesis and male sterility in pepper (unpublished data). Therefore, we verified the interaction between CaWD40-91 and CaDYT1 proteins. The results indicated that CaWD40-91 interacted with the CaDYT1 and CaAN1 proteins, resulting in the formation of a novel MBW complex (CaAN1-CaDYT1-CaWD40-91) (Fig. 6B and C), which may regulate synergistically anthocyanin accumulation and genic male sterility of pepper.

Fig. 6 Subcellular localization and interaction. (A) Subcellular localization of CaWD40-91. Scale bars represent 50 μm. (B) Interactions of CaWD40-91 with CaAN1 and CaDYT1 via a yeast two-hybrid assay. (C) The dual-luciferase reporter gene assay.

Mutation in CaDYT1 causes male sterility in pepper

We previously cloned the CaDYT1 gene by crossing the dyt1 mutant with wild type (WT) to obtain the F2 genetic population (unpublished data). The sequencing results are shown in Fig. 7A. The second exon of the CaDYT1 gene had a 7 bp deletion in the dyt1 mutant, resulting in a frameshift mutation. Phenotypic analysis showed that the anthers of the dyt1 mutant were thinner and yellower compared with WT (Fig. 7B). Scanning electron microscopy (SEM) showed a round and full WT anther, with a closely arranged convex surface structure. Several pollen grains are produced following anther dehiscence. In contrast, dyt1 anthers were small and folded, no pollen grains were observed, and only impurities and starch grains were found (Fig. 7B).

Fig. 7 Phenotypic analysis of the wild type (WT) and dyt1 mutant. (A) Mutation site detection between WT and dyt1. (B) The phenotype of anther and pollen grain in WT and dyt1. Anther phenotype is shown on the left, and SEM analysis of anther and pollen grain is shown on the right

Discussion

Diversity and conservation of CaWD40 proteins in pepper

In plants, WD40 is not directly involved in recognizing target gene promoters, but it plays a role in the MBW complexes with MYB and bHLH proteins [9, 15]. The MBW complex is important in the anthocyanin biosynthesis pathway [17]. In the MBW complex, the WD40 protein is usually located at the center of the triadic structure. It may protect the MBW complex by preventing other transcriptional regulators from binding to MYB or bHLH [15]. The functions of WD40 are diverse. In addition to anthocyanin synthesis, WD40 proteins are critical for pollen germination [36], drought stress [37], RNA processing [38], regulation of plant biological clock [39], skin and fur development [40], and other processes.

In this study, 269 CaWD40s were identified in the Zunla pepper genome. Interestingly, 237 and 257 CaWD40s were found in CM334 and Zhangshugang genome, respectively. Different pepper cultivars may lead to the difference in CaWD40 numbers in the three genomes [31, 56, 57]. Finally, the 269 CaWD40 members from the Zunla genome were analyzed. These genes were divided into six main branches based on their gene structure and motif compositions. The CaWD40 members within the branch showed high similarity in domain compositions and sequences, suggesting a conserved gene function.

The WD40 gene family has been reported in many species. The number of members varies greatly between species, such as 207 in tomato [41], 237 in Arabidopsis [3], 200 in rice [26], 191 in cucumber [27], 743 in Triticum aestivum [25], 164 in barley [29], and 220 in Peach [28]. Based on published genomic databases, the genome sizes of pepper, tomato, and Arabidopsis are predicted to be 3.5 Gb, 0.95 Gb, and 0.125 Gb, respectively. The above results indicate that the number of WD40s is not related to the genome size of a species. We found a high polarization in physical and chemical properties: protein length ranges from 100 aa (CaWD40-85, CaWD40-212) to 3,595 aa (CaWD40-87), and molecular weights ranged from 10.75 kDa (CaWD40-85) to 399.13 kDa (CaWD40-87). These results are consistent with previously reported studies [25, 26, 28, 29].

Nineteen pairs (40 CaWD40s) were identified as tandem duplication events, and these genes were unevenly distributed in 10 chromosomes (Fig. 1). Tandem repeat genes from a pair also sometimes clustered on the different phylogenetic branches (Fig. 2), suggesting functional divergence in the CaWD40 gene family. Sequence characteristics revealed that the number of exons and introns in the 269 CaWD40 gene family varied greatly, that is, from intronless to 30 introns (Fig. S1B and C). However, some CaWD40s possessed similar exon–intron and motif compositions (CaWD40-69 and CaWD40-266) (Fig. S1B and C). Overall, CaWD40 proteins were structurally dynamic, which could determine the functional diversity of the WD40 gene. In humans, WD40s are involved in multiple cellular networks, many of which have been implicated in diseases [42]. Two proteins, WDR5 and EED, are involved in chromatin complexes [42]. In Arabidopsis, several WD40 proteins are related to gametogenesis, seed, and endosperm development [19, 20]. The WD40 gene family is a superfamily of plant species. Some supergene families, such as WRKY, bHLH, and MYB, are usually accompanied by structural and sequence similarities and differences in the evolution process [19, 43–46].

CaWD40-91 is a potential candidate involved in anthocyanin biosynthesis and pollen fertility

In plants, flavonoids are synthesized through a conserved metabolic pathway, producing several secondary metabolites (SMs), including flavones, anthocyanins, and proanthocyanidins [47]. These SMs have pivotal roles in growth and development as they provide plants with different pigments that scavenge reactive oxide species (ROS) [32], defend against UV damage [48], or mediate plant-microbe interactions [49]. The SM biosynthetic pathway is among the most extensively studied in plants. Several structural genes related to anthocyanin biosynthesis, including CHS, CHI, F3H, F3’5’H, DFR, ANS, and UFGT, are conserved in most plants [47, 49, 50]. In recent years, several reports suggest a close association between flavonoids and pollen fertility [32, 48, 51–53].

Wang et al. [17] showed that CaTTG1, CaAN1, and CaGL3 formed an MBW complex, which regulated anthocyanin synthesis. In Arabidopsis, TTG1 can bind to AtMYB123 and AtbHLH42 transcription factors to regulate the biosynthesis of the proanthocyanidins pigments in the seed coat [13, 14]. DYSFUNCTIONAL TAPETUM 1 (DYT1), acting as an upstream regulator of tapetum development, is required for pollen formation in Arabidopsis and pepper [54, 55]. Our recent study suggests that the cha1 mutant (CaTTG1) exhibits partially aborted pollen grains (unpublished). In this study, CaWD40-91, a paralog protein of CaTTG1 (named CaWD40-80 in this paper), was confirmed to interact with CaAN1 and CaDYT1. In general, genes that are highly homologous in sequence and structure tend to have similar functions. More interestingly, we recently found that DYT1 could bind to DFR synergistically and regulate anthocyanins and male sterility in pepper (unpublished). Based on the above results, CaWD40-91, CaAN1, and CaDYT1 may form an MBW complex, which can regulate the formation of anthocyanins and pollen fertility.

