Next Article in Journal
Deficit Irrigation and Nitrogen Application Rate Influence Growth and Yield of Four Potato Cultivars (Solanum tuberosum L.)
Previous Article in Journal
The Influence of Bud Positions on the Changes in Carbohydrates and Nitrogen in Response to Hydrogen Cyanamide During Budbreak in Low-Chill Kiwifruit
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 11
Issue 7
10.3390/horticulturae11070848
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification and Analysis of the CCT Gene Family Contributing to Photoperiodic Flowering in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

by
Wei Fu
†,
Xinyu Jia
†,
Shanyu Li
,
Yang Zhou
,
Xinjie Zhang
,
Lisi Jiang
* and
Lin Hao
*
College of Life Science, Shenyang Normal University, Shenyang 110034, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(7), 848; https://doi.org/10.3390/horticulturae11070848
Submission received: 13 June 2025 / Revised: 9 July 2025 / Accepted: 16 July 2025 / Published: 17 July 2025
(This article belongs to the Special Issue Optimized Light Management in Controlled-Environment Horticulture)
Browse Figures
Versions Notes

Abstract

Photoperiod sensitivity significantly affects the reproductive process of plants. The CONSTANS, CONSTANS-LIKE, and TOC1 (CCT) genes play pivotal roles in photoperiod sensitivity and regulating flowering time. However, the function of the CCT gene in regulating flowering varies among different species. Further research is needed to determine whether it promotes or delays flowering under long-day (LD) or short-day (SD) conditions. CCT MOTIF FAMILY (CMF) belongs to one of the three subfamilies of the CCT gene and has been proven to be involved in the regulation of circadian rhythms and flowering time in cereal crops. In this study, 60 CCT genes in Chinese cabbage were genome-wide identified, and chromosomal localization, gene duplication events, gene structure, conserved domains, co-expression networks, and phylogenetic tree were analyzed by bioinformatics methods. The specific expression patterns of the BrCMF gene in different tissues, as well as the transcriptome and RT-qPCR results under different photoperiodic conditions, were further analyzed. The results showed that BrCMF11 was significantly upregulated in ebm5 under LD conditions, suggesting that BrCMF11 promoted flowering under LD conditions in Chinese cabbage. These findings revealed the function of the BrCCT gene family in photoperiod flowering regulation and provided a prominent theoretical foundation for molecular breeding in Chinese cabbage.

1. Introduction

Flowering is a key switch from vegetative to reproductive growth in flowering plants and acts as a prerequisite for crop production in agriculture [1]. Controlling flowering time has a significant impact on crop yield, as well as the adaptability of plants to diverse environmental conditions [2]. Flowering time is regulated by both endogenous and environmental signals, including the photoperiodic pathway, vernalization pathway, autonomous pathway, and gibberellin pathway [3]. The CCT domain gene has long been shown to control flowering time by suppressing the expression of some important photoperiodic regulators [4,5,6]. Originally, the CCT (CO, COL, and TOC1) motif was described as an approximately 45 amino acid-long conserved region in Arabidopsis thaliana [7,8]. Based on differences in motif composition, the CCT family genes were further classified into three subfamilies: CONSTANS-like (COL), PREUDORESPONSE REGULATOR (PRR), and CMF gene families. COL subfamily genes possess CCT and B-box domains. PRR subfamily genes possess CCT and response-regulator domains. CMF subfamily genes possess only one CCT domain [4,8].
To date, the CCT gene family has been genome-wide identified and analyzed in many species. Among these species, it has been proven that the CCT gene can control the flowering time and play a role in the regulation of circadian rhythms and photoperiodic adaptation. In Arabidopsis Thaliana, AtCCTs are important regulators of the photoperiodic flowering pathway, among which AtCO was the first cloned CCT gene in Arabidopsis to control flowering time and is also a COL subfamily gene [9,10]; CO protein binds to the FLOWERING LOCUS T (FT) promoter to accelerate flowering under LD photoperiods [11,12,13]. In rice (Oryza sativa), 41 CCT gene family members have been identified and divided into three subfamilies, of which 13 CCT family genes have been shown to respond to flowering pathways. For instance, the first flowering gene Heading Date 1 (Hd1) is the orthologue of CO that inhibits flowering under LD conditions and promotes flowering under SD conditions [14,15]; additionally, OsCOL4 was identified as a constitutive repressor regulating the upstream process of EH domain-containing protein 1 (Ehd1) to control flowering time [16]. In maize (Zea mays L.), 53 CCT family genes have been identified, 15 of which are significantly associated with the regulation of flowering time. Furthermore, ZmCOL3 acts as a flowering repressor, with overexpression delaying flowering time by approximately 4 days under both LD and SD conditions [17]; and CACTA-like transposable element (TE) was detected in the ZmCCT promoter, dramatically reducing flowering time by repressing ZmCCT expression to lower photoperiod sensitivity and facilitate maize adaptation to LD environments [18]. In the wheat (Triticum aestivum L.) genome, 127 TaCCTs have been identified, some of which, along with other photoperiodic genes (such as VRN2 and VRN1), are involved in the regulation of vernalization and flowering [19,20]. A total of 27 SlCCTs and 29 SmCCTs were identified in the tomato (Solanum lycopersicum L.) and eggplant (Solanum melongena L.) genomes, respectively, with both species containing significantly more G-box (light-responsive) elements than other cis-acting elements, suggesting their potential roles in light adaptation [21]. In Medicago truncatula, a total of 36 MtCCTs were identified, including 22 MtCCTs with typical circadian rhythmic variations, indicating their different responses to light [2]. In Chinese white pear (Pyrus bretschneideri Rehd.), a total of 42 PbCCTs were identified, among which some PbCCTs were sensitive to light induction, suggesting that PbCCTs are involved in adaptation to light environments; specifically, PbPRR2 was shown to negatively regulate photosynthetic performance under the enhanced red light environment [10]. In addition, the CCT gene also plays a role in responding to abiotic stress. In Brassica napus, a total of 87 CCT genes were identified at the whole-genome level, among which PRR subfamily genes are particularly important in regulating growth, development, and coping with abiotic stress [22]. In soybean (Glycine max), 19 GmCMF genes were identified, and GmCMF04 and GmCMF06 were, respectively, down-regulated and up-regulated after exogenous application of hormone SA and under drought stress [23]. The CMF gene belongs to one of the subfamilies of the CCT gene family. The CMF subfamily genes in cereal crops will delay the flowering process under LD conditions [24]. Additionally, in rice, OsCMF8 and OsCMF1 have been demonstrated to influence flowering time, plant height, and grain yield, and in barley, HvCMF7 and HvCMF3 have been shown to play a role in chloroplast translation regulation [25]. Therefore, the CMF genes of the CCT subfamily play a role in regulating various physiological processes. However, the CCT gene family contributing to photoperiodic flowering has not been genome-wide identified in Chinese cabbage, and the functions and specific mechanisms of its subfamily CMF in Chinese cabbage have not been fully reported.
The photoperiodic response serves as a critical regulatory mechanism governing the floral transition in B. rapa, exerting significant influence on both crop productivity and phenotypic quality. In the present study, bioinformatics methods were used to conduct a whole-genome analysis of CCT family genes in B. rapa under different photoperiod conditions. The chromosome distribution, gene structure, and conserved motifs of the BrCCT gene family were analyzed. Tissue-specific expression and RT-qPCR expression, along with transcriptome expression of BrCMF subfamily genes, were investigated. This study could help to deepen the understanding of the functions of the BrCCT gene family in photoperiod and flowering regulation and establish a foundation for further study.

