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Article

Integrated Phylogenomic and Expression Analyses Reveal Lineage-Specific Loss of the Mβ Subfamily and Regulatory Diversification of MADS-Box Genes in Pepper

by
Jiajun Zhu
1,2,†,
Shibo Meng
1,2,†,
Jia Liu
1,
Ting Zhang
3,
Yuan Cheng
1,
Meiying Ruan
1,
Qingjing Ye
1,
Rongqing Wang
1,
Zhuping Yao
1,
Guozhi Zhou
1,
Zhimiao Li
1,
Chenxu Liu
1 and
Hongjian Wan
1,*
1
State Key Laboratory for Quality and Safety of Agro-Products, Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2
School of Horticulture, Anhui Agricultural University, Hefei 230036, China
3
Institute of Vegetables, Quzhou Academy of Agricultural and Forestry Sciences, Quzhou 324000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2026, 15(4), 620; https://doi.org/10.3390/plants15040620
Submission received: 21 January 2026 / Revised: 9 February 2026 / Accepted: 13 February 2026 / Published: 15 February 2026
(This article belongs to the Special Issue Plant Stress Responses: Molecular Genetics and Enzyme Regulation)
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Abstract

MADS-box transcription factors are key regulators of plant development and environmental responses. Here, we performed an integrated phylogenomic and expression analysis of the MADS-box gene family in Capsicum annuum, identifying 97 members that fall into 52 Type I and 45 Type II genes. Comparative phylogeny, exon–intron organization, conserved motifs, and chromosomal mapping allowed classification into 15 subfamilies. Gene duplication analysis revealed that segmental duplication has been a major driver of family expansion. Expression profiling across multiple tissues, together with promoter cis-element prediction and stress-responsive transcriptome data, demonstrated that Type II genes exhibit broad and dynamic expression patterns, particularly under ABA treatment and temperature stress. A key finding of this study is the complete absence of the Mβ lineage, a Type I subfamily typically associated with gametophyte and endosperm development in other angiosperms. No Mβ-like sequences were detected in the pepper genome, and Type I genes overall showed extremely low expression, suggesting that the Mβ lineage has undergone lineage-specific evolutionary loss and that its functions may be compensated by other Type I members or by expanded Type II regulatory modules. Together, this study provides the first evidence for the evolutionary disappearance of the Mβ subfamily in Capsicum and offers a comprehensive resource for dissecting the developmental and stress-responsive roles of MADS-box genes in pepper.

1. Introduction

Transcription factors (TFs) are sequence-specific DNA-binding proteins that fine-tune downstream gene expression and thereby regulate nearly all biological processes [1]. Among these, the MADS-box family represents one of the most ancient and widely distributed TF families in eukaryotes [2]. The acronym “MADS” originates from the first four members identified: MCM1 in yeast [3], AGAMOUS in Arabidopsis [4], DEFICIENS in snapdragon [5], and SRF in humans [6]. All MADS-box proteins share a highly conserved 58–60 aa DNA-binding MADS domain at the N-terminus. Based on domain organisation and phylogenetic relationships, plant MADS-box genes are classified into two monophyletic lineages: Type I and Type II [7]. Type I genes typically contain a single exon and encode proteins with an SRF-like MADS domain, whereas Type II genes harbour 2–12 exons and possess four characteristic domains—the MADS (M), Intervening (I), Keratin-like (K) and C-terminal (C) regions. Type I genes, also known as M-type, can be further subdivided into Mα, Mβ and Mγ, while Type II genes comprise the MIKCc and MIKC* subclades [8]. Recent analyses have, however, proposed the reassignment of MIKC* genes into the Type I lineage [9]. Among all groups, MIKCc genes are the best characterised due to their repeated implication in developmental regulation, with 39 members in Arabidopsis [10], 38 in rice [11] and 50 in tomato [12].
In plants, MADS-box TFs orchestrate a wide array of processes related to growth, development and environmental adaptation [13]. Type I genes predominantly function in female gametophyte development (e.g., AGL80, AGL61) and seed formation (e.g., PHE1/2, AGL23, AGL28, AGL40, AGL62) [14,15,16,17], whereas the Type II lineage has been studied extensively due to its key roles in reproductive development. The canonical ABCDE model exemplifies their influence on floral organ identity: A-class genes (AP1) specify sepals and petals [18]; B-class genes (AP3, PI) determine petals and stamens [19]; the C-class gene AG specifies stamens and carpels [20]; the D-class gene STK regulates ovule identity [21]; and E-class SEP1–4 genes interact with other classes to specify all floral organs [22]. Beyond floral patterning, MIKCc members regulate flowering time (SOC1, FLC, SVP, AGL24) [23,24,25,26,27,28,29,30], fruit ripening (RIN) [31,32], embryogenesis (TT16) [33], and root development (AGL12, AGL44) [34,35].
Plant growth and development are further shaped by hormonal cues and environmental factors. For example, PsAGL9 participates in the GA-mediated release of bud dormancy in peony, influencing floral initiation [36]. In cucumber, the CsAGL16–CsGRF1 regulatory module promotes axillary bud outgrowth by modulating ABA metabolism via CsCYP707A4 [37]. ABA itself inhibits vegetative growth in parthenocarpic plants and affects fruit development in the absence of fertilization [38]. In Arabidopsis, vernalization-induced epigenetic repression of FLC releases AGL24 from inhibition, thereby promoting flowering [25]. In monocots, MADS-box genes also mediate abiotic stress responses: OsMADS18, OsMADS22, OsMADS26, and OsMADS27 are induced by cold and dehydration in rice, whereas OsMADS87 responds strongly to heat [39]. Similarly, MADS-box genes in alfalfa are upregulated under cold stress [40]. In tomato, loss of SlMADS23-like compromises cold tolerance, indicating its positive role in stress adaptation [41]. Despite such extensive knowledge from model plants and major crops, the MADS-box gene repertoire in pepper (Capsicum spp.) remains poorly characterised, and its potential involvement in development and stress responses is largely unexplored.
Here, we conducted a comprehensive genome-wide survey of the MADS-box gene family in pepper. A total of 97 CaMADS genes were identified, all encoding proteins with a canonical MADS domain and exhibiting clear orthology to known plant MADS-box members. We systematically analysed their physicochemical properties, phylogenetic classification, exon–intron structures, conserved motifs and chromosomal distribution. Furthermore, we investigated gene duplication patterns, promoter cis-regulatory elements, and organ-specific expression profiles. Finally, we quantified transcript abundance under high- and low-temperature treatments as well as exogenous ABA application. This integrative dataset elucidates the evolutionary history of the MADS-box family in pepper, identifies promising candidates associated with developmental and stress-responsive pathways, and provides a robust foundation for functional dissection of MADS-mediated regulatory networks in Capsicum.

2. Results

2.1. Genome-Wide Identification of Pepper MADS-Box Genes

Using profile hidden Markov model (HMM) searches (Pfam PF00319 and PF01486) combined with SMART-based domain verification, we identified 97 non-redundant MADS-box loci from the C. annuum genome and designated them CaMADS1 through CaMADS97 based on chromosomal order (Table 1). The physicochemical characteristics of all proteins are summarised in Table 1. The predicted proteins range from 94 aa (CaMADS32) to 407 aa (CaMADS2), with molecular masses of 10.7–46.7 kDa and theoretical isoelectric points ranging from pI 5.07 (CaMADS3) to pI 10.27 (CaMADS17). Subcellular-localisation prediction using WoLF PSORT indicated that 72 CaMADS proteins are nuclear-localised, 18 are predicted to localise to chloroplasts, 5 to the cytoplasm, and only CaMADS4 and CaMADS23 to mitochondria, consistent with their expected regulatory functions.