Conclusions

From bioinformatics analysis, we identified 269, 237, and 257 CaWD40 genes in the Zunla, CM334, and Zhangshugang genomes, respectively. A total of 269 candidates from the Zunla genome were analyzed, and they were named CaWD40-1 to CaWD40-269 according to their arrangement on the chromosomes. The protein length and physico-chemical properties varied greatly. Protein lengths ranged from 100 to 3,595 aa, and molecular weights ranged from 10.75 kDa to 399.13 kDa. The analysis revealed 245 CaWD40 proteins clustered unevenly along chromosomes, with 24 CaWD40s remaining unlinked to chromosomes. Further analysis revealed 19 tandem duplicate pairs that clustered in different branches of the phylogenetic tree. Sequence analysis indicated that the numbers of exon (1–31) and intron (0–30) varied greatly in the CaWD40 gene family, and the composition of conserved motifs was diverse. However, several CaWD40s showed highly conserved sequence characteristics, expression patterns, and biological function. Finally, we found that CaWD40-91 showed high homology with CaTTG1 based on phylogenetic relationship, gene structure, and domain composition, with further speculations that they may share similar functions. A recent report revealed that CaTTG1 could interact with CaGL3 and CaAN1 to form an MBW complex, which could regulate anthocyanin synthesis in pepper [17]. In this study, we found that CaWD40-91 interacted with CaAN1 but not with CaGL3. Interestingly, we observed that CaWD40-91 could interact with CaDYT1 from the Y2H assay. Moreover, CaDYT1 activated CaDFR to synergistically regulate anthocyanin synthesis and male sterility. Therefore, we speculate that CaWD40-91 can interact with CaAN1 and CaDYT1 to control pigment accumulation and sterility in pepper.

Methods

Retrieval and identification of WD40 genes in pepper

In this study, the candidate WD40 proteins were retrieved as follows: first, the protein, nucleotide, and genome sequences of the Zunla, CM334, and Zhangshugang genomes were downloaded from the Sol Genomics Network (https://solgenomics.net/organism/Capsicum_annuum/genome) [56, 57] and Pepper Genomics Database (http://ted.bti.cornell.edu/cgi-bin/pepper/index) [31], respectively. Second, the HMM of WD40 protein (PF00400) was downloaded from the Pfam database (http://pfam-legacy.xfam.org/) [58]. Furthermore, the HMMER (v3.0) software [59] was used to search the predicted WD40 proteins using the cut-off value of the default parameter. Finally, the WD40 domain of all potential candidates was examined in InterPro (https://www.ebi.ac.uk/) [60] and SMART database (http://smart.embl-heidelberg.de/smart/batch.pl) [61], and those without the complete WD40 domain were manually deleted.

Sequence analysis and structural characteristics

Protein length, molecular weight, and theoretical pIs of CaWD40 proteins were analyzed using the Expasy tool (https://web.expasy.org/compute_pi/) [62]. The supposed subcellular localization of CaWD40 proteins was predicted using the online tool WOLF PSORT (https://wolfpsort.hgc.jp) [63]. The protein sequences were submitted to the MEME program (https://meme-suite.org/meme/tools/meme) [64] to assess conserved motifs.

Chromosome localization, tandem duplication, and synteny analysis

Chromosome locations and gene position in pepper were obtained by searching the Sol Genomics Network. Chromosome mapping of the CaWD40 gene family was visualized using MG2C (http://mg2c.iask.in/mg2c_v2.1) [65]. Tandem duplication events were further confirmed using the following criteria: (1) the alignment length had a coverage rate of more than 70% of the full length of the CaWD40 genes; (2) the identity of the aligned region was over 70%, (3) and an array of two or more genes was less than 100 kb distance. The genome sequence was downloaded and annotation files of three different pepper species from NCBI (https://www.ncbi.nlm.nih.gov/assembly/?term=pepper) were obtained. TBtools-II [66] was used to analyze the synteny of CaWD40 genes among the three pepper genomes.

Phylogenetic analysis

The phylogenetic tree was generated in the following three steps: first, the CaWD40 protein sequences were imported into Clustal X to produce a multiple sequence alignment file. Second, the alignment result was used to build an unrooted tree using MEGA11 with a bootstrap of 1000 replicates and neighbor-joining (NJ) methods [67]. Third, the newly produced phylogenetic tree was visualized using the Interactive Tree of Life online website (https://itol.embl.de/) [68].

RNA-Seq analysis of CaWD40 genes

Transcriptome sequencing (RNA-seq) data of development was used to explore the distribution of gene expression in pepper (the elite Capsicum line 6421) [69] to gain insight into the expression profiles of the CaWD40 gene family in different tissues across periods. Expression levels were determined in the following tissues and stages: leaf tissues were sampled at 2, 5, 10, 15, 20, 25, 30, 40, 50 and 60 days after emergence, and marked correspondingly as L1, L2, L3, L4, and L5, L6, and L7, L8, L9, and AL; floral buds were sampled at 0.25, 0.35, 0.5, 0.8, 1.0, 1.2, and 1.7 cm, and marked correspondingly as F1, F2, F3, F4, and F5, F6, and F7, F8, and F9; petals, stamens, and ovaries with stigmas were sampled in fully blossomed flowers, and marked correspondingly as P10, STA10 and O10; fruits were collected at 3, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 days after flowering (DAF), and marked correspondingly as FST0, FST1, G1, G2, G3, G4, G5, G6, G7, G8, G9, G10, G11; seed samples were collected at 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 DAF, and marked correspondingly as ST1, ST2, S3, S4, S5, S6, S7, S8, S9, S10, S11; placenta samples were collected at 20, 25, 30, 35, 40, 45, 50, 55 and 60 DAF, and marked correspondingly as T3, T4, T5, T6, T7, T8, T9, T10, and T11. Stems and roots were marked AS and AR, respectively. The treatment methods of all samples were based on those published by Liu et al. [69]. All data of CaWD40 genes were normalized (log2(FPKM + 1)), and a heatmap was drawn using TBtools-II (v2.019) [66].

RNA extraction and RT-qPCR analysis

Capsicum line 6421 was grown at 27 ℃/22 ℃ day/night cycles under a 16/8 h (light/dark) photoperiod. Total RNA was extracted using the TransZol Up (TransGen, China) and reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, China). RT-qPCR sample contained 2 × ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) and was performed on a LightCycle® 96 Real-Time PCR System (Roche, Switzerland).

The UBI-3 gene was used as a reference gene [70]. Gene-specific primers were designed. These are listed in Table S5. Three biological repeats and three technical repeats of RT-qPCR were set, and the relative expression of each gene was calculated using the 2−ΔΔCt method.