2. Materials and Methods

2.1. Plant Materials, Growth Conditions, and Stress Treatments

The Chinese cabbage DH (doubled haploid) line seedling ‘FT’ and photoperiod-sensitive mutant ebm5 were prepared for this research. The emb5 mutant flowered significantly earlier than ‘FT’, and it was created by mutagenesis of germinating seeds of Chinese cabbage ‘FT’ with 0.8% EMS aqueous solution. It harbors a deletion mutation in the 12th exon of the mutant gene, resulting in a premature termination of translation [26]. The seeds were cultured in pots containing a soil:vermiculite mixture (3:1) in a growth chamber with controlled and different photoperiod environments (LD condition 16/8 h at 25/15 °C for day/night, SD condition 8/16 h at 25/15 °C for day/night, and relative humidity 55–60%). The leaves of the seedlings with consistent growth and healthy conditions at the ebm5 first flowering stage were collected after the above treatments. In addition, samples of other tissues such as roots, stems, flowers, buds, and pods of seedlings ‘FT’ were collected to facilitate the investigation of the expression of genes in different tissues. All samples were collected in three biological replicates, frozen in liquid nitrogen, and stored at −80 °C for subsequent RNA extraction experiments, transcriptome sequencing, and RT-qPCR.

2.2. Identification of the CCT Gene Family in Chinese Cabbage

The whole-genome DNA sequences, CDS sequences, amino acid sequences, and GFF3 format genome annotation information of CCT genes in Chinese cabbage, Arabidopsis, and rice were, respectively, downloaded from the Brassica database (BRAD, http://brassicadb.cn (accessed on 12 August 2024)), the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/download/index.jsp (accessed on 12 August 2024)), and Rice Genome Annotation Project (RGAP, http://rice.uga.edu/ (accessed on 12 August 2024)) [27]. Based on the GFF3 file, the multi-transcript genes were filtered, and the longest mRNA was selected as the representative sequence of the gene. Additionally, the CCT domains’ HMM (Hidden Markov Model) profiles (PF06203, PF00643, and PF00072) were initially acquired from Pfam (http://pfam.xfam.org/ (accessed on 13 August 2024)), and the HMMER (version 3.1b2) software package was used to detect the protein sequences containing CCT domains in Arabidopsis, Chinese cabbage, and rice [28]. Then, InterProScan (version 5.61–93.0) was used to align the protein sequences of the three species to verify the CCT functional domain [29]. And the molecular weight, isoelectric point (pI), amino acid length, and chromosome location information of the target protein sequence were calculated. The protein sequence of the CCT gene was analyzed for subcellular localization using the CELLO (http://cello.life.nctu.edu.tw/ (accessed on 15 August 2024)) online website.

2.3. Analysis of the Gene Location, Duplication Relationship, and Collinearity

The specific location information of CCT family genes on chromosomes was extracted from the GFF3 file of the Chinese cabbage genome. Possible duplicate gene types were identified using DupGen_finder-unique software [30]. Meanwhile, homologous duplication events between genes were identified based on the BLASTP comparison results. The MCScanX software of TBtools 2.0 (Multiple Collinearity Scan toolkit) was used to analyze the collinear relationship of Arabidopsis thaliana, Brassica rapa, and Oryza sativa [31]. The MCScanX parameter is -s 5.

2.4. Analysis of Gene Structure and Motif Structure

The position information of introns/exons of Chinese cabbage CCT family genes was extracted from the GFF3 file, and the structure prediction analysis of CCT genes was performed using the TBtools (version 0.665) software [32]. To explore the conserved sequence characteristics, the MEME Suite web server (https://meme-suite.org/meme/doc/meme.html (accessed on 23 August 2024)) was used to set the number of conserved motifs to 10, the maximum motif width to 50, and the minimum motif width to 6, while keeping other parameters at default settings. Finally, the obtained gene structure data, the protein conserved domain data, and the evolutionary tree data of the Chinese cabbage CCT protein were integrated and plotted on one figure through TBtools (version 0.665).

2.5. Co-Expression Network Analysis of the CCT Family Genes

Protein-protein interaction (PPI) analysis was conducted utilizing the STRING (Search3 Tool for the Retrieval of Interacting Genes/Proteins) developed by EMBL (European Molecular Biology Laboratory). The PPI pairs containing CCT family genes were screened, and the figure was drawn using Cytoscape 3.10.1.

2.6. Multiple Sequence Alignment and Phylogenetic Analysis

Using the Muscle algorithm, multiple sequence alignments on 38 AtCCT proteins from Arabidopsis, 37 OsCCT proteins from rice, and 60 BrCCT proteins from Chinese cabbage were conducted. The alignments were then visualized using the online tool ESPript 3 (http://espript.ibcp.fr/ESPript/ESPript/ (accessed on 26 August 2024)) [33]. With the MEGA7.0 software, the neighbor-joining (NJ) clustering algorithm was applied based on the results of the multiple sequence alignments to construct a phylogenetic tree for the CCT gene family. The number of bootstrap replicates was set to 1000 to assess the branch confidence, and all other parameters were maintained at the default settings of the program.