2.2. Phylogenetic Classification of Pepper MADS-Box Genes

To clarify the evolutionary relationships between pepper and Arabidopsis MADS-box proteins, an unrooted neighbour-joining phylogeny was constructed using full-length sequences of 107 AtMADS and 97 CaMADS proteins (Figure 1). These genes were separated into the two canonical plant MADS-box lineages: Type I (52 genes) and Type II (45 genes). Type I genes further grouped into Mα, Mβ, Mγ, and MIKC* subclades; however, no CaMADS gene clustered with the Arabidopsis Mβ clade. In total, 47 CaMADS genes belonged to the Mα subclade, four to MIKC*, and only one to Mγ. Within Type II, representatives of all major angiosperm subfamilies were detected, including SEP, AGL6, AP1, MAF/FLC, AGL17, AG, SOC1, GGM13, AGL12, SVP, AGL15, and PI/AP3. The MAF/FLC subfamily contained the largest number of pepper members (7 genes), whereas AGL6, GGM13, AGL12, and Mγ were each represented by a single gene, illustrating uneven subfamily expansion since the divergence of pepper and Arabidopsis.

2.3. Lineage-Specific Loss of the Mβ Subfamily in Capsicum

To determine whether the absence of Mβ-type genes in pepper reflects a true lineage-specific event rather than an annotation artifact, we performed a comprehensive phylogenomic comparison from C. annuum and A. thaliana. Full-length MADS-box protein sequences from C. annuum and A. thaliana were incorporated into a maximum-likelihood phylogeny (Figure 1). As expected, Arabidopsis formed well-supported Mβ clades, consistent with previous reports identifying Mβ-type genes in Arabidopsis, rice, and tomato (Table S1) [10,11,12]. Strikingly, no CaMADS sequence clustered within the Mβ lineage, confirming its complete absence in pepper.
To further validate this pattern, we extended the analysis to additional species. Consistent with our observations in chili peppers, no Mβ subfamily genes were detected in melons (Cucumis melo), sugar beets (Beta vulgaris), or lettuce (Lactuca sativa) either (Table S1) [42,43,44]. These findings indicate that the loss of Mβ genes is not unique to pepper but represents a recurrent phenomenon that has independently arisen in several angiosperm lineages. In contrast, other crop species, such as tomato and rice, retain intact Mβ clades, demonstrating that Mβ loss is lineage-dependent rather than universally conserved.

2.4. Gene Structure and Conserved-Motif Analyses of Pepper MADS-Box Genes

To assess structural variation and evolutionary patterns, exon–intron structures of all 97 CaMADS genes were visualised using GSDS 2.0 (Figure 2A,B). Intron numbers ranged from 0 to 11. Similarly to Arabidopsis, Type I genes were largely intronless; only six genes (CaMADS2, CaMADS3, CaMADS23, CaMADS25, CaMADS29, CaMADS33) contained introns, potentially reflecting retrotransposition or independent intron gain/loss. In contrast, 66.7% of Type II genes (30/45) contained more than five introns, and paralogous gene pairs exhibited highly similar exon–intron structures. Ten conserved motifs (1–10) were identified using MEME and annotated via SMART (Figure 3 and Figure S1). Motif 1 (50 aa), corresponding to the canonical MADS domain, was detected in more than 90% of CaMADS proteins. This motif was highly conserved across both Type I and Type II members, consistent with its essential role in DNA binding and transcriptional regulation. Motif 5 was shared by both Type I and Type II proteins, whereas motifs 8 and 9 represented K-domain motifs restricted to MIKC proteins. Motif 4 was absent from five MIKC members (CaMADS1, 3, 25, 33, 48), and motif 8 was missing in 13 MIKC genes. Motif 10 constituted a secondary MADS-like signature primarily associated with Type I proteins, although two MIKC genes (CaMADS64, 82) also harboured it. Motifs 2, 3, 6 and 7 were specific to Type I proteins.

2.5. Chromosomal Localization and Synteny of Pepper MADS-Box Genes

All 97 CaMADS genes were successfully mapped onto the 12 chromosomes of the pepper genome, and their physical positions are illustrated in Figure 4. The CaMADS genes showed a highly uneven chromosomal distribution, with substantial variation in gene density among chromosomes. Chromosome 8 (Chr08) harbored the largest number of CaMADS genes (33 genes), accounting for more than one-third of the total family members. Notably, 26 of these genes were clustered within a relatively narrow genomic region of approximately 2 Mb, forming a pronounced gene-dense cluster (Figure 4). Such local enrichment strongly suggests that tandem duplication events have contributed to the expansion of the MADS-box gene family on this chromosome. In addition, Chr12 contained 12 CaMADS genes, whereas Chr10 carried only a single CaMADS locus, indicating marked asymmetry in chromosomal gene distribution. Chr07, Chr08, and Chr12 thus represent major hotspots for CaMADS gene accumulation in the pepper genome.
To further investigate the contribution of large-scale duplication events, segmental duplication analysis was performed using MCScanX. A total of 14 segmentally duplicated CaMADS gene pairs were identified (Figure 5A; Table S2). These duplicated pairs were distributed across seven chromosomes and mainly involved members of the AP1 (6 pairs), SEP (3 pairs), SOC1 (2 pairs), AGL17 (2 pairs), and SVP (1 pair) subclades, indicating that segmental duplication has played a key role in the expansion of Type II MADS-box genes in pepper. Among these chromosomes, Chr02 and Chr12 contained the highest numbers of duplicated gene pairs.
Furthermore, inter-species synteny analysis between pepper (Solanum lycopersicum) and tomato revealed 59 orthologous CaMADS–SlMADS gene pairs forming conserved syntenic blocks (Figure 5B). The extensive collinearity observed between the two genomes indicates a high degree of conservation in chromosomal organization of MADS-box genes between pepper and tomato, consistent with their close evolutionary relationship within the Solanaceae family.

2.6. Cis-Element Characterization of Pepper MADS-Box Promoter

To investigate the potential regulatory mechanisms associated with the pepper MADS-box gene family, the 2 kb promoter region upstream of each CaMADS gene was scanned using the PlantCARE database (Figure 6A). After removing ubiquitous core promoter elements such as the CAAT-box and TATA-box, a total of 2222 putative cis-acting elements were identified. These elements were classified into five major functional categories: light-responsive, hormone-responsive, environmental stress-related, development-related, and protein-binding sites (Figure 6B; Table S3).
Light-responsive elements constituted the most abundant category, with 1400 occurrences (63%), including well-characterized motifs such as Box 4 and G-box. The second most prevalent category consisted of hormone-responsive elements (375; 16.9%), which included abscisic acid-responsive elements (ABRE), gibberellin-responsive motifs (P-box), and salicylic acid-responsive TCA-elements. Environmental stress-related elements formed the third major group (346; 15.6%), represented by anaerobic induction elements (ARE) and low-temperature-responsive motifs (LTR). Development-related elements (38; 3.5%) included circadian control elements (Circadian), endosperm-specific motifs (GCN4_motif), and meristem-associated elements (CAT-box). Protein-binding sites were the least frequent category, with 22 elements (1%) detected across all promoters.