Y2H assays

Using specific primer pairs, the coding sequences of CaWD40-91, CaAN1, CaDYT1, and CaGL3 were amplified and inserted into pGBKT7 and pGADT7 vectors, respectively (Table S5). Four plasmids, pGBKT7-CaWD40-91, pGADT7-CaAN1, pGADT7-CaDYT1, and pGADT7-CaGL3, were extracted and co-transformed into Y2HGold receptor cells (WEIDI, China). All transformation products were plated and grown on SD/-Trp/-Leu medium for three days. Protein-protein interactions were detected on the SD/-Trp/-Leu/-His/-Ade selective medium following the instructions in the Matchmaker® Gold Yeast Two-Hybrid System User Manual.

Dual-luciferase reporter assays

The primers for Nluc-CaWD40-91, Cluc-CaAN1, and Cluc-CaDYT1 were designed (Table S5). CaWD40-91 was cloned and inserted into the pCambia1300-Nluc. CaAN1, and CaDYT1 were cloned and inserted into pCambia1300-Cluc linearized by KpnI and SalI. These recombinant plasmids were transferred into Agrobacterium tumefaciens GV3101. The plasmids were delivered into tobacco leaves, and a fluorescence signal was captured three days later using the Vilber Fusion FX7 Spectra device system (Vilber Bio Imaging, Paris, France). Nluc-EV and Cluc-EV were used as controls.

Subcellular localization

The full-length ORF sequences of CaWD40-91 without the termination codon were cloned into the pCAMBIA1300-GFP vector and transformed into Agrobacterium tumefaciens GV3101. Arabidopsis histone H2B-mCherry was used as a nuclear marker. CaWD40-91 fusion constructs and H2B marker were co-transformed into tobacco (Nicotiana benthamiana) leaves in a ratio of 1:1. After three days, the fluorescence signals were observed and captured using a confocal laser scanning microscope (Zeiss LSM510 META, Germany).

SEM analysis

Floral buds were sampled and fixed with a fixative solution. Samples were washed with 0.1 M PB (pH 7.4) thrice for 15 min each. The samples were transferred into 1% OsO4 in 0.1 M PB (pH 7.4) for 1–2 h at room temperature. Subsequently, samples were washed in 0.1 M PB (pH 7.4) thrice for 15 min each. Then, they were transferred to 30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100% ethanol for 15 min each. Finally, the samples were incubated in isoamyl acetate for 15 min and dried using a Critical Point Dryer. The dried samples were attached to metallic stubs using carbon stickers and sputter-coated with gold for 30 s. Samples were observed and photographs were captured with a scanning electron microscope (SU8100, Hitachi, Japan).

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Author contributions

F.L. & L.P gave the general idea of the paper. J.W & M.W were responsible for processing data and drawing figures. P.T & Q.H finished the experiment. P.T write the paper and J. H revised the context. All authors read and approved the final manuscript.

Funding

This research was supported by grants from the Science and Technology Innovation Program of Hunan Province, China (2021NK1006 and 2021JC0007).

Data availability

All methods using plant material were carried out in accordance with relevant guidelines and regulations in this paper. The data used and/or analyzed during the current study are obtained from the Sol Genomics Network (www.solgenomics.net/organism/Capsicum_annuum/genome) (Accession numbers: GCA_000512255.1, GCA_000710875.1); pepper Genomics Database (www.ted.bti.cornell.edu/cgi-bin/pepper/index) (Accession numbers: GCA_030867735.1).

Declarations

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Abbreviations

RT-qPCR Reverse transcription-Quantitative real-time PCR

HMM Hidden Markov Model

DAF Days after flowering

SMs Secondary metabolites

MWs Molecular weights

pIs Isoelectric point

NJ Neighbor-Joining

Y2H Yeast two-hybrid

SMART Simple Modular Architecture Research Tool

MEME Multiple Em for Motif Elicitation

LUC Luciferase

SEM Scanning electron microscopy

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peng Tang and Luzhao Pan contributed equally to this work.
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References