2.7. RNA Isolation, cDNA Synthesis, Transcriptome, and Quantitative Real-Time PCR Analysis

RNA extraction was carried out using the RNA Easy Fast Plant Tissue RNA Rapid Extraction Kit (TIANGEN, Beijing, China), and cDNA synthesis was performed using the Prime Script RT Reagent Kit (TaKaRa, Kusatsu, Japan). The high-throughput sequencing service for RNA samples was entrusted to Shanghai Baiqu Biomedical Technology Co., Ltd. The cDNA library construction and sequencing process were executed using the Illumina NovaseqTM 6000 testing system. To obtain high-quality, clean reads, Cutadapt (version 1.9) was used to further filter the reads. Subsequently, quantitative analysis of gene expression levels was conducted on the Lightcycler 96 RT-qPCR platform (Roche, Basel, Switzerland). Specific primers were designed using the NCBI database and synthesized by Shanghai Shenggong Technology Co., Ltd. (Shanghai, China). Actin was used as the reference gene, and the relative expression levels of genes were calculated by the 2−ΔΔCT method. The specific primer sequence information and the housekeeping gene information are shown in Supplementary Table S1.

3. Results

3.1. Flowering Time of Chinese Cabbage Under LD and SD Conditions

Under different photoperiod conditions, the bolting and flowering time of ‘FT’ and photoperiod-sensitive mutant ebm5 showed significant differences (Figure 1a). Under LD conditions, the bolting and flowering time of mutant ebm5 was 16 and 15 days earlier than that of wild type ‘FT’, respectively (Figure 1b). Under SD conditions, ebm5 bolting and flowering time were 29 and 30 days earlier than the wild type ‘FT’ (Figure 1b), but the flowering time was longer than that under LD conditions. Under the same photoperiod condition, ebm5 always had earlier bolting and flowering compared to ‘FT’. The actual phenotypic records confirmed the trait of the mutant ebm5 having an earlier flowering time.

3.2. Genome-Wide Identification and Physicochemical Property Analysis of the BrCCT Gene Family

To explore the relationship between the genome of Chinese cabbage and the regulatory mechanism of its flowering time, the CCT gene family contributing to photoperiodic flowering was selected for genome-wide identification and analysis. A total of 60 BrCCTs were identified in the Chinese cabbage genome (Table 1). According to sequence alignment, evolutionary relationship of gene, and conserved domain composition, these genes were divided into three subfamilies: BrCMFs (28 members), BrCOLs (23 members), and BrPRRs (9 members). They were named BrCMF1-28, BrCOL1-23, and BrPRR1-9 in ascending order based on their chromosomal positions.
Further analysis of the physicochemical properties of the encoded proteins (Table 1) showed that the length of the proteins encoded by BrCCTs ranged from 128 amino acids (BrCMF5) to 760 amino acids (BrPRR1), and the corresponding molecular weight ranged from 14,454.51 Da (BrCMF5) to 82,882.73 Da (BrPRR1). In addition, the theoretical isoelectric point (pI) value spanned from 4.41 (BrCMF15) to 10.50 (BrCMF5), with 73.3% (44/60) of proteins showing the pI value lower than 7, meaning that the majority of CCT proteins were rich in acidic amino acids.

3.3. Chromosomal Localization Analysis of the BrCCT Gene Family

Based on the chromosome annotation information of the B. rapa genome, the lengths of the identified BrCCTs were determined and subsequently mapped onto the chromosomes. As shown in Figure 2, 60 BrCCTs were distributed on 10 chromosomes of B. rapa; however, their distribution was uneven. Chromosomes A02 and A10 contained the largest number of CCTs, each containing 11 BrCCTs; A03 and A05 contained the second most CCTs, each containing 7 BrCCTs; while chromosomes A04, A06, and A07 contained the least number of CCTs, with only 3 BrCCTs each. The uneven distribution of BrCCTs on B. rapa chromosomes suggested that gene duplication or loss might have occurred during the evolutionary processes. Chromosomes containing more BrCCTs might have undergone more frequent gene duplication events, leading to BrCCT enrichment on these chromosomes.

3.4. Gene Duplication Analysis of the BrCCT Gene Family

Gene duplication is considered to be one of the primary driving forces in the evolution of genomes and genetic systems, as well as a major mechanism for generating new gene functions and establishing new evolutionary processes. The homology between pairwise BrCCTs was analyzed by the BLASTP comparison method. As shown in Figure 3, the identification of 52 homologous pairs among 60 BrCCTs provided direct evidence that gene duplication events had significantly contributed to the diversification and expansion of the BrCCT gene family.
Gene collinearity analysis can provide important clues for understanding the evolutionary history of species and gene function. Collinearity analysis was performed to identify collinear relationships of CCT genes between Brassica rapa and Arabidopsis thaliana, as well as between Brassica rapa and Oryza sativa. As shown in Figure 4, there was strong collinearity between Brassica rapa and Arabidopsis thaliana CCTs, whereas the collinearity between Brassica rapa and Oryza sativa CCTs was weak, with only five collinear gene pairs identified. These results indicated that the relationship between Brassica rapa and Arabidopsis thaliana was relatively closer, having a higher degree of conservation in the structure and function of the CCT gene; It reflected the maintenance of core functions in the Brassicaceae family during their evolutionary process. The lower collinearity between Brassica rapa and Oryza sativa highlighted the profound differentiation between monocotyledonous and dicotyledonous plants. And there might be more gene family expansion events between Brassica rapa and Arabidopsis thaliana.

3.5. Gene Structure Analysis of the BrCCT Gene Family

The diversity of gene structure has an important impact on gene evolution. To better understand the structural characteristics of BrCCTs, the present study conducted a detailed analysis of exon/intron positions within the CCT family genes in B. rapa, focusing on their conservation and diversity. As illustrated in Figure 5, there were significant gene structural differences among the three subfamilies in B. rapa, while the gene structure within each subfamily showed remarkable consistency with minimal internal variation. The results in Figure 5a,c showed that the number of exons and introns varied significantly among BrCCTs. The number of exons varied from as few as one (BrCOL22 or BrCOL2) to as many as ten (BrPRR8). The variation range of exon number in the PRR subfamily was 4–10; the number of exons in the CMF subfamily varied from 2 to 7; while the number of exons in the COL subfamily varied from 1 to 4. Concretely, of sixty BrCCTs, four BrCCTs (BrPRR6, BrCMF5, BrCMF15, BrCMF23) contained 5 exons; seven BrCCTs (BrPRR2, BrPRR3, BrPRR7, BrCMF16, BrCMF21, BrCMF22, BrCMF25) contained 6 exons; the six BrCCTs (BrPRR5, BrCMF1, BrCMF3, BrCMF12, BrCMF17, and BrCMF19) contained 7 exons. In addition, the number of introns varied from 1 (BrCOL11 or BrCOL18) to 10 (BrPRR8), and the intron of BrCMF4 was much longer than that of other BrCCTs. With the evolution of the BrCCT gene family, the number of introns and exons in BrCCTs increased or decreased; the diversity of gene structure indicated that the BrCCT gene family might have experienced some non-essential functional changes in the process of evolution.