2.7. Expression Profiling of Pepper MADS-Box Genes Across Different Organs

To characterize the organ-specific expression patterns of pepper MADS-box genes, RNA-seq datasets covering multiple developmental contexts—including leaves, flowers, and fruits (pericarp, seeds, and placenta)—were analyzed. As most Type I MADS-box genes showed little or no detectable expression across tissues, subsequent analyses focused on the 45 Type II members, which represent the major transcriptionally active fraction of the family. Hierarchical clustering of Type II CaMADS genes revealed distinct expression modules with clear organ-associated patterns (Figure 7; Table S4). One group of genes exhibited preferential expression in vegetative tissues, particularly leaves, including CaMADS68, CaMADS72, CaMADS78, CaMADS89, and CaMADS91, suggesting potential roles in leaf development or vegetative growth regulation. A second module showed flower-enriched expression, with CaMADS73 and CaMADS74 being almost exclusively expressed during floral development, consistent with conserved functions of MADS-box genes in floral organ specification.
A third prominent expression module was associated with fruit development. Several genes, including CaMADS62, CaMADS65, CaMADS66, CaMADS69, and CaMADS77, displayed strong and sustained expression in pericarp, seeds, and placenta, implying roles in fruit growth and reproductive development. Notably, CaMADS65 showed minimal expression at early fruit stages but was strongly induced during later developmental stages, suggesting involvement in fruit maturation rather than early fruit set. In addition, a subset of genes, such as CaMADS62, CaMADS66, CaMADS69, and CaMADS77, exhibited high transcript levels during both floral and fruit development, indicating potential regulatory roles spanning the flower-to-fruit transition. Although a small number of genes showed pronounced organ-preferential expression, the majority of Type II CaMADS genes were expressed across multiple organs, reflecting their pleiotropic regulatory functions. In contrast, five genes (CaMADS1, CaMADS71, CaMADS75, CaMADS79, and CaMADS81) displayed extremely low transcript abundance across all sampled organs, suggesting limited or highly specialized transcriptional activity under the conditions examined. Overall, this organ-resolved expression atlas highlights both broadly expressed and organ-preferential CaMADS genes, providing a structured framework for identifying candidate regulators of vegetative growth, floral development, and fruit formation in pepper.

2.8. Expression Patterns of Pepper MADS-Box Genes Under Temperature Stress

Based on the cis-element analysis, we hypothesized that a subset of pepper MADS-box genes may be responsive to temperature-related abiotic stress. To examine this possibility, we analyzed their expression patterns under heat and cold stress conditions using time-course transcriptome data from leaf and root tissues collected at 0, 0.5, 1, 3, 6, 12, and 24 h after treatment. Hierarchical clustering of expression profiles revealed that CaMADS genes could be broadly classified into distinct temperature-responsive expression patterns rather than strictly stress-specific responses (Figure 8; Tables S5 and S6). Under heat stress, several genes displayed clear induction or repression in a tissue-dependent manner (Figure 8A). In leaves, a group of genes exhibited increased transcript abundance, whereas a smaller subset showed reduced expression. In roots, a partially overlapping but distinct set of genes responded to heat treatment, indicating organ-dependent regulation of temperature responses.
A similar overall expression architecture was observed under cold stress (Figure 8B). Multiple CaMADS genes showed transcriptional induction in leaves but repression in roots, while others displayed moderate or transient responses. Notably, CaMADS56, CaMADS86, and CaMADS91 exhibited highly consistent expression patterns under both heat and cold stress, characterized by upregulation in leaves and downregulation in roots across multiple time points. This convergence suggests that these genes may participate in a shared temperature-stress response module rather than functioning exclusively in heat- or cold-specific signaling pathways. Collectively, these results indicate that pepper MADS-box genes respond to temperature stress in an organ-dependent manner and that heat and cold treatments elicit broadly similar transcriptional response patterns. The identification of genes with consistent regulation under both temperature extremes highlights candidate CaMADS members potentially involved in general thermal stress adaptation.

2.9. Expression Patterns of Pepper MADS-Box Genes Under ABA Treatment

To investigate the potential involvement of pepper MADS-box genes in hormone-mediated regulation, transcriptome datasets from ABA-treated plants were analyzed to examine expression dynamics in leaf and root tissues (Figure 9; Table S7). Hierarchical clustering revealed that CaMADS genes responded to ABA treatment in a time- and organ-dependent manner, forming distinct response patterns rather than uniform transcriptional changes. In leaves, a major group of CaMADS genes displayed ABA-induced expression, with transcript levels generally peaking at 6 h or 12 h after treatment. Within this group, CaMADS69 showed particularly strong induction, suggesting high sensitivity to ABA signaling. In contrast, another subset of genes exhibited sustained repression following ABA treatment, indicating differential regulatory roles within the family. In roots, ABA-responsive expression patterns were broadly similar in structure but differed in gene composition and magnitude. Several CaMADS genes showed pronounced induction, again with peak expression typically occurring at 6 h or 12 h, whereas others were consistently downregulated throughout the treatment period. Notably, CaMADS67 exhibited the strongest ABA-induced expression in roots, highlighting potential organ-specific regulatory roles in ABA responses.
Despite these dynamic responses, a small subset of genes (CaMADS1, CaMADS79, and CaMADS83) showed no detectable transcript accumulation in either leaves or roots under any treatment condition, suggesting limited transcriptional activity or highly restricted expression domains under the conditions examined. Overall, these results demonstrate that ABA elicits diverse and temporally structured transcriptional responses among CaMADS genes, with clear organ-dependent differences. The identification of early- and late-responsive expression modules provides a framework for prioritizing candidate MADS-box genes involved in ABA-mediated stress signaling and developmental regulation in pepper.

3. Discussion

Previous studies have demonstrated that MADS-box transcription factors play essential roles in plant growth, development, and adaptation to adverse environmental conditions [45,46,47]. However, the characteristics and regulatory features of MADS-box genes in pepper remain poorly understood. Therefore, a comprehensive investigation of the pepper MADS-box gene family and its expression patterns under different abiotic stresses can provide deeper insight into developmental and stress-response mechanisms and facilitate molecular breeding in pepper.

3.1. Family Characteristics of Pepper MADS-Box Genes

In this study, 97 MADS-box genes were identified in the pepper genome and named CaMADS1–CaMADS97 according to their chromosomal positions (Table 1). This number differs from the 72 genes previously reported by Gan et al. [48], likely reflecting differences in genome assembly quality. Across plants, the size of the MADS-box family varies considerably—Arabidopsis (107) [10], rice (75) [11], maize (211) [49], wheat (117) [50], and tomato (131) [12]—and pepper possesses notably fewer Type I members (52) than tomato. This suggests lineage-specific differences in duplication and post-duplication gene retention. In contrast, the number of Type II genes (45) is similar between the two species.
Exon–intron structure analysis (Figure 2) showed that Type I genes are largely intronless, consistent with extensive intron loss during evolution, whereas Type II genes contain 1–11 introns, reflecting their more complex architecture. Similar structural patterns in Arabidopsis and pea [10,51] indicate strong evolutionary conservation. Nonetheless, several closely related gene pairs displayed divergent exon–intron structures, implying additional lineage-specific structural changes. Conserved motif analysis revealed that members within the same subfamily share similar motif compositions (Figure 3), whereas distinct motif patterns among subfamilies support their functional diversification.
Both tandem and segmental duplications contributed to family expansion in pepper. MADS-box genes were distributed across all 12 chromosomes (Figure 4), with chromosome 8 harboring 33 genes, likely resulting from tandem duplication. Tandem clusters were also found on chromosomes 7 and 12. Additionally, 14 segmentally duplicated gene pairs were identified (Figure 5), confirming that segmental duplication has played a major role in CaMADS family expansion, consistent with patterns observed in Salvia miltiorrhiza and Cucurbita foetidissima [52,53].