1. Neer EJ Schmidt CJ Nambudripad R Smith TF The ancient regulatory-protein family of WD-repeat proteins Nature 1994 371 297 300 10.1038/371297a0 8090199
Neer EJ, Schmidt CJ, Nambudripad R, Smith TF. The ancient regulatory-protein family of WD-repeat proteins. Nature. 1994;371:297–300.8090199 10.1038/371297a0
2. Smith TF Gaitatzes C Saxena K Neer EJ The WD repeat: a common architecture for diverse functions Trends Biochem Sci 1999 24 181 5 10.1016/S0968-0004(99)01384-5 10322433
Smith TF, Gaitatzes C, Saxena K, Neer EJ. The WD repeat: a common architecture for diverse functions. Trends Biochem Sci. 1999;24:181–5.10322433 10.1016/S0968-0004(99)01384-5
3. Nocker SV Ludwig P The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function BMC Genomics 2003 4 50 61 10.1186/1471-2164-4-50 14672542
Nocker SV, Ludwig P. The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function. BMC Genomics. 2003;4:50–61.14672542 10.1186/1471-2164-4-50
4. Mishra AK Puranik S Prasad M Structure and regulatory networks of WD40 protein in plants J Plant Biochem Biotechnol 2012 21 32 9 10.1007/s13562-012-0134-1
Mishra AK, Puranik S, Prasad M. Structure and regulatory networks of WD40 protein in plants. J Plant Biochem Biotechnol. 2012;21:32–9.10.1007/s13562-012-0134-1
5. Sondek J Lambright DG Hamm HE Sigler PB Bohm A Crystal structure of a G-protein beta gamma dimer at 2.1A resolution Nature 1996 379 369 74 10.1038/379369a0 8552196
Sondek J, Lambright DG, Hamm HE, Sigler PB, Bohm A. Crystal structure of a G-protein beta gamma dimer at 2.1A resolution. Nature. 1996;379:369–74.8552196 10.1038/379369a0
6. Xu C Min J Structure and function of WD40 domain proteins Protein Cell 2011 2 202 14 10.1007/s13238-011-1018-1 21468892
Xu C, Min J. Structure and function of WD40 domain proteins. Protein Cell. 2011;2:202–14.21468892 10.1007/s13238-011-1018-1
7. Haar ET Musacchio A Harrison SC Kirchhausen T Atomic structure of clathrin: a β propeller terminal domain joins an alpha zigzag linker Cell 1998 95 563 73 10.1016/S0092-8674(00)81623-2 9827808
Haar ET, Musacchio A, Harrison SC, Kirchhausen T. Atomic structure of clathrin: a β propeller terminal domain joins an alpha zigzag linker. Cell. 1998;95:563–73.9827808 10.1016/S0092-8674(00)81623-2
8. Chen M Zhang B Li C Kulaveerasingam H Chew FT Yu H TRANSPARENT TESTA GLABRA1 regulates the Accumulation of seed storage reserves in Arabidopsis Plant Physiol 2015 169 391 402 10.1104/pp.15.00943 26152712
Chen M, Zhang B, Li C, Kulaveerasingam H, Chew FT, Yu H. TRANSPARENT TESTA GLABRA1 regulates the Accumulation of seed storage reserves in Arabidopsis. Plant Physiol. 2015;169:391–402.26152712 10.1104/pp.15.00943
9. Jain BP Pandey S WD40 repeat proteins: signalling Scaffold with diverse functions Protein J 2018 37 391 406 10.1007/s10930-018-9785-7 30069656
Jain BP, Pandey S. WD40 repeat proteins: signalling Scaffold with diverse functions. Protein J. 2018;37:391–406.30069656 10.1007/s10930-018-9785-7
10. Song JJ Garlick JD Kingston RE Structural basis of histone H4 recognition by p55 Genes Dev 2008 10 1313 8 10.1101/gad.1653308
Song JJ, Garlick JD, Kingston RE. Structural basis of histone H4 recognition by p55. Genes Dev. 2008;10:1313–8.10.1101/gad.1653308
11. Higa LA Zhang H Stealing the spotlight: CUL4-DDB1 ubiquitin ligase docks WD40-repeat proteins to destroy Cell Div 2007 2 5 10.1186/1747-1028-2-5 17280619
Higa LA, Zhang H. Stealing the spotlight: CUL4-DDB1 ubiquitin ligase docks WD40-repeat proteins to destroy. Cell Div. 2007;2:5.17280619 10.1186/1747-1028-2-5
12. Tan L Salih H Htet N Azeem F Zhan R Genomic analysis of WD40 protein family in the mango reveals a TTG1 protein enhances root growth and abiotic tolerance in Arabidopsis Sci Rep 2021 11 2266 10.1038/s41598-021-81969-z 33500544
Tan L, Salih H, Htet N, Azeem F, Zhan R. Genomic analysis of WD40 protein family in the mango reveals a TTG1 protein enhances root growth and abiotic tolerance in Arabidopsis. Sci Rep. 2021;11:2266.33500544 10.1038/s41598-021-81969-z
13. Gonzalez A Zhao M Leavitt JM Lloyd AM Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings Plant J 2008 53 814 27 10.1111/j.1365-313X.2007.03373.x 18036197
Gonzalez A, Zhao M, Leavitt JM, Lloyd AM. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 2008;53:814–27.18036197 10.1111/j.1365-313X.2007.03373.x
14. Zhao M Morohashi K Hatlestad G Grotewold E Lloyd A The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci Development 2008 135 1991 9 10.1242/dev.016873 18434419
Zhao M, Morohashi K, Hatlestad G, Grotewold E, Lloyd A. The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci. Development. 2008;135:1991–9.18434419 10.1242/dev.016873
15. Baudry A Heim MA Dubreucq B Caboche M Weisshaar B Lepiniec L TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana Plant J 2004 39 366 80 10.1111/j.1365-313X.2004.02138.x 15255866
Baudry A, Heim MA, Dubreucq B, Caboche M, Weisshaar B, Lepiniec L. TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J. 2004;39:366–80.15255866 10.1111/j.1365-313X.2004.02138.x
16. Xu W Dubos C Lepiniec L Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes Trends Plant Sci 2015 20 176 85 10.1016/j.tplants.2014.12.001 25577424
Xu W, Dubos C, Lepiniec L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci. 2015;20:176–85.25577424 10.1016/j.tplants.2014.12.001
17. Wang J, Dai Y, Pan L, Chen Y, Dai L, Ma Y, Zhou X, Miao W, Hamid MR, Zou X, Liu F, Xiong C. Fine mapping and identification of CaTTG1, a candidate gene that regulates the hypocotyl anthocyanin accumulation in Capsicum annuum L. Hortic Plant J. 2023.
18. Cai J Huang H Xu X Zhu G An Arabidopsis WD40 repeat-containing protein XIW1 promotes salt inhibition of seed germination Plant Signal Behav 2020 15 1712542 10.1080/15592324.2020.1712542 31914847