3.6. Conserved Motif Analysis of the BrCCT Gene Family

In the sequences of 60 BrCCTs, a total of 6 conserved motifs were identified and named Motif 1 to Motif 6, respectively. Based on the analysis in Figure 5a,b, of the 60 BrCCTs, 57 BrCCTs contained Motif 1, and 59 BrCCTs contained Motif 3. In the BrCMF subfamily, BrCMF15 only contained Motif 1; all the other 27 BrCMFs contained Motif 1 and Motif 3; in addition, BrCMF1 also contained Motif 5. The Motif composition was different in other subfamilies but was similar within the same subfamily. For instance, all BrPRRs contained Motif 4 and Motif 6; all BrCOLs contained Motif 2, and more than half of BrCOLs contained Motif 5. The variation of motif composition among different subfamilies indicated that the CCT gene family had internal diversification during evolution. At the same time, the similarity of conserved motifs found in each subfamily indicated that the members of the same subfamily were composed of similar conserved domains, which might have similar biological functions.

3.7. Co-Expression Network Analysis of the BrCCT Gene Family

In order to obtain the interaction between BrCCT genes and other genes, the co-expression network was established. In general, the more target genes a gene regulates, the more important its position in the regulatory network. As shown in Figure 6, in the BrCMF subfamily, only BrCMF5 and BrCMF19 regulated multiple genes; in the BrCOL subfamily, BrCOL2, BrCOL5, and BrCOL23 regulated multiple genes; while in the BrPRR subfamily, all genes could regulate multiple other genes. These results indicated that different BrCCTs were specific in their regulatory functions and that BrPRRs were at the core of the regulatory network and played a potential key role in the physiological and biochemical metabolism regulated by BrCCTs.

3.8. Multiple Sequence Alignment and Phylogenetic Tree Analysis of the BrCCT Gene Family

Multiple sequence alignment of BrCCT amino acid sequences was performed. The results (Figure S1) showed that the similarity of many amino acid sites was higher than 0.7 (marked in red), which was considered to be highly similar, indicating that protein sequences were highly conserved in BrCCTs, thereby expressing similar biological functions.
In order to deeply understand the evolution process within the BrCCT gene family, the obtained 60 CCT protein sequences of B. rapa were compared with 38 A. thaliana CCT protein sequences and 37 O. sativa CCT protein sequences. Based on sequence similarity, the CCT phylogenetic tree was constructed after multisequence comparison analysis. As shown in Figure 7, the CCT family genes of B. rapa, A. thaliana, and O. sativa were all distributed in three subfamilies: COL, CMF, and PRR, showing high homology, indicating that the CCT family genes were highly conserved in the process of species differentiation. At the same time, it demonstrated that the CCT family genes of the three species might perform similar molecular functions in the physiological processes such as photoperiod perception, flowering time regulation, and circadian rhythm maintenance. The phylogenetic tree (Figure 7) also showed that BrCCT family genes are unevenly distributed in the three subfamilies, and the BrCMF subfamily contained the largest number of genes, with 28 genes, suggesting that BrCCT family genes might have experienced gene duplication events during the evolution process, which was consistent with the analysis results in Section 3.4, where it was shown that gene duplication events significantly promoted the diversification and expansion of the BrCCT gene family.

3.9. Transcriptome Analysis and Expression Analysis in Different Tissues of BrCMF Genes

Comparing the FPKM values of wild-type ‘FT’ and mutant ebm5 in transcripts of three subfamilies, it was found that the number of genes with increased expression of mutant ebm5 relative to wild-type ‘FT’ in the CMF subfamily accounted for the highest proportion, so the CMF subfamily was selected for subsequent analysis. To verify the expression of BrCMF members in wild-type ‘FT’ and photoperiod-sensitive mutant ebm5 under LD conditions, RNA-seq sequencing was performed on leaves. Most of the results in Figure 8b were basically consistent with the results of RT-qPCR, but the expression levels of some genes were slightly different from RT-qPCR, possibly owing to the inconsistency of environmental conditions and sampling time during Chinese cabbage seedling culture.
The expression of 28 BrCMF subfamily genes in six tissues of root, stem, leaf, flower, bud, and pod was analyzed to explore the tissue-specific expression of BrCMF subfamily genes. The results were shown in Figure 8a, in root tissues, 27 BrCMFs were highly expressed; in stem tissues, BrCMF4, BrCMF10, BrCMF16, and BrCMF28 were highly expressed; in leaf tissues, BrCMF4, BrCMF7, BrCMF25, and BrCMF28 were highly expressed; in flower tissues, BrCMF1, BrCMF15, BrCMF22, and BrCMF23 with higher expression levels, especially the expression level of BrCMF1 was abnormally significant among all BrCMF genes; in bud tissues, the expression levels of BrCMF1, BrCMF18, and BrCMF28 were higher; in pod tissues, the expression levels of BrCMF1, BrCMF7, and BrCMF10 were higher. The above analysis demonstrated that BrCMF subfamily genes were expressed in six tissues of B. rapa, but the expression levels were significantly different.