3.2. Organ-Specific and Temperature-Responsive Roles of Pepper MADS-Box Genes

Across plant species, MADS-box genes exhibit substantial variation in spatial expression, reflecting their diverse regulatory roles in growth and development. In peanut, most MADS-box genes are predominantly expressed in floral organs, whereas others accumulate in roots, suggesting functions in reproductive development or nutrient uptake and ion transport [54]. Similar patterns are widely observed: in cucumber, several AG- and AP1-subfamily members are highly expressed in flowers, while multiple additional MADS-box genes show root-enriched expression [55]; in tomato, many Type II MADS-box genes are strongly expressed during flower and fruit development, whereas a subset displays root-specific expression [12]. Consistent with these reports, our transcriptome analyses revealed pronounced tissue specificity among CaMADS genes (Figure 7). Five genes (CaMADS68, CaMADS72, CaMADS78, CaMADS89, and CaMADS91) were preferentially expressed in leaves, whereas CaMADS73 and CaMADS74 transcripts were detected exclusively in floral tissues. In addition, CaMADS62, CaMADS65, CaMADS66, CaMADS69, and CaMADS77 showed strong expression in pericarp, seeds, and placenta, indicating potential functions in fruit growth and maturation. These patterns underscore the broad developmental involvement and functional diversification of the MADS-box family in pepper.
Beyond developmental roles, accumulating evidence demonstrates that MADS-box genes also participate in abiotic stress adaptation [39,56]. In litchi, multiple MADS-box genes-including LcMADS16, LcMADS17, LcMADS19, LcMADS20, LcMADS21, LcMADS24, LcMADS37, and LcMADS61—are strongly induced by heat and cold stress [57]. Low temperature significantly regulates the expression of AtAGL91 in Arabidopsis [58]. In cucumber, TaMADS63 and TaMADS41 are markedly downregulated under high temperatures [55], while in Chinese cabbage, BrMADS063 and BrMADS100 are responsive to heat and cold, respectively [59]. Low temperature also triggers upregulation of TM5 (SEP), TM6 (DEF), and TAG1 (AG) in tomato, contributing to stress-induced floral abnormalities [60,61]. In the present study, we observed similar temperature-dependent expression dynamics among CaMADS genes. Under heat stress, six genes were upregulated in leaves and five were downregulated in roots, while cold stress induced five genes in leaves and repressed four in roots. Remarkably, CaMADS56, CaMADS86, and CaMADS91 displayed a consistent regulatory pattern—upregulated in leaves but downregulated in roots—under both heat and cold stress conditions. This convergence suggests that these genes may participate in a shared temperature-stress response module, rather than acting in strictly heat- or cold-specific pathways.
Such similar transcriptional responses to opposite temperature extremes have been reported for other stress-responsive transcription factors and are often interpreted as components of common stress signaling networks, including ABA-dependent pathways, oxidative stress responses, or general cellular homeostasis mechanisms. The organ-dependent regulation observed here further implies that CaMADS genes may contribute to organ-specific thermal adaptation strategies, potentially coordinating growth restraint or developmental adjustment in leaves while modulating stress sensitivity in roots.
It is important to emphasize that the present expression data alone do not establish direct functional roles in temperature tolerance. Rather, these results identify candidate CaMADS genes—particularly CaMADS56, CaMADS86, and CaMADS91—that may act as integrators of developmental and temperature-stress signals. Functional validation through genetic or molecular approaches will be required to elucidate their precise regulatory roles. Overall, the organ-specific and temperature-responsive expression patterns reported here provide a valuable framework for understanding how MADS-box transcription factors integrate environmental cues with developmental programs in pepper.

3.3. Functional Implications of the Mβ Subfamily Loss in Pepper

One of the most striking findings of this study is the complete absence of Mβ-type MADS-box genes in pepper. Although Type I MADS-box genes (Mα, Mβ, Mγ) are generally less well-characterized than Type II genes, previous studies provide valuable clues regarding their biological roles. In several angiosperms, Mβ genes tend to exhibit extremely low transcript abundance and show highly restricted—often gametophyte- or endosperm-specific—expression patterns [62]. Functional analyses in Arabidopsis and monocots suggest that Mβ members may contribute to early reproductive processes such as female gametophyte development, endosperm formation, and early embryo patterning, although their roles appear partially redundant and less essential than those of other Type I or Type II MADS-box genes [63,64]. This weak functional constraint has been proposed to underlie the high evolutionary turnover of Mβ genes, making them more susceptible to pseudogenization or lineage-specific loss.
The complete loss of Mβ genes in pepper therefore likely reflects relaxed purifying selection on this subfamily, implying that Mβ-mediated developmental modules are not required for successful reproduction in Capsicum. Importantly, our phylogenomic framework shows that Mα and Mγ genes are retained in pepper, whereas Mβ is selectively lost—indicating a non-random contraction of the Type I lineage. Such a pattern raises the possibility that functional compensation may have occurred: Mα or Mγ genes, or even Type II genes, may have adopted regulatory roles traditionally associated with Mβ in species where Mβ is present. Compensation within Type I clades has been previously observed in Arabidopsis, where Mα and Mγ gene pairs exhibit partially overlapping expression domains, and may buffer the loss or weakening of Mβ [65].
Our expression analyses support this interpretation. Compared with Type II CaMADS genes, Type I members—including Mα and the single Mγ gene—showed minimal or undetectable transcript accumulation across vegetative, floral, and fruit tissues. This expression landscape suggests that pepper development relies predominantly on Type II regulatory modules, whereas Type I genes contribute little to global transcriptional output. The near-silencing of Mα and Mγ genes also implies that these lineages may have undergone functional reduction, making the presence or absence of Mβ largely inconsequential for pepper development. Together, these genomic and transcriptomic observations indicate that the Mβ subfamily likely represents a dispensable regulatory component in Capsicum, consistent with its independent loss in several other dicot species [42,43,44].
The absence of Mβ genes also raises broader evolutionary questions. The pepper genome has undergone substantial structural rearrangements and selective pressures related to domestication, which may have further accelerated the erosion of weakly expressed or redundant Type I genes. Given that Mβ genes often participate in reproductive processes sensitive to dosage imbalance, their loss in pepper may reflect adaptive optimization of reproductive pathways under domestication or ecological specialization. Future functional studies—particularly in early reproductive tissues, ovules, and gametophytes—will be essential to determine whether latent or cryptic regulatory roles of Mβ have been reassigned to Mα, Mγ, or Type II MADS-box genes in pepper.