Cai J, Huang H, Xu X, Zhu G. An Arabidopsis WD40 repeat-containing protein XIW1 promotes salt inhibition of seed germination. Plant Signal Behav. 2020;15:1712542.31914847 10.1080/15592324.2020.1712542
19. Yan J Ma Z Xu X Guo AY Evolution, functional divergence and conserved exon-intron structure of bHLH/PAS gene family Mol Genet Genomics 2014 289 25 36 10.1007/s00438-013-0786-0 24202550
Yan J, Ma Z, Xu X, Guo AY. Evolution, functional divergence and conserved exon-intron structure of bHLH/PAS gene family. Mol Genet Genomics. 2014;289:25–36.24202550 10.1007/s00438-013-0786-0
20. Bjerkan KN Jung-Roméo S Jürgens G Grini GPE Arabidopsis WD REPEAT DOMAIN55 interacts with DNA DAMAGED BINDING PROTEIN1 and is required for apical patterning in the embryo Plant Cell 2012 24 1013 33 10.1105/tpc.111.089425 22447688
Bjerkan KN, Jung-Roméo S, Jürgens G, Grini GPE, Arabidopsis. WD REPEAT DOMAIN55 interacts with DNA DAMAGED BINDING PROTEIN1 and is required for apical patterning in the embryo. Plant Cell. 2012;24:1013–33.22447688 10.1105/tpc.111.089425
21. Shi DQ Liu J Xiang YH Ye D Sundaresan V Yang WC SLOW WALKER1, essential for gametogenesis in Arabidopsis, encodes a WD40 protein involved in 18S ribosomal RNA biogenesis Plant Cell 2005 17 2340 54 10.1105/tpc.105.033563 15980260
Shi DQ, Liu J, Xiang YH, Ye D, Sundaresan V, Yang WC. SLOW WALKER1, essential for gametogenesis in Arabidopsis, encodes a WD40 protein involved in 18S ribosomal RNA biogenesis. Plant Cell. 2005;17:2340–54.15980260 10.1105/tpc.105.033563
22. Grigorieva G Shestakov S Transformation in the cyanobacterium Synechocystis sp.6803 FEMS Microbiol Lett 1982 13 367 70 10.1111/j.1574-6968.1982.tb08289.x
Grigorieva G, Shestakov S. Transformation in the cyanobacterium Synechocystis sp.6803. FEMS Microbiol Lett. 1982;13:367–70.10.1111/j.1574-6968.1982.tb08289.x
23. Janda L Tichý P Spízek J Petrícek M A deduced thermomonospora curvata protein containing serine/threonine protein kinase and WD-repeat domains J Bacteriol 1996 178 1487 9 10.1128/jb.178.5.1487-1489.1996 8631732
Janda L, Tichý P, Spízek J, Petrícek M. A deduced thermomonospora curvata protein containing serine/threonine protein kinase and WD-repeat domains. J Bacteriol. 1996;178:1487–9.8631732 10.1128/jb.178.5.1487-1489.1996
24. Zou XD Hu XJ Ma J Li T Ye ZQ Wu YD Genome-wide analysis of WD40 protein family in human Sci Rep 2016 6 39262 10.1038/srep39262 27991561
Zou XD, Hu XJ, Ma J, Li T, Ye ZQ, Wu YD. Genome-wide analysis of WD40 protein family in human. Sci Rep. 2016;6:39262.27991561 10.1038/srep39262
25. Hu R Xiao J Gu T Yu X Zhang Y Chang J Yang G He G Correction to: genome-wide identification and analysis of WD40 proteins in wheat (Triticum aestivum L) BMC Genomics 2018 19 852 65 10.1186/s12864-018-5252-2 30497365
Hu R, Xiao J, Gu T, Yu X, Zhang Y, Chang J, Yang G, He G. Correction to: genome-wide identification and analysis of WD40 proteins in wheat (Triticum aestivum L). BMC Genomics. 2018;19:852–65.30497365 10.1186/s12864-018-5252-2
26. Ouyang Y Huang X Lu Z Yao J Genomic survey, expression profile and co-expression network analysis of OsWD40 family in rice BMC Genomics 2012 13 100 13 10.1186/1471-2164-13-100 22429805
Ouyang Y, Huang X, Lu Z, Yao J. Genomic survey, expression profile and co-expression network analysis of OsWD40 family in rice. BMC Genomics. 2012;13:100–13.22429805 10.1186/1471-2164-13-100
27. Li Q Zhao P Li J Zhang C Wang L Ren Z Genome-wide analysis of the WD-repeat protein family in cucumber and Arabidopsis Mol Genet Genomics 2014 289 103 24 10.1007/s00438-013-0789-x 24292651
Li Q, Zhao P, Li J, Zhang C, Wang L, Ren Z. Genome-wide analysis of the WD-repeat protein family in cucumber and Arabidopsis. Mol Genet Genomics. 2014;289:103–24.24292651 10.1007/s00438-013-0789-x
28. Feng R Zhang C Ma R Cai Z Lin Y Yu M Identification and characterization of WD40 superfamily genes in peach Gene 2019 710 291 306 10.1016/j.gene.2019.06.010 31185283
Feng R, Zhang C, Ma R, Cai Z, Lin Y, Yu M. Identification and characterization of WD40 superfamily genes in peach. Gene. 2019;710:291–306.31185283 10.1016/j.gene.2019.06.010
29. Chen L Cui Y Yao Y An L Bai Y Li X Yao X Wu K Genome-wide identification of WD40 transcription factors and their regulation of the MYB-bHLH-WD40 (MBW) complex related to anthocyanin synthesis in Qingke (Hordeum vulgare L. var. Nudum hook. F) BMC Genomics 2023 24 166 10.1186/s12864-023-09240-5 37016311
Chen L, Cui Y, Yao Y, An L, Bai Y, Li X, Yao X, Wu K. Genome-wide identification of WD40 transcription factors and their regulation of the MYB-bHLH-WD40 (MBW) complex related to anthocyanin synthesis in Qingke (Hordeum vulgare L. var. Nudum hook. F). BMC Genomics. 2023;24:166.37016311 10.1186/s12864-023-09240-5
30. Carolina CG Barfuss MHJ Sehr EM Barboza GE Rosabelle S Moscone EA Friedrich E Phylogenetic relationships, diversification and expansion of Chili peppers (Capsicum, Solanaceae) Ann Botany 2016 118 35 51 10.1093/aob/mcw079 27245634
Carolina CG, Barfuss MHJ, Sehr EM, Barboza GE, Rosabelle S, Moscone EA, Friedrich E. Phylogenetic relationships, diversification and expansion of Chili peppers (Capsicum, Solanaceae). Ann Botany. 2016;118:35–51.27245634 10.1093/aob/mcw079
31. Liu F Zhao J Sun H Xiong C Sun X Wang X Wang Z Jarret R Wang J Tang B Xu H Hu B Suo H Yang B Ou L Li X Zhou S Yang S Liu Z Yuan F Pei Z Ma Y Dai X Wu S Fei Z Zou X Genomes of cultivated and wild Capsicum species provide insights into pepper domestication and population differentiation Nat Commun 2023 14 5487 10.1038/s41467-023-41251-4 37679363