3.10. Expression Profiles Analysis of BrCMF Genes Under LD and SD Conditions

To understand the expression patterns of BrCMFs under different photoperiodic conditions, the present study selected 28 BrCMFs for RT-qPCR. As shown in Figure 9, compared to the wild type ‘FT’, in the photoperiod sensitive mutant ebm5, the expression levels of BrCMF6, BrCMF10, BrCMF21, and BrCMF27 were significantly increased under LD conditions. In the SD condition, the expression levels of BrCMF5, BrCMF6, BrCMF18, and BrCMF21 in mutant ebm5 were significantly upregulated (Figure 9). In general, under LD/SD, both BrCMF6 and BrCMF21 were significantly upregulated in mutant ebm5, suggesting that the expression of these two genes was photoperiod-insensitive. The expression of BrCMF3, BrCMF12–15 in mutant ebm5 was not significantly different under LD, but significantly decreased under SD, which may play a role in the delayed flowering of SD in Chinese cabbage. However, the LD expression level of BrCMF11 was significantly increased in ebm5 and significantly decreased in SD, indicating that this gene is sensitive to photoperiodic response to flowering. The expression of BrCMF11 in mutant ebm5 was significantly upregulated under LD compared to ‘FT’, which may promote flowering under LD in Chinese cabbage.

4. Discussion

Chinese cabbage (Brassica rapa L. ssp. pekinensis), an important leafy vegetable belonging to the Brassicaceae family, is widely cultivated worldwide, and it shares a close genomic relationship with Arabidopsis [34]. Whole-genome data from Chinese cabbage enable comprehensive characterization and functional annotation of genes in this important crop species. The CCT gene family is widely present in plants and regulates essential plant processes, including flowering time, circadian rhythms, growth development, and stress responses [25]. With the availability of whole-genome data for many plants, CCT gene families have been comprehensively analyzed in the genomes of Chinese white pear, wheat, soybean, rice, foxtail millet, and pigeonpea [13,22,23,35,36,37]. So far, the CCT gene regulating photoperiodic flowering in Chinese cabbage has not been fully studied. The present study selected potential CCT genes from the Chinese cabbage genome database and systematically identified and analyzed these genes using bioinformatics methods.
In this study, a total of 60 BrCCTs were genome-wide identified. More than 70 percent (44/60) of BrCCT proteins had theoretical isoelectric point (pI) values below 7, suggesting that most BrCCT proteins were rich in acidic amino acids and might function in acidic subcellular environments. Whole genome duplication events had important effects on the structural evolution of eukaryotic genomes [38]. The results of gene duplication in the present study showed that 52 homologous gene pairs were formed, indicating that BrCCTs might have been duplicated between different chromosomes during evolution. The structure of BrCCT genes showed diversity characteristics; the number of exons in the 60 BrCCTs ranged from 1 to 10, and the number of introns was also constantly changing. This suggested that the BrCCT genes might experience exon and intron loss or insertion events during evolution. Notably, most BrCCTs were composed of 2 to 4 exons, and this structural feature was similar to the CCT gene structure observed in other crops. Six different conserved motifs were identified, which provided the key basis for the classification of CCT genes. In the same subfamily, the gene structure and conserved motifs were similar, but there were significant differences among different subfamilies. Additionally, multiple sequence alignment was widely employed in comparative genomics and protein studies to elucidate conserved structural and functional elements in biological sequences [39]. In this study, there were two highly conserved amino acid sites and many similar sites in BrCCTs. These conserved sites might have similar and important biological functions. In phylogenetic analysis, the CCT family genes of B. rapa, A. thaliana, and O. sativa were all distributed in three subfamilies: COL, CMF, and PRR, showing high homology. Among the 60 CCTs of B. rapa, the CMF subfamily had the most members.
The tissue-specific expression of BrCMF genes reflected the complexity of the BrCCT genes’ regulatory network. In RT-qPCR expression analysis, some BrCMFs showed different expression patterns in response to LD and SD conditions. For instance, BrCMF8, BrCMF11, BrCMF20, and BrCMF27 increased their expression levels under LD conditions, but decreased their expression levels under SD conditions. Transcriptome analysis verified the expression of BrCMFs under LD conditions. In transcriptome analysis, the expression levels of many BrCMFs were similar in photoperiod sensitive mutant ebm5 and wild type ‘FT’, and the expression levels of BrCMF10 and BrCMF21 were up-regulated in photoperiod sensitive mutant ebm5 compared with those in wild type ‘FT’, which was basically consistent with the results of RT-qPCR. Therefore, it was reasonable to speculate that some BrCMFs might be involved in the flowering process of B. rapa, such as the development of flowering organs, the determination of flowering time, and the regulation of flowering duration. Under LD/SD conditions, the difference in flowering time between ‘FT’ and ebm5 was associated with the expression pattern of the BrCMF gene. Under LD conditions, the expression differences of BrCMF subfamily genes were significant. In the photoperiod-sensitive mutant ebm5, compared with the wild-type ‘FT’, the expression levels of BrCMF6, BrCMF10, BrCMF21, and BrCMF27 significantly increased under LD, and the expression levels of BrCMF5, BrCMF6, BrCMF18, and BrCMF21 significantly increased under SD. These research results indicated that the BrCMF subfamily genes were involved in regulating the flowering mechanism of Chinese cabbage under different photoperiods. Members of the CCT gene family are central to the molecular mechanisms governing photoperiod sensing and floral transition in plants. This study revealed the potential function of the BrCMF gene family in the photoperiod regulation of flowering and provided valuable information for subsequent functional studies and molecular breeding. For instance, increasing the expression of BrCMF11 in Chinese cabbage may accelerate the flowering time of the plant. The functional validation experiment of the gene will be the focus of future research.

5. Conclusions

In short, 60 BrCCTs were genome-wide identified in Chinese cabbage and were divided into three subfamilies according to sequence alignment and evolutionary relationship. Structural difference analysis indicated that the BrCCT gene family might have diverse functions during evolution. Expression profiles analysis showed that BrCMF11 was significantly upregulated in ebm5 under LD, indicating that BrCMF11 might promote early flowering under LD conditions in Chinese cabbage. All the results presented in this study demonstrated that the BrCCT gene family constituted an essential component of the photoperiodic flowering regulatory network and laid a foundation for further study of the functional mechanism of CCT genes in photoperiodic flowering in Chinese cabbage.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11070848/s1: Table S1. Chinese cabbage CCT primer and housekeeping genes; Table S2. Differential expression genes; Figure S1. The multisequence alignment of the BrCCT protein family.