3.4. Integration of Phylogenomic, Structural, and Expression Evidence Supports the Evolutionary Disappearance of the Mβ Lineage in Capsicum

The integration of phylogenomic, structural, chromosomal, and expression analyses provides convergent evidence that the Mβ lineage has been evolutionarily lost in pepper. Phylogenetic reconstruction based on 204 full-length MADS-box proteins from Capsicum and Arabidopsis revealed no CaMADS genes clustering with the canonical Mβ clade, despite robust resolution of other Type I and Type II subfamilies. Consistently, exon–intron structure analyses showed that pepper Type I MADS-box genes possess highly simplified architectures characteristic of rapid evolutionary turnover, yet no pseudogenized, truncated, or partially retained Mβ-like gene structures were detected elsewhere in the genome. Chromosomal distribution analysis further failed to uncover isolated remnants or degenerated copies, supporting an ancient and complete lineage loss rather than recent pseudogenization.
Expression profiling adds a regulatory perspective to this evolutionary scenario. While Type II MADS-box genes exhibit broad and dynamic expression across developmental stages and stress conditions, most Type I genes show extremely low transcriptional activity. Such weak expression patterns may predispose certain sublineages, including Mβ, to evolutionary elimination due to functional redundancy or limited selective constraint. Notably, similar absences of the Mβ lineage have been reported in several unrelated angiosperm families, including Asteraceae, Amaranthaceae, and Cucurbitaceae, suggesting that Mβ loss represents a recurrent evolutionary outcome rather than a Capsicum-specific anomaly. Collectively, these findings indicate that MADS-box gene evolution in pepper involved not only changes in gene content but also extensive regulatory reorganization, ultimately favoring the retention and diversification of functionally essential Type II subfamilies.

4. Materials and Methods

4.1. Genome-Wide Identification of Pepper MADS-Box Genes

The C. annuum reference genome (release 2.0) and corresponding annotation files were retrieved from the Pepper Genome Platform (http://ted.bti.cornell.edu/cgi-bin/pepper/search, accessed on 17 March 2025). Two Pfam hidden Markov models—PF00319 (SRF-TF MADS domain) and PF01486 (K-box)—were downloaded from PlantTFDB (https://planttfdb.gao-lab.org/, accessed on 20 March 2025). HMMER v3.0 (hmmsearch, E-value ≤ 1 × 10−2) was used to scan the pepper proteome. All candidate sequences were subsequently validated using SMART to confirm the presence of the MADS and/or K-box domains. Partial or redundant sequences were removed, resulting in 97 high-confidence CaMADS genes. Molecular weight, theoretical isoelectric point, and protein length were calculated using ProtParam (http://web.expasy.org/protparam/, accessed on 28 March 2025), and subcellular localisation was predicted with WoLF PSORT (http://www.genscript.com/wolf-psort.html, accessed on 7 April 2025).

4.2. Phylogenetic and Sequence Analyses

A total of 107 Arabidopsis thaliana MADS-box protein sequences were obtained from TAIR (https://www.arabidopsis.org, accessed on 11 April 2025). Protein sequences from pepper and Arabidopsis were aligned using MAFFT (L-INS-i algorithm), and alignments were manually curated in DNAMAN. Conserved domains were inspected using NCBI-CDD and PfamScan. A neighbour-joining phylogenetic tree was constructed in MEGA v11 with 1000 bootstrap replicates, and visualised in iTOL v6. CaMADS genes were assigned to subfamilies based on their clustering with the well-established Arabidopsis groups.

4.3. Gene Structure and Conserved Motif Characterisation

Genomic DNA and CDS sequences were extracted from the pepper genome. Exon–intron structures were illustrated using GSDS 2.0. Conserved motifs were identified de novo using MEME (parameters: motif width 6–60 aa; zero or more repetitions). Motifs were annotated by comparison with SMART and SoftBerry databases.

4.4. Chromosomal Localisation and Synteny Analysis

All 97 CaMADS loci were mapped onto the 12 pseudochromosomes of pepper using MG2C v2.1. Segmental duplication events were identified with MCScanX under default settings. For interspecific synteny analysis, the pepper genome was compared with tomato (SL4.0) using MCScanX, and syntenic blocks were visualised with TBtools (Version 2.121).

4.5. Promoter Cis-Element Prediction

For each CaMADS gene, the 2 kb region upstream of the translation start site was extracted and analysed using PlantCARE. Identified cis-elements were grouped into functional categories, including hormone-responsive, stress-responsive and developmental elements. Visualisation was performed in TBtools, and final figures were refined using Adobe Illustrator.

4.6. RNA-Seq Data Source and Experimental Background

RNA-seq data analyzed in this study were obtained from PepperHub (http://lifenglab.hzau.edu.cn/PepperHub/index.php, accessed on 5 July 2025), comprising three transcriptome series: (i) multi-organ, (ii) temperature-stress, and (iii) hormone-treatment datasets. No new plant growth, stress, or hormone treatment experiments were performed by the authors.
According to the original experimental design, pepper seedlings were grown under controlled environmental conditions and subjected to temperature and hormone treatments prior to RNA extraction and sequencing. Detailed descriptions of plant growth conditions, stress treatments, hormone concentrations, and sampling procedures are provided in the original study [66], which is cited here to facilitate interpretation of the expression data.

5. Conclusions

In this study, we performed a genome-wide characterization of the pepper MADS-box gene family and identified 97 members, including 52 Type I and 45 Type II genes. We systematically analyzed their phylogeny, gene structures, conserved motifs, chromosomal distribution, and expression patterns. Gene duplication events were found to be major contributors to family expansion, and pronounced differences in intron number distinguished Type I and Type II genes. The CaMADS family displayed clear organ-specific and stress-responsive expression patterns. Importantly, our analyses provide the first integrated phylogenomic and expression-based evidence that the Mβ subfamily is completely absent from the Capsicum genome, indicating a lineage-specific gene-loss event. This finding highlights a unique evolutionary trajectory of Type I MADS-box genes in pepper and offers a framework for future investigations into functional compensation among Type I lineages and their roles in reproductive and stress-related processes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants15040620/s1, Figure S1: Ten conserved motifs identified in CaMADS-box genes. Table S1: Loss of the Mβ Subfamily in Different Plant Species. Table S2: Segmental duplicate genes in the CaMADS-box gene family. Table S3: Analysis of Cis-acting Elements in the Promoter Regions of CaMADS-box Genes. Table S4: Expression Profile of Type II CaMADS-box Gene Family in Different Tissues. Table S5: Expression Profiling of Type II CaMADS-box Genes in Response to Heat Stress. Table S6: Expression of Type II CaMADS-box Genes in Response to Cold Stress. Table S7: Expression of Type II CaMADS-box Genes in Response to Abscisic Acid Treatment. Table S8. Expression of Type II CaMADS-box Genes in Response to Abscisic Acid Treatment.

Author Contributions

Conceptualization and supervision, H.W.; writing—original draft, J.Z.; S.M.; J.L.; reviewed and modified the manuscript, T.Z., Y.C., R.W., M.R., Q.Y., G.Z., Z.Y., Z.L. and C.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by National Natural Science Foundation of China (32341044, 32472751), Zhejiang Provincial major Agricultural Science and Technology Projects of New Varieties Breeding (2021C02065), China Agriculture Research System of MOF and MARA (CARS-23-G44), National Key Research and Development Program of China (2023YFD1201504), Local Cooperation Projects of Zhejiang Academy of Agricultural Sciences (2024R23A77C03, 2025R23A77C02).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author(s).