Liu F, Zhao J, Sun H, Xiong C, Sun X, Wang X, Wang Z, Jarret R, Wang J, Tang B, Xu H, Hu B, Suo H, Yang B, Ou L, Li X, Zhou S, Yang S, Liu Z, Yuan F, Pei Z, Ma Y, Dai X, Wu S, Fei Z, Zou X. Genomes of cultivated and wild Capsicum species provide insights into pepper domestication and population differentiation. Nat Commun. 2023;14:5487.37679363 10.1038/s41467-023-41251-4
32. Zhang Z Liu Y Yuan Q Xiong C Xu H Hu B Suo H Yang S Hou X Yuan F The bHLH1-DTX35/DFR module regulates pollen fertility by promoting flavonoid biosynthesis in Capsicum annuum L Hortic Res 2022 9 3458 69 10.1093/hr/uhac172
Zhang Z, Liu Y, Yuan Q, Xiong C, Xu H, Hu B, Suo H, Yang S, Hou X, Yuan F, et al. The bHLH1-DTX35/DFR module regulates pollen fertility by promoting flavonoid biosynthesis in Capsicum annuum L. Hortic Res. 2022;9:3458–69.10.1093/hr/uhac172
33. Byun J Kim TG Lee JH Li N Jung S Kang BC Identification of CaAN3 as a fruit-specific regulator of anthocyanin biosynthesis in pepper (Capsicum annuum) Theor Appl Genet 2022 135 2197 211 10.1007/s00122-022-04106-y 35536305
Byun J, Kim TG, Lee JH, Li N, Jung S, Kang BC. Identification of CaAN3 as a fruit-specific regulator of anthocyanin biosynthesis in pepper (Capsicum annuum). Theor Appl Genet. 2022;135:2197–211.35536305 10.1007/s00122-022-04106-y
34. Liu J Ai X Wang Y Lu Q Li T Wu L Sun L Shen H Fine mapping of the Ca3GT gene controlling anthocyanin biosynthesis in mature unripe fruit of Capsicum annuum L Theor Appl Genet 2020 133 2729 42 10.1007/s00122-020-03628-7 32564095
Liu J, Ai X, Wang Y, Lu Q, Li T, Wu L, Sun L, Shen H. Fine mapping of the Ca3GT gene controlling anthocyanin biosynthesis in mature unripe fruit of Capsicum annuum L. Theor Appl Genet. 2020;133:2729–42.32564095 10.1007/s00122-020-03628-7
35. Chen R Yang C Gao H Shi C Zhang Z Lu G Shen X Tang Y Li F Lu Y Ouyang B Induced mutation in ELONGATED HYPOCOTYL5 abolishes anthocyanin accumulation in the hypocotyl of pepper Theor Appl Genet 2022 135 3455 68 10.1007/s00122-022-04192-y 35963933
Chen R, Yang C, Gao H, Shi C, Zhang Z, Lu G, Shen X, Tang Y, Li F, Lu Y, Ouyang B. Induced mutation in ELONGATED HYPOCOTYL5 abolishes anthocyanin accumulation in the hypocotyl of pepper. Theor Appl Genet. 2022;135:3455–68.35963933 10.1007/s00122-022-04192-y
36. Kim YJ Kim MH Hong WJ Moon S Kim EJ Silva J Lee J Lee S Kim ST Park SK GORI, encoding the WD40 domain protein, is required for pollen tube germination and elongation in rice Plant J 2021 105 1645 64 10.1111/tpj.15139 33345419
Kim YJ, Kim MH, Hong WJ, Moon S, Kim EJ, Silva J, Lee J, Lee S, Kim ST, Park SK, et al. GORI, encoding the WD40 domain protein, is required for pollen tube germination and elongation in rice. Plant J. 2021;105:1645–64.33345419 10.1111/tpj.15139
37. Tian G Wang S Wu J Allelic variation of TaWD40-4B.1 contributes to drought tolerance by modulating catalase activity in wheat Nat Commun 2023 1200 36901 6
Tian G, Wang S, Wu J, et al. Allelic variation of TaWD40-4B.1 contributes to drought tolerance by modulating catalase activity in wheat. Nat Commun. 2023;1200:36901–6.
38. Liu H Xiu ZH Yang HH Ma ZX Yang DL Wang HQ Tan BC Maize Shrek1 encodes a WD40 protein that regulates pre-rRNA processing in ribosome biogenesis Plant Cell 2022 34 4028 44 10.1093/plcell/koac216 35867001
Liu H, Xiu ZH, Yang HH, Ma ZX, Yang DL, Wang HQ, Tan BC. Maize Shrek1 encodes a WD40 protein that regulates pre-rRNA processing in ribosome biogenesis. Plant Cell. 2022;34:4028–44.35867001 10.1093/plcell/koac216
39. Airoldi CA Hearn TJ Brockington SF TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation Nat Plants 2019 5 1145 53 10.1038/s41477-019-0544-3 31712761
Airoldi CA, Hearn TJ, Brockington SF, et al. TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation. Nat Plants. 2019;5:1145–53.31712761 10.1038/s41477-019-0544-3
40. Wang Z Yang Z Li F Updates on molecular mechanisms in the development of branched trichome in Arabidopsis and nonbranched in cotton Plant Biotechnol J 2019 17 1706 22 10.1111/pbi.13167 31111642
Wang Z, Yang Z, Li F. Updates on molecular mechanisms in the development of branched trichome in Arabidopsis and nonbranched in cotton. Plant Biotechnol J. 2019;17:1706–22.31111642 10.1111/pbi.13167
41. Yan C Yang T Wang B Yang H Wang J Yu Q Genome-wide identification of the WD40 Gene Family in Tomato (Solanum lycopersicum L) Genes (Basel) 2023 14 1273 10.3390/genes14061273 37372453
Yan C, Yang T, Wang B, Yang H, Wang J, Yu Q. Genome-wide identification of the WD40 Gene Family in Tomato (Solanum lycopersicum L). Genes (Basel). 2023;14:1273.37372453 10.3390/genes14061273
42. Schapira M Tyers M Torrent M Arrowsmith CH WD40 repeat domain proteins: a novel target class? Nat Rev Drug Discov 2017 16 773 86 10.1038/nrd.2017.179 29026209
Schapira M, Tyers M, Torrent M, Arrowsmith CH. WD40 repeat domain proteins: a novel target class? Nat Rev Drug Discov. 2017;16:773–86.29026209 10.1038/nrd.2017.179
43. Stracke R Werber M Weisshaar B The R2R3-MYB gene family in Arabidopsis thaliana Curr Opin Plant Biol 2001 4 447 56 10.1016/S1369-5266(00)00199-0 11597504
Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol. 2001;4:447–56.11597504 10.1016/S1369-5266(00)00199-0
44. Li X Xue C Li J Qiao X Li L Yu L Huang Y Wu J Genome-wide identification, evolution and functional divergence of MYB Transcription Factors in Chinese White Pear (Pyrus Bretschneideri) Plant Cell Physiol 2016 57 824 47 10.1093/pcp/pcw029 26872835
Li X, Xue C, Li J, Qiao X, Li L, Yu L, Huang Y, Wu J. Genome-wide identification, evolution and functional divergence of MYB Transcription Factors in Chinese White Pear (Pyrus Bretschneideri). Plant Cell Physiol. 2016;57:824–47.26872835 10.1093/pcp/pcw029
45. Liu J Wang X Chen Y Liu Y Wu Y Ren S Li L Identification, evolution and expression analysis of WRKY gene family in Eucommia ulmoides Genomics 2021 113 3294 309 10.1016/j.ygeno.2021.05.011 34022347