Author Contributions

Methodology, W.F., X.J., S.L., Y.Z. and X.Z.; formal analysis, X.J. and L.H.; investigation, X.J., S.L., Y.Z. and X.Z.; data curation, X.J. and L.J.; writing—original draft preparation, W.F. and X.J.; writing—reviewing and editing, W.F. and L.H.; supervision, L.H.; project administration, W.F. and L.H.; funding acquisition, W.F., L.J. and L.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant No. 32202482, Grant No. 42307186, Grant No. 31572213), and the Liaoning Provincial universities Basic Research funds special fund (LJ202410166039, LJ202410166041).

Data Availability Statement

The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CCTCONSTANS, CONSTANS-LIKE and TOC1
LDlong-day
SDshort-day
CMFCCT MOTIF FAMILY
COLCONSTANS-like
PRRPREUDORESPONSE REGULATOR
FTFLOWERING LOCUS T
Hd1Heading Date 1
Ehd1EH domain-containing protein 1
TEtransposable element
DHdoubled haploid
TAIRthe Arabidopsis Information Resource
RGAPRice Genome Annotation Project
PPIProtein-protein interaction
aaAmino acids
pIIsoelectric point
MWMolecular weight
DaDalton
MbMegabases
UTRsUntranslated regions
CDSCoding DNA Sequence