Conflicts of Interest

The authors declare no conflicts of interest.

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Plants 15 00620 g001
Figure 1. Phylogenetic analysis and classification of MADS-box transcription factors in pepper (C. annuum). The phylogenetic tree was constructed using the full-length protein sequences of the MADS-box genes identified in the pepper genome. The analysis clearly classified the pepper MADS-box genes into distinct subfamilies according to the established Arabidopsis-based nomenclature. Different subfamilies were distinguished by color, with major groups such as SOC1, AGL17, and AGL15 clearly resolved. The systematic naming (e.g., the CaMADSSE prefix) indicates the presumed orthologous relationships between pepper MADS-box genes and their Arabidopsis counterparts.
Figure 1. Phylogenetic analysis and classification of MADS-box transcription factors in pepper (C. annuum). The phylogenetic tree was constructed using the full-length protein sequences of the MADS-box genes identified in the pepper genome. The analysis clearly classified the pepper MADS-box genes into distinct subfamilies according to the established Arabidopsis-based nomenclature. Different subfamilies were distinguished by color, with major groups such as SOC1, AGL17, and AGL15 clearly resolved. The systematic naming (e.g., the CaMADSSE prefix) indicates the presumed orthologous relationships between pepper MADS-box genes and their Arabidopsis counterparts.
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Figure 2. Gene structure analysis of pepper MADS-box genes. (A) Gene structures of Type I (M) MADS-box genes. (B) Gene structures of Type II (MIKC) MADS-box genes. Coding sequences (CDS) are represented by yellow boxes, introns by black lines, and upstream/downstream non-coding regions by blue boxes. Intron phases are indicated by numbers 0, 1, and 2.
Figure 2. Gene structure analysis of pepper MADS-box genes. (A) Gene structures of Type I (M) MADS-box genes. (B) Gene structures of Type II (MIKC) MADS-box genes. Coding sequences (CDS) are represented by yellow boxes, introns by black lines, and upstream/downstream non-coding regions by blue boxes. Intron phases are indicated by numbers 0, 1, and 2.
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Figure 3. Conserved motif analysis of MADS-box genes in pepper (C. annuum). The protein sequences of the identified pepper MADS-box genes were analyzed using the MEME suite to identify conserved motifs. A total of ten distinct motifs, designated as motifs 1–10, are represented by colored boxes. Each horizontal line corresponds to a single MADS-box protein (e.g., CAMADS4, CAMADS5, etc.), with the schematic illustrating the type, order, and relative position of the conserved motifs within each protein. The specific distribution pattern of these motifs is closely associated with the phylogenetic classification of the genes, suggesting functional divergence among different subfamilies. Notably, Motif 1 is universally present across nearly all members, likely representing the highly conserved DNA-binding MADS-domain.
Figure 3. Conserved motif analysis of MADS-box genes in pepper (C. annuum). The protein sequences of the identified pepper MADS-box genes were analyzed using the MEME suite to identify conserved motifs. A total of ten distinct motifs, designated as motifs 1–10, are represented by colored boxes. Each horizontal line corresponds to a single MADS-box protein (e.g., CAMADS4, CAMADS5, etc.), with the schematic illustrating the type, order, and relative position of the conserved motifs within each protein. The specific distribution pattern of these motifs is closely associated with the phylogenetic classification of the genes, suggesting functional divergence among different subfamilies. Notably, Motif 1 is universally present across nearly all members, likely representing the highly conserved DNA-binding MADS-domain.
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Figure 4. Chromosomal distribution of MADS-box genes in pepper (C. annuum). The physical locations of all identified MADS-box genes are mapped onto the 12 pepper chromosomes. The chromosome numbers are indicated at the top of each bar (Chr1–Chr12). Each gene (e.g., CAM.0854, CAM.0855, etc.) is represented by a specific marker and its position corresponds to its physical location on the chromosome in megabases (Mb), as shown on the scale to the left. The distribution of genes across the chromosomes is uneven, with some chromosomes harboring dense clusters of MADS-box genes while others contain only a few. This genomic localization provides a foundation for understanding the evolution of this gene family and their potential roles in chromosome-based genetic studies.
Figure 4. Chromosomal distribution of MADS-box genes in pepper (C. annuum). The physical locations of all identified MADS-box genes are mapped onto the 12 pepper chromosomes. The chromosome numbers are indicated at the top of each bar (Chr1–Chr12). Each gene (e.g., CAM.0854, CAM.0855, etc.) is represented by a specific marker and its position corresponds to its physical location on the chromosome in megabases (Mb), as shown on the scale to the left. The distribution of genes across the chromosomes is uneven, with some chromosomes harboring dense clusters of MADS-box genes while others contain only a few. This genomic localization provides a foundation for understanding the evolution of this gene family and their potential roles in chromosome-based genetic studies.
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Plants 15 00620 g005
Figure 5. Collinearity and homology analysis of MADS-box genes. (A) Duplicated genes in the MADS-box gene family. Blue boxes represent chromosomes, and red lines indicate segmental duplication linear relationships among MADS-box genes. The heatmap on chromosomes denotes the density of MADS-box genes, with the color scale ranging from 1.00 to 7.00. (B) Collinearity analysis of MADS-box genes on chromosomes of pepper and tomato. Gray lines represent all collinear gene pairs, while red lines specifically indicate the collinear relationships of pepper MADS-box genes. Chromosomes of pepper and tomato are shown as orange and green bars, respectively, labeled from Chr01 to Chr12.
Figure 5. Collinearity and homology analysis of MADS-box genes. (A) Duplicated genes in the MADS-box gene family. Blue boxes represent chromosomes, and red lines indicate segmental duplication linear relationships among MADS-box genes. The heatmap on chromosomes denotes the density of MADS-box genes, with the color scale ranging from 1.00 to 7.00. (B) Collinearity analysis of MADS-box genes on chromosomes of pepper and tomato. Gray lines represent all collinear gene pairs, while red lines specifically indicate the collinear relationships of pepper MADS-box genes. Chromosomes of pepper and tomato are shown as orange and green bars, respectively, labeled from Chr01 to Chr12.
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Plants 15 00620 g006
Figure 6. Cis-acting elements in the promoter regions of pepper MADS-box gene family members (A) and their proportions (B). (A) Different colored dots represent various types of cis-acting elements, with the legend on the right detailing their functional categories (e.g., light-responsive elements, hormone-related elements, environmental stress-responsive elements, development-related elements, and protein binding sites). The x-axis indicates the promoter sequence length (0–2000 bp). (B) A pie chart showing the proportion of different cis-acting element types. Light-responsive elements account for 63%, hormone-related elements 16.9%, environmental stress-responsive elements 15.6%, development-related elements 3.5%, and protein binding sites 1%.
Figure 6. Cis-acting elements in the promoter regions of pepper MADS-box gene family members (A) and their proportions (B). (A) Different colored dots represent various types of cis-acting elements, with the legend on the right detailing their functional categories (e.g., light-responsive elements, hormone-related elements, environmental stress-responsive elements, development-related elements, and protein binding sites). The x-axis indicates the promoter sequence length (0–2000 bp). (B) A pie chart showing the proportion of different cis-acting element types. Light-responsive elements account for 63%, hormone-related elements 16.9%, environmental stress-responsive elements 15.6%, development-related elements 3.5%, and protein binding sites 1%.
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Plants 15 00620 g007
Figure 7. Organ-specific expression patterns of MADS-box genes in pepper (C. annuum). The heatmap displays the expression profiles (likely based on RNA-seq data, represented by FPKM or TPM values) of identified MADS-box genes across various tissues and developmental stages. Expression levels are color-coded, with the scale indicating low (e.g., blue) to high (e.g., red) expression. The analyzed tissues and stages include: leaf (L1 to L9, representing 9 developmental stages), flower (F1 to F9, representing 9 developmental stages; with specific floral organs P10 for petal, O10 for ovary with stigma, and STA10 for stamen at stage 10), pericarp (G1 to G11, representing 11 developmental stages), seed (S1 to S11, representing 11 developmental stages), and placenta (T1 to T11, representing 11 developmental stages). The hierarchical clustering of genes (rows) reveals groups with similar expression patterns, suggesting potential functional specializations in specific tissues or developmental processes, such as floral organ identity, fruit development, and seed formation.