Liu J, Wang X, Chen Y, Liu Y, Wu Y, Ren S, Li L. Identification, evolution and expression analysis of WRKY gene family in Eucommia ulmoides. Genomics. 2021;113:3294–309.34022347 10.1016/j.ygeno.2021.05.011
46. Feller A Machemer K Braun EL Grotewold E Evolutionary and comparative analysis of MYB and bHLH plant transcription factors Plant J 2011 66 94 116 10.1111/j.1365-313X.2010.04459.x 21443626
Feller A, Machemer K, Braun EL, Grotewold E. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J. 2011;66:94–116.21443626 10.1111/j.1365-313X.2010.04459.x
47. Gou JY Felippes FF Liu CJ Weigel D Wang JW Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor Plant Cell 2011 23 1512 22 10.1105/tpc.111.084525 21487097
Gou JY, Felippes FF, Liu CJ, Weigel D, Wang JW. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell. 2011;23:1512–22.21487097 10.1105/tpc.111.084525
48. Xue JS Qiu S Jia XL Shen SY Shen CW Wang S Xu P Tong Q Lou YX Yang NY Cao JG Hu JF Shen H Zhu RL Murray JD Chen WS Yang ZN Stepwise changes in flavonoids in spores/pollen contributed to terrestrial adaptation of plants Plant Physiol 2023 193 627 42 10.1093/plphys/kiad313 37233029
Xue JS, Qiu S, Jia XL, Shen SY, Shen CW, Wang S, Xu P, Tong Q, Lou YX, Yang NY, Cao JG, Hu JF, Shen H, Zhu RL, Murray JD, Chen WS, Yang ZN. Stepwise changes in flavonoids in spores/pollen contributed to terrestrial adaptation of plants. Plant Physiol. 2023;193:627–42.37233029 10.1093/plphys/kiad313
49. Buer CS Imin N Djordjevic MA Flavonoids: new roles for old molecules J Integr Plant Biol 2010 52 98 111 10.1111/j.1744-7909.2010.00905.x 20074144
Buer CS, Imin N, Djordjevic MA. Flavonoids: new roles for old molecules. J Integr Plant Biol. 2010;52:98–111.20074144 10.1111/j.1744-7909.2010.00905.x
50. Lepiniec LC Debeaujon I Routaboul JM Baudry A Pourcel L Nesi N Caboche M Genetics and biochemistry of seed flavonoids Annu Rev Plant Biol 2006 57 405 30 10.1146/annurev.arplant.57.032905.105252 16669768
Lepiniec LC, Debeaujon I, Routaboul JM, Baudry A, Pourcel L, Nesi N, Caboche M. Genetics and biochemistry of seed flavonoids. Annu Rev Plant Biol. 2006;57:405–30.16669768 10.1146/annurev.arplant.57.032905.105252
51. Grunewald S Marillonnet S Hause G Haferkamp I Neuhaus HE Veß A Hollemann T Vogt T The tapetal major facilitator NPF2.8 is required for accumulation of flavonol glycosides on the pollen surface in Arabidopsis thaliana Plant Cell 2020 32 1727 48 10.1105/tpc.19.00801 32156687
Grunewald S, Marillonnet S, Hause G, Haferkamp I, Neuhaus HE, Veß A, Hollemann T, Vogt T. The tapetal major facilitator NPF2.8 is required for accumulation of flavonol glycosides on the pollen surface in Arabidopsis thaliana. Plant Cell. 2020;32:1727–48.32156687 10.1105/tpc.19.00801
52. Muhlemann JK Younts TLB Muday GK Flavonols control pollen tube growth and integrity by regulating ROS homeostasis during high-temperature stress Proc Natl Acad Sci U S A 2018 115 E11188 97 10.1073/pnas.1811492115 30413622
Muhlemann JK, Younts TLB, Muday GK. Flavonols control pollen tube growth and integrity by regulating ROS homeostasis during high-temperature stress. Proc Natl Acad Sci U S A. 2018;115:E11188–97.30413622 10.1073/pnas.1811492115
53. Rutley N Miller G Wang F Harper JF Miller G Lieberman-Lazarovich M Enhanced reproductive thermotolerance of the tomato high pigment 2 mutant is associated with increased accumulation of flavonols in pollen Front Plant Sci 2021 12 672368 10.3389/fpls.2021.672368 34093629
Rutley N, Miller G, Wang F, Harper JF, Miller G, Lieberman-Lazarovich M. Enhanced reproductive thermotolerance of the tomato high pigment 2 mutant is associated with increased accumulation of flavonols in pollen. Front Plant Sci. 2021;12:672368.34093629 10.3389/fpls.2021.672368
54. Cui J You C Zhu E Huang Q Ma H Chang F Feedback regulation of DYT1 by interactions with downstream bHLH factors promotes DYT1 Nuclear localization and Anther Development Plant Cell 2016 28 1078 93 10.1105/tpc.15.00986 27113773
Cui J, You C, Zhu E, Huang Q, Ma H, Chang F. Feedback regulation of DYT1 by interactions with downstream bHLH factors promotes DYT1 Nuclear localization and Anther Development. Plant Cell. 2016;28:1078–93.27113773 10.1105/tpc.15.00986
55. Cheng Q Wang P Liu, Jinqiu, Wu L Zhang Z Theoretical Appl Genet Int J Breed Res Cell Genet 2018 131 1861 72
Cheng Q, Wang P, Liu, Jinqiu, Wu L, Zhang Z, Theoretical. Appl Genet Int J Breed Res Cell Genet. 2018;131:1861–72.
56. Qin C, Yu C, Shen Y, Fang X, Chen L, Min J, Cheng J, Zhao S, Xu M, Luo Y, Yang Y, Wu Z, Mao L, Wu H, Ling-Hu C, Zhou H, Lin H, González-Morales S, Trejo-Saavedra DL, Tian H, Tang X, Zhao M, Huang Z, Zhou A, Yao X, Cui J, Li W, Chen Z, Feng Y, Niu Y, Bi S, Yang X, Li W, Cai H, Luo X, Montes-Hernández S, Leyva-González MA, Xiong Z, He X, Bai L, Tan S, Tang X, Liu D, Liu J, Zhang S, Chen M, Zhang L, Zhang L, Zhang Y, Liao W, Zhang Y, Wang M, Lv X, Wen B, Liu H, Luan H, Zhang Y, Yang S, Wang X, Xu J, Li X, Li S, Wang J, Palloix A, Bosland PW, Li Y, Krogh A, Rivera-Bustamante RF, Herrera-Estrella L, Yin Y, Yu J, Hu K, Zhang Z. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Volume 111. Proc Natl Acad Sci U S A; 2014. pp. 5135–40.
57. Kim S Park M Yeom SI Kim YM Lee JM Lee HA Seo E Choi J Cheong K Kim KT Jung K Lee GW Oh SK Bae C Kim SB Lee HY Kim SY Kim MS Kang BC Jo YD Yang HB Jeong HJ Kang WH Kwon JK Shin C Lim JY Park JH Huh JH Kim JS Kim BD Cohen O Paran I Suh MC Lee SB Kim YK Shin Y Noh SJ Park J Seo YS Kwon SY Kim HA Park JM Kim HJ Choi SB Bosland PW Reeves G Jo SH Lee BW Cho HT Choi HS Lee MS Yu Y Do Choi Y Park BS van Deynze A Ashrafi H Hill T Kim WT Pai HS Ahn HK Yeam I Giovannoni JJ Rose JK Sørensen I Lee SJ Kim RW Choi IY Choi BS Lim JS Lee YH Choi D Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species Nat Genet 2014 46 270 8 10.1038/ng.2877 24441736