References

  1. Blümel, M.; Dally, N.; Jung, C. Flowering time regulation in crops—What did we learn from Arabidopsis? Curr. Opin. Biotechnol. 2015, 32, 121–129. [Google Scholar] [CrossRef] [PubMed]
  2. Ma, L.; Yi, D.; Yang, J.; Liu, X.; Pang, Y. Genome-wide identification, expression analysis and functional study of CCT gene family in Medicago truncatula. Plants 2020, 9, 513. [Google Scholar] [CrossRef] [PubMed]
  3. Pajoro, A.; Biewers, S.; Dougali, E.; Leal Valentim, F.; Mendes, M.A.; Porri, A.; Coupland, G.; Van de Peer, Y.; van Dijk, A.D.; Colombo, L.; et al. The (r)evolution of gene regulatory networks controlling Arabidopsis plant reproduction: A two-decade history. J. Exp. Bot. 2014, 65, 4731–4745. [Google Scholar] [CrossRef] [PubMed]
  4. Cockram, J.; Thiel, T.; Steuernagel, B.; Stein, N.; Taudien, S.; Bailey, P.C.; O’Sullivan, D.M. Genome dynamics explain the evolution of flowering time CCT domain gene families in the Poaceae. PLoS ONE 2012, 7, e45307. [Google Scholar] [CrossRef] [PubMed]
  5. Liu, C.; Qu, X.; Zhou, Y.; Song, G.; Abiri, N.; Xiao, Y.; Liang, F.; Jiang, D.; Hu, Z.; Yang, D. Osprr37 confers an expanded regulation of the diurnal rhythms of the transcriptome and photoperiodic flowering pathways in rice. Plant Cell Environ. 2018, 41, 630–645. [Google Scholar] [CrossRef] [PubMed]
  6. Yan, W.; Liu, H.; Zhou, X.; Li, Q.; Zhang, J.; Lu, L.; Liu, T.; Liu, H.; Zhang, C.; Zhang, Z.; et al. Natural variation in ghd7.1 plays an important role in grain yield and adaptation in rice. Cell Res. 2013, 23, 969–971. [Google Scholar] [CrossRef] [PubMed]
  7. Strayer, C.; Oyama, T.; Schultz, T.F.; Raman, R.; Somers, D.E.; Más, P.; Panda, S.; Kreps, J.A.; Kay, S.A. Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog. Science 2000, 289, 768–771. [Google Scholar] [CrossRef] [PubMed]
  8. Zhang, L.; Li, Q.; Dong, H.; He, Q.; Liang, L.; Tan, C.; Han, Z.; Yao, W.; Li, G.; Zhao, H.; et al. Three CCT domain-containing genes were identified to regulate heading date by candidate gene-based association mapping and transformation in rice. Sci. Rep. 2015, 5, 7663. [Google Scholar] [CrossRef] [PubMed]
  9. An, H.; Roussot, C.; Suárez-López, P.; Corbesier, L.; Vincent, C.; Piñeiro, M.; Hepworth, S.; Mouradov, A.; Justin, S.; Turnbull, C.; et al. Constans acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis. Development 2004, 131, 3615–3626. [Google Scholar] [CrossRef] [PubMed]
  10. Liu, Z.; Liu, J.L.; An, L.; Wu, T.; Yang, L.; Cheng, Y.S.; Nie, X.S.; Qin, Z.Q. Genome-wide analysis of the CCT gene family in chinese white pear (Pyrus bretschneideri rehd.) and characterization of pbprr2 in response to varying light signals. BMC Plant Biol. 2022, 22, 81. [Google Scholar] [CrossRef] [PubMed]
  11. Tiwari, S.B.; Shen, Y.; Chang, H.C.; Hou, Y.; Harris, A.; Ma, S.F.; McPartland, M.; Hymus, G.J.; Adam, L.; Marion, C.; et al. The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element. New Phytol. 2010, 187, 57–66. [Google Scholar] [CrossRef] [PubMed]
  12. Suárez-López, P.; Wheatley, K.; Robson, F.; Onouchi, H.; Valverde, F.; Coupland, G. CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis. Nature 2001, 410, 1116–1120. [Google Scholar] [CrossRef] [PubMed]
  13. Robson, F.; Costa, M.M.; Hepworth, S.R.; Vizir, I.; Piñeiro, M.; Reeves, P.H.; Putterill, J.; Coupland, G. Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants. Plant J. 2001, 28, 619–631. [Google Scholar] [CrossRef] [PubMed]
  14. Zhang, J.; Hu, Y.; Xu, L.-H.; He, Q.; Fan, X.-W.; Xing, Y.-Z. The CCT domain-containing gene family has large impacts on heading date, regional adaptation, and grain yield in rice. J. Integr. Agric. 2017, 16, 2686–2697. [Google Scholar] [CrossRef]
  15. Yano, M.; Katayose, Y.; Ashikari, M.; Yamanouchi, U.; Monna, L.; Fuse, T.; Baba, T.; Yamamoto, K.; Umehara, Y.; Nagamura, Y.; et al. Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell 2000, 12, 2473–2484. [Google Scholar] [CrossRef] [PubMed]
  16. Lee, Y.S.; Jeong, D.H.; Lee, D.Y.; Yi, J.; Ryu, C.H.; Kim, S.L.; Jeong, H.J.; Choi, S.C.; Jin, P.; Yang, J.; et al. Oscol4 is a constitutive flowering repressor upstream of Ehd1 and downstream of osphyB. Plant J. 2010, 63, 18–30. [Google Scholar] [CrossRef] [PubMed]
  17. Jin, M.; Liu, X.; Jia, W.; Liu, H.; Li, W.; Peng, Y.; Du, Y.; Wang, Y.; Yin, Y.; Zhang, X.; et al. Zmcol3, a CCT gene represses flowering in maize by interfering with the circadian clock and activating expression of ZmCCT. J. Integr. Plant Biol. 2018, 60, 465–480. [Google Scholar] [CrossRef] [PubMed]
  18. Yang, Q.; Li, Z.; Li, W.; Ku, L.; Wang, C.; Ye, J.; Li, K.; Yang, N.; Li, Y.; Zhong, T.; et al. CACTA-like transposable element in ZmCCT attenuated photoperiod sensitivity and accelerated the postdomestication spread of maize. Proc. Natl. Acad. Sci. USA 2013, 110, 16969–16974. [Google Scholar] [CrossRef] [PubMed]
  19. Zhang, H.; Jiao, B.; Dong, F.; Liang, X.; Zhou, S.; Wang, H. Genome-wide identification of CCT genes in wheat (Triticum aestivum L.) and their expression analysis during vernalization. PLoS ONE 2022, 17, e0262147. [Google Scholar] [CrossRef] [PubMed]
  20. Yang, Y.; Zhang, X.; Wu, L.; Zhang, L.; Liu, G.; Xia, C.; Liu, X.; Kong, X. Transcriptome profiling of developing leaf and shoot apices to reveal the molecular mechanism and co-expression genes responsible for the wheat heading date. BMC Genom. 2021, 22, 468. [Google Scholar] [CrossRef] [PubMed]
  21. Cai, L.; Xiang, R.; Jiang, Y.; Li, W.; Yang, Q.; Gan, G.; Li, W.; Yu, C.; Wang, Y. Genome-wide identification and expression profiling analysis of the CCT gene family in Solanum lycopersicum and Solanum melongena. Genes 2024, 15, 1385. [Google Scholar] [CrossRef] [PubMed]
  22. Yu, L.; Xia, J.; Jiang, R.; Wang, J.; Yuan, X.; Dong, X.; Chen, Z.; Zhao, Z.; Wu, B.; Zhan, L.; et al. Genome-wide identification and characterization of the CCT gene family in rapeseed (Brassica napus L.). Int. J. Mol. Sci. 2024, 25, 5301. [Google Scholar] [CrossRef] [PubMed]
  23. Mengarelli, D.A.; Zanor, M.I. Genome-wide characterization and analysis of the CCT motif family genes in soybean (Glycine max). Planta 2021, 253, 15. [Google Scholar] [CrossRef] [PubMed]
  24. Liu, H.; Zhou, X.; Li, Q.; Wang, L.; Xing, Y. CCT domain-containing genes in cereal crops: Flowering time and beyond. Theor. Appl. Genet. 2020, 133, 1385–1396. [Google Scholar] [CrossRef] [PubMed]
  25. Yang, C.Y.; Yan, W.Y.; Chang, H.Y.; Sun, C.W. Arabidopsis CIA2 and CIL have distinct and overlapping functions in regulating chloroplast and flower development. Plant Direct 2022, 6, e380. [Google Scholar] [CrossRef] [PubMed]
  26. Fu, W.; Huang, S.; Gao, Y.; Zhang, M.; Qu, G.; Wang, N.; Liu, Z.; Feng, H. Role of BrSDG8 on bolting in Chinese cabbage (Brassica rapa). Theor. Appl. Genet. 2020, 133, 2937–2948. [Google Scholar] [CrossRef] [PubMed]
  27. Chen, H.; Wang, T.; He, X.; Cai, X.; Lin, R.; Liang, J.; Wu, J.; King, G.; Wang, X. Brad v3.0: An upgraded Brassicaceae database. Nucleic Acids Res. 2022, 50, D1432–D1441. [Google Scholar] [CrossRef] [PubMed]
  28. Potter, S.C.; Luciani, A.; Eddy, S.R.; Park, Y.; Lopez, R.; Finn, R.D. HMMER web server: 2018 update. Nucleic Acids Res. 2018, 46, W200–W204. [Google Scholar] [CrossRef] [PubMed]
  29. Jones, P.; Binns, D.; Chang, H.Y.; Fraser, M.; Li, W.; McAnulla, C.; McWilliam, H.; Maslen, J.; Mitchell, A.; Nuka, G.; et al. InterProscan 5: Genome-scale protein function classification. Bioinformatics 2014, 30, 1236–1240. [Google Scholar] [CrossRef] [PubMed]