Figure 7. Organ-specific expression patterns of MADS-box genes in pepper (C. annuum). The heatmap displays the expression profiles (likely based on RNA-seq data, represented by FPKM or TPM values) of identified MADS-box genes across various tissues and developmental stages. Expression levels are color-coded, with the scale indicating low (e.g., blue) to high (e.g., red) expression. The analyzed tissues and stages include: leaf (L1 to L9, representing 9 developmental stages), flower (F1 to F9, representing 9 developmental stages; with specific floral organs P10 for petal, O10 for ovary with stigma, and STA10 for stamen at stage 10), pericarp (G1 to G11, representing 11 developmental stages), seed (S1 to S11, representing 11 developmental stages), and placenta (T1 to T11, representing 11 developmental stages). The hierarchical clustering of genes (rows) reveals groups with similar expression patterns, suggesting potential functional specializations in specific tissues or developmental processes, such as floral organ identity, fruit development, and seed formation.
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Plants 15 00620 g008
Figure 8. Expression patterns of pepper MADS-box genes under heat and cold stress. (A) Heatmap showing the expression profiles of MADS-box genes in pepper under heat stress. (B) Heatmap showing the expression profiles under cold stress. For both panels, samples were collected from leaf (L) and root (R) tissues at 0, 0.5, 1, 3, 6, 12, and 24 h after treatment. Sample abbreviations are defined as follows: C0, control (0 h); H1–H6, heat stress at the indicated time points; F1–F6, cold stress at the indicated time points; L, leaf; R, root. Thus, for example, HL3 represents leaf tissue sampled 3 h after heat treatment, and FR6 represents root tissue sampled 24 h after cold treatment. Expression levels (FPKM values) were log2-transformed and visualized using a color scale, with red indicating higher expression and blue indicating lower expression. Hierarchical clustering was applied to group genes with similar temporal expression patterns, allowing the identification of genes potentially involved in early or late temperature-stress responses in an organ-specific manner.
Figure 8. Expression patterns of pepper MADS-box genes under heat and cold stress. (A) Heatmap showing the expression profiles of MADS-box genes in pepper under heat stress. (B) Heatmap showing the expression profiles under cold stress. For both panels, samples were collected from leaf (L) and root (R) tissues at 0, 0.5, 1, 3, 6, 12, and 24 h after treatment. Sample abbreviations are defined as follows: C0, control (0 h); H1–H6, heat stress at the indicated time points; F1–F6, cold stress at the indicated time points; L, leaf; R, root. Thus, for example, HL3 represents leaf tissue sampled 3 h after heat treatment, and FR6 represents root tissue sampled 24 h after cold treatment. Expression levels (FPKM values) were log2-transformed and visualized using a color scale, with red indicating higher expression and blue indicating lower expression. Hierarchical clustering was applied to group genes with similar temporal expression patterns, allowing the identification of genes potentially involved in early or late temperature-stress responses in an organ-specific manner.
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Plants 15 00620 g009
Figure 9. Expression patterns of pepper MADS-box genes in response to abscisic acid (ABA) treatment. The heatmap displays the expression profiles of MADS-box genes in leaf (L) and root (R) tissues of pepper following ABA treatment at 0, 0.5, 1, 3, 6, 12, and 24 h. Sample abbreviations are defined as follows: C0, control (0 h); A1–A6, ABA treatment at the indicated time points; L, leaf; R, root. Thus, for example, AL3 represents leaf tissue sampled 3 h after ABA treatment, whereas AR6 represents root tissue sampled 24 h after ABA treatment. Expression values (FPKM values) were log2-transformed and visualized using a color scale, with red indicating higher expression and blue indicating lower expression. Hierarchical clustering was performed to group genes with similar temporal expression patterns, allowing the identification of early-responsive, late-responsive, and persistently induced or repressed gene clusters in response to ABA signaling.
Figure 9. Expression patterns of pepper MADS-box genes in response to abscisic acid (ABA) treatment. The heatmap displays the expression profiles of MADS-box genes in leaf (L) and root (R) tissues of pepper following ABA treatment at 0, 0.5, 1, 3, 6, 12, and 24 h. Sample abbreviations are defined as follows: C0, control (0 h); A1–A6, ABA treatment at the indicated time points; L, leaf; R, root. Thus, for example, AL3 represents leaf tissue sampled 3 h after ABA treatment, whereas AR6 represents root tissue sampled 24 h after ABA treatment. Expression values (FPKM values) were log2-transformed and visualized using a color scale, with red indicating higher expression and blue indicating lower expression. Hierarchical clustering was performed to group genes with similar temporal expression patterns, allowing the identification of early-responsive, late-responsive, and persistently induced or repressed gene clusters in response to ABA signaling.
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Table 1. Information of MADS-box genes identified in C. annuum.
Table 1. Information of MADS-box genes identified in C. annuum.
Gene NameGene LocusProteinSubcellular LocalizationType
Length (aa)MW (Da)pI
CaMADS1Caz05g0159011212,813.979.66ChloroplastType II
CaMADS2Caz05g1529040746,730.366.29NucleusType I
CaMADS3Caz04g0568031135,625.895.07NucleusType I
CaMADS4Caz08g1975020923,394.0210MitochondrionType I
CaMADS5Caz12g0924016818,914.989.49NucleusType II
CaMADS6Caz08g2007019922,646.169.36NucleusType I
CaMADS7Caz08g1974021123,101.389.1NucleusType I
CaMADS8Caz08g1970021123,169.378.72NucleusType I
CaMADS9Caz08g2010019422,023.329.43NucleusType I
CaMADS10Caz08g2005018420,756.886.98CytoplasmType I
CaMADS11Caz08g2004021123,156.559.28NucleusType I
CaMADS12Caz08g1957021123,341.789.44NucleusType I
CaMADS13Caz03g1433015617,899.415.83ChloroplastType I
CaMADS14Caz12g2004022325,044.628.31ChloroplastType I
CaMADS15Caz08g2006021123,224.649.16NucleusType I
CaMADS16Caz08g1967022325,456.249.49ChloroplastType I
CaMADS17Caz07g1489020422,879.610.27NucleusType I
CaMADS18Caz03g2612017019,204.918.93ChloroplastType I
CaMADS19Caz08g1976016218,186.999.43NucleusType I
CaMADS20Caz08g1964022325,377.099.4NucleusType I
CaMADS21Caz08g2002021123,292.669.24NucleusType I
CaMADS22Caz12g2000022325,037.649.28ChloroplastType I
CaMADS23Caz08g2011032036,097.529.58MitochondrionType I
CaMADS24Caz08g1956024026,222.19.24ChloroplastType I
CaMADS25Caz07g1461020623,083.236.32ChloroplastType I
CaMADS26Caz08g2199019221,509.049.54ChloroplastType I
CaMADS27Caz08g0816014716,633.895.16CytoplasmType I
CaMADS28Caz08g2009021924,881.149.51ChloroplastType I
CaMADS29Caz01g2263035941,180.575.31NucleusType I
CaMADS30Caz08g1136015717,819.276.37NucleusType I
CaMADS31Caz07g1488021023,660.639.85ChloroplastType I
CaMADS32Caz08g197109410,727.289.51ChloroplastType I
CaMADS33Caz07g1462014816,830.165.54NucleusType I
CaMADS34Caz03g1139021524,407.759ChloroplastType I
CaMADS35Caz09g0631016118,258.445.29NucleusType I
CaMADS36Caz08g2014017119,866.959.84NucleusType I
CaMADS37Caz08g1968017119,493.649.62ChloroplastType I
CaMADS38Caz08g1965021223,600.359.91ChloroplastType I
CaMADS39Caz11g1664017119,026.69ChloroplastType I
CaMADS40Caz09g0638017520,018.678.9NucleusType I
CaMADS41Caz08g2013019020,723.536.91NucleusType I
CaMADS42Caz08g2008019020,771.576.91NucleusType I
CaMADS43Caz11g0603017119,141.869.15ChloroplastType I
CaMADS44Caz08g1138017018,709.929.87NucleusType I
CaMADS45Caz08g1134016918,652.879.87NucleusType I
CaMADS46Caz04g1346018921,657.546.75ChloroplastType I
CaMADS47Caz09g0520017419,947.457.85NucleusType I
CaMADS48Caz11g1031027531,086.115.85NucleusType II
CaMADS49Caz08g1962020122,991.199.49NucleusType I
CaMADS50Caz04g2382019021,321.476.83NucleusType I
CaMADS51Caz01g0607017019,623.449.05CytoplasmType I
CaMADS52Caz09g1931016419,020.799.57NucleusType I
CaMADS53Caz08g1139016317,938.129.64NucleusType I
CaMADS54Caz01g3587030735,034.498.54NucleusType I
CaMADS55Caz08g0468018020,566.798.94CytoplasmType I
CaMADS56Caz03g0830024728,669.198.98NucleusType II
CaMADS57Caz11g1879024228,364.418.96NucleusType II
CaMADS58Caz01g1323025528,998.98.85NucleusType II
CaMADS59Caz03g0829024528,226.988.94NucleusType II
CaMADS60Caz02g0677024228,198.269.3NucleusType II
CaMADS61Caz06g1063025128,947.569.1NucleusType II
CaMADS62Caz01g2242024027,748.488.87NucleusType II
CaMADS63Caz09g1948024027,620.187.78NucleusType II
CaMADS64Caz02g2881024828,508.399.32NucleusType II
CaMADS65Caz11g1878024327,949.467.06NucleusType II
CaMADS66Caz02g1313026430,486.269.16NucleusType II
CaMADS67Caz11g1040024127,570.278.82NucleusType II
CaMADS68Caz12g1053020624,073.356.27NucleusType II
CaMADS69Caz07g1905025529,317.919.31NucleusType II
CaMADS70Caz05g1003017920,730.819.46NucleusType II
CaMADS71Caz12g0868023427,304.189.81NucleusType II
CaMADS72Caz01g1324021825,173.89.22NucleusType II
CaMADS73Caz06g1883021525,129.728.74NucleusType II
CaMADS74Caz08g0559020924,652.138.49NucleusType II
CaMADS75Caz04g0755023426,962.579.25NucleusType II
CaMADS76Caz01g0425021324,626.39.38NucleusType II
CaMADS77Caz02g2883023326,809.347.65NucleusType II
CaMADS78Caz01g1325022425,801.79.26NucleusType II
CaMADS79Caz06g2413034839,868.369.68NucleusType II
CaMADS80Caz04g0810024427,325.265.39NucleusType II
CaMADS81Caz02g2514023327,135.949.32NucleusType II
CaMADS82Caz08g1935024227,698.469.03NucleusType II
CaMADS83Caz04g1477030334,992.626.13NucleusType II
CaMADS84Caz03g1323018321,301.629.96NucleusType II
CaMADS85Caz12g2420019322,523.818.98NucleusType II
CaMADS86Caz04g0239022626,096.779.37NucleusType II
CaMADS87Caz02g2281022526,150.729.57NucleusType II
CaMADS88Caz01g2447028732,406.756.98NucleusType II
CaMADS89Caz08g1879026830,043.289.57NucleusType II
CaMADS90Caz12g0923020223,488.218.29NucleusType II
CaMADS91Caz10g1929021623,990.558.56NucleusType II
CaMADS92Caz12g0931023226,455.689.38CytoplasmType II
CaMADS93Caz04g1633020123,181.926.45NucleusType II
CaMADS94Caz12g0932019522,516.028.74NucleusType II
CaMADS95Caz12g0927020423,902.528.26NucleusType II
CaMADS96Caz12g0920019222,341.658.4NucleusType II
CaMADS97Caz12g0921019222,106.579.46NucleusType II
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MDPI and ACS Style