Kim S, Park M, Yeom SI, Kim YM, Lee JM, Lee HA, Seo E, Choi J, Cheong K, Kim KT, Jung K, Lee GW, Oh SK, Bae C, Kim SB, Lee HY, Kim SY, Kim MS, Kang BC, Jo YD, Yang HB, Jeong HJ, Kang WH, Kwon JK, Shin C, Lim JY, Park JH, Huh JH, Kim JS, Kim BD, Cohen O, Paran I, Suh MC, Lee SB, Kim YK, Shin Y, Noh SJ, Park J, Seo YS, Kwon SY, Kim HA, Park JM, Kim HJ, Choi SB, Bosland PW, Reeves G, Jo SH, Lee BW, Cho HT, Choi HS, Lee MS, Yu Y, Do Choi Y, Park BS, van Deynze A, Ashrafi H, Hill T, Kim WT, Pai HS, Ahn HK, Yeam I, Giovannoni JJ, Rose JK, Sørensen I, Lee SJ, Kim RW, Choi IY, Choi BS, Lim JS, Lee YH, Choi D. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat Genet. 2014;46:270–8.24441736 10.1038/ng.2877
58. El-Gebali S Mistry J Bateman A Eddy SR Luciani A Potter SC The pfam protein families database in 2019 Nucleic Acids Res 2019 47 D427 32 10.1093/nar/gky995 30357350
El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, et al. The pfam protein families database in 2019. Nucleic Acids Res. 2019;47:D427–32.30357350 10.1093/nar/gky995
59. Wheeler TJ Eddy SR Nhmmer: DNA homology search with profile HMMs Bioinformatics 2013 29 2487 9 10.1093/bioinformatics/btt403 23842809
Wheeler TJ, Eddy SR. Nhmmer: DNA homology search with profile HMMs. Bioinformatics. 2013;29:2487–9.23842809 10.1093/bioinformatics/btt403
60. Paysan-Lafosse T Blum M Chuguransky S Grego T Pinto BL Salazar GA Bileschi ML Bork P Bridge A Colwell L Gough J Haft DH Letunić I Marchler-Bauer A Mi H Natale DA Orengo CA Pandurangan AP Rivoire C Sigrist CJA Sillitoe I Thanki N Thomas PD Tosatto SCE Wu CH Bateman A InterPro in 2022 Nucleic Acids Res 2023 51 D418 27 10.1093/nar/gkac993 36350672
Paysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, Bileschi ML, Bork P, Bridge A, Colwell L, Gough J, Haft DH, Letunić I, Marchler-Bauer A, Mi H, Natale DA, Orengo CA, Pandurangan AP, Rivoire C, Sigrist CJA, Sillitoe I, Thanki N, Thomas PD, Tosatto SCE, Wu CH, Bateman A. InterPro in 2022. Nucleic Acids Res. 2023;51:D418–27.36350672 10.1093/nar/gkac993
61. Letunic I Bork P 20 years of the SMART protein domain annotation resource Nucleic Acids Res 2018 46 D493 6 10.1093/nar/gkx922 29040681
Letunic I, Bork P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 2018;46:D493–6.29040681 10.1093/nar/gkx922
62. Artimo P Jonnalagedda M Arnold K Baratin D Csardi G de Castro E ExPASy: SIB bioinformatics resource portal Nucleic Acids Res 2012 40 W597 603 10.1093/nar/gks400 22661580
Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 2012;40:W597–603.22661580 10.1093/nar/gks400
63. Horton P Park K-J Obayashi T Fujita N Harada H Adams-Collier CJ Nakai K WoLF PSORT: protein localization predictor Nucleic Acids Res 2007 35 585 7 10.1093/nar/gkm259
Horton P, Park K-J, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, Nakai K. WoLF PSORT: protein localization predictor. Nucleic Acids Res. 2007;35:585–7.10.1093/nar/gkm259
64. Bailey TL Elkan C Fitting a mixture model by expectation maximization to discover motifs in biopolymers Proc Int Conf Intell Syst Mol Biol 1994 2 28 36 7584402
Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol. 1994;2:28–36.7584402
65. Chao JT Kong YZ Wang Q Sun YH Gong DP Lv J MapGene2Chrom, a tool to draw gene physical map based on Perl and SVG languages Yi Chuan 2015 37 91 7 25608819
Chao JT, Kong YZ, Wang Q, Sun YH, Gong DP, Lv J, et al. MapGene2Chrom, a tool to draw gene physical map based on Perl and SVG languages. Yi Chuan. 2015;37:91–7.25608819
66. Chen C Chen H Zhang Y Thomas HR Frank MH He Y Xia R TBtools: an integrative Toolkit developed for interactive analyses of big Biological Data Mol Plant 2020 13 1194 202 10.1016/j.molp.2020.06.009 32585190
Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: an integrative Toolkit developed for interactive analyses of big Biological Data. Mol Plant. 2020;13:1194–202.32585190 10.1016/j.molp.2020.06.009
67. Tamura K Stecher G Kumar S MEGA11: Molecular Evolutionary Genetics Analysis Version 11 Mol Biol Evol 2021 38 3022 7 10.1093/molbev/msab120 33892491
Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. 2021;38:3022–7.33892491 10.1093/molbev/msab120
68. Letunic I Bork P Interactive tree of life (iTOL) v5: an online tool for phylogenetic tree display and annotation Nucleic Acids Res 2021 49 W293 6 10.1093/nar/gkab301 33885785
Letunic I, Bork P. Interactive tree of life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49:W293–6.33885785 10.1093/nar/gkab301
69. Liu F Yu H Deng Y Zheng J Liu M Ou L Yang B Dai X Ma Y Feng S He S Li X Zhang Z Chen W Zhou S Chen R Liu M Yang S Wei R Li H Li F Ouyang B Zou X PepperHub, an Informatics Hub for the Chili Pepper Research Community Mol Plant 2017 10 1129 32 10.1016/j.molp.2017.03.005 28343897
Liu F, Yu H, Deng Y, Zheng J, Liu M, Ou L, Yang B, Dai X, Ma Y, Feng S, He S, Li X, Zhang Z, Chen W, Zhou S, Chen R, Liu M, Yang S, Wei R, Li H, Li F, Ouyang B, Zou X. PepperHub, an Informatics Hub for the Chili Pepper Research Community. Mol Plant. 2017;10:1129–32.28343897 10.1016/j.molp.2017.03.005
70. Wan H Yuan W Ruan M Ye Q Wang R Li Z Zhou G Yao Z Zhao J Liu S Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper (Capsicum annuum L) Biochem Biophys Res Commun 2011 416 24 30 10.1016/j.bbrc.2011.10.105 22086175
Wan H, Yuan W, Ruan M, Ye Q, Wang R, Li Z, Zhou G, Yao Z, Zhao J, Liu S. Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper (Capsicum annuum L). Biochem Biophys Res Commun. 2011;416:24–30.22086175 10.1016/j.bbrc.2011.10.105