  30. Qiao, X.; Li, Q.; Yin, H.; Qi, K.; Li, L.; Wang, R.; Zhang, S.; Paterson, A.H. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol. 2019, 20, 38. [Google Scholar] [CrossRef] [PubMed]
  31. Wang, Y.; Tang, H.; Debarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.H.; Jin, H.; Marler, B.; Guo, H.; et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef] [PubMed]
  32. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant. 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  33. Edgar, R.C. Muscle: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [CrossRef] [PubMed]
  34. Nie, S.; Zhang, M.; Zhang, L. Genome-wide identification and expression analysis of calmodulin-like (CML) genes in Chinese cabbage (Brassica rapa L. Ssp. Pekinensis). BMC Genom. 2017, 18, 842. [Google Scholar] [CrossRef] [PubMed]
  35. Peng, X.; Tun, W.; Dai, S.F.; Li, J.Y.; Zhang, Q.J.; Yin, G.Y.; Yoon, J.; Cho, L.H.; An, G.; Gao, L.Z. Genome-wide analysis of CCT transcript factors to identify genes contributing to photoperiodic flowering in Oryza rufipogon. Front. Plant Sci. 2021, 12, 736419. [Google Scholar] [CrossRef] [PubMed]
  36. Li, C.; Ma, J.; Wang, G.; Li, H.; Wang, H.; Wang, G.; Jiang, Y.; Liu, Y.; Liu, G.; Liu, G.; et al. Exploring the SiCCT gene family and its role in heading date in foxtail millet. Front. Plant Sci. 2022, 13, 863298. [Google Scholar] [CrossRef] [PubMed]
  37. Tribhuvan, K.U.; Kaila, T.; Srivastava, H.; Das, A.; Kumar, K.; Durgesh, K.; Joshi, R.; Singh, B.K.; Singh, N.K.; Gaikwad, K. Structural and functional analysis of CCT family genes in pigeonpea. Mol. Biol. Rep. 2022, 49, 217–226. [Google Scholar] [CrossRef] [PubMed]
  38. Chapman, B.A.; Bowers, J.E.; Schulze, S.R.; Paterson, A.H. A comparative phylogenetic approach for dating whole genome duplication events. Bioinformatics 2004, 20, 180–185. [Google Scholar] [CrossRef] [PubMed]
  39. Pirovano, W.; Heringa, J. Multiple sequence alignment. Methods Mol. Biol. 2008, 452, 143–161. [Google Scholar] [PubMed]
Horticulturae 11 00848 g001
Figure 1. (a) Phenotype of the Chinese cabbage ‘FT’ and ebm5 under LD and SD conditions. Bar = 1 cm (b) Flowering time of the Chinese cabbage ‘FT’ and ebm5 under LD and SD conditions. The *** on the column indicates the significance of the difference compared with the control (p < 0.001).
Figure 1. (a) Phenotype of the Chinese cabbage ‘FT’ and ebm5 under LD and SD conditions. Bar = 1 cm (b) Flowering time of the Chinese cabbage ‘FT’ and ebm5 under LD and SD conditions. The *** on the column indicates the significance of the difference compared with the control (p < 0.001).
Horticulturae 11 00848 g001
Horticulturae 11 00848 g002
Figure 2. The chromosomal distribution of CCT genes in B. rapa. The chromosomal distribution of BrCCT genes across Chinese cabbage chromosomes. The BrCCT gene locations are marked on the right side of the chromosome. Chromosome lengths are scaled in megabases (Mb).
Figure 2. The chromosomal distribution of CCT genes in B. rapa. The chromosomal distribution of BrCCT genes across Chinese cabbage chromosomes. The BrCCT gene locations are marked on the right side of the chromosome. Chromosome lengths are scaled in megabases (Mb).
Horticulturae 11 00848 g002
Horticulturae 11 00848 g003
Figure 3. Gene duplication analysis of CCT genes in B. rapa. A schematic representation of gene duplication events among BrCCT genes. The ten Chinese cabbage chromosomes are arranged in a circle. The colored lines indicate duplicated gene pairs.
Figure 3. Gene duplication analysis of CCT genes in B. rapa. A schematic representation of gene duplication events among BrCCT genes. The ten Chinese cabbage chromosomes are arranged in a circle. The colored lines indicate duplicated gene pairs.
Horticulturae 11 00848 g003
Horticulturae 11 00848 g004
Figure 4. Collinearity analysis of CCT genes between B. rapa and two representative plant species (A. thaliana and O. sativa). Gray lines in the background represent collinear blocks in B. rapa and other genomes. The collinear gene pairs with CCT genes between different species were highlighted by the green lines.
Figure 4. Collinearity analysis of CCT genes between B. rapa and two representative plant species (A. thaliana and O. sativa). Gray lines in the background represent collinear blocks in B. rapa and other genomes. The collinear gene pairs with CCT genes between different species were highlighted by the green lines.
Horticulturae 11 00848 g004
Horticulturae 11 00848 g005
Figure 5. Phylogenetic tree, compositions of the conserved protein motifs, and gene structures of BrCCT genes in B. rapa. (a) A phylogenetic tree was constructed based on 60 BrCCT proteins divided into three groups. (b) Conserved motifs of the BrCCT proteins; the conservative motif of the BrCCT gene is predicted by the MEME method, the numbers (1–6) in the colored rectangles indicate motif 1–6, and the length of the rectangle indicates the size of the motif; (c) Exons-introns and untranslated regions (UTRs) of BrCCT genes. Green boxes indicate exons; black horizontal lines indicate introns; blue boxes represent UTRs. The length of the protein can be estimated using the scale at the bottom.
Figure 5. Phylogenetic tree, compositions of the conserved protein motifs, and gene structures of BrCCT genes in B. rapa. (a) A phylogenetic tree was constructed based on 60 BrCCT proteins divided into three groups. (b) Conserved motifs of the BrCCT proteins; the conservative motif of the BrCCT gene is predicted by the MEME method, the numbers (1–6) in the colored rectangles indicate motif 1–6, and the length of the rectangle indicates the size of the motif; (c) Exons-introns and untranslated regions (UTRs) of BrCCT genes. Green boxes indicate exons; black horizontal lines indicate introns; blue boxes represent UTRs. The length of the protein can be estimated using the scale at the bottom.
Horticulturae 11 00848 g005
Horticulturae 11 00848 g006
Figure 6. Co-expression network of CCT genes in B. rapa. The green ellipses represent BrPRR subfamily genes, the red ellipses represent BrCOL subfamily genes, the blue ellipses represent BrCMF subfamily genes, while the grey ellipses represent genes co-expressed with BrCCT.
Figure 6. Co-expression network of CCT genes in B. rapa. The green ellipses represent BrPRR subfamily genes, the red ellipses represent BrCOL subfamily genes, the blue ellipses represent BrCMF subfamily genes, while the grey ellipses represent genes co-expressed with BrCCT.
Horticulturae 11 00848 g006
Horticulturae 11 00848 g007
Figure 7. Phylogenetic tree of B. rapa, A. thaliana, and O. sativa CCT proteins. The phylogenetic tree was constructed using the Maximum Likelihood method based on the amino acid sequences of CCT genes. Bootstrap values (shown on the branches) represent the confidence levels from 1000 replicates. The CCT genes are classified into three subfamilies (CMF, COL, and PRR) and highlighted in distinct colors. CMF subfamily is shown in blue, COL subfamily in red, and PRR subfamily in green. The various icons represent the CCT genes in diverse species, with red stars representing B. rapa, blue circles representing A. thaliana, and green squares representing O. sativa.
Figure 7. Phylogenetic tree of B. rapa, A. thaliana, and O. sativa CCT proteins. The phylogenetic tree was constructed using the Maximum Likelihood method based on the amino acid sequences of CCT genes. Bootstrap values (shown on the branches) represent the confidence levels from 1000 replicates. The CCT genes are classified