Zhu, J.; Meng, S.; Liu, J.; Zhang, T.; Cheng, Y.; Ruan, M.; Ye, Q.; Wang, R.; Yao, Z.; Zhou, G.; et al. Integrated Phylogenomic and Expression Analyses Reveal Lineage-Specific Loss of the Mβ Subfamily and Regulatory Diversification of MADS-Box Genes in Pepper. Plants 2026, 15, 620. https://doi.org/10.3390/plants15040620

AMA Style

Zhu J, Meng S, Liu J, Zhang T, Cheng Y, Ruan M, Ye Q, Wang R, Yao Z, Zhou G, et al. Integrated Phylogenomic and Expression Analyses Reveal Lineage-Specific Loss of the Mβ Subfamily and Regulatory Diversification of MADS-Box Genes in Pepper. Plants. 2026; 15(4):620. https://doi.org/10.3390/plants15040620

Chicago/Turabian Style

Zhu, Jiajun, Shibo Meng, Jia Liu, Ting Zhang, Yuan Cheng, Meiying Ruan, Qingjing Ye, Rongqing Wang, Zhuping Yao, Guozhi Zhou, and et al. 2026. "Integrated Phylogenomic and Expression Analyses Reveal Lineage-Specific Loss of the Mβ Subfamily and Regulatory Diversification of MADS-Box Genes in Pepper" Plants 15, no. 4: 620. https://doi.org/10.3390/plants15040620

APA Style

Zhu, J., Meng, S., Liu, J., Zhang, T., Cheng, Y., Ruan, M., Ye, Q., Wang, R., Yao, Z., Zhou, G., Li, Z., Liu, C., & Wan, H. (2026). Integrated Phylogenomic and Expression Analyses Reveal Lineage-Specific Loss of the Mβ Subfamily and Regulatory Diversification of MADS-Box Genes in Pepper. Plants, 15(4), 620. https://doi.org/10.3390/plants15040620

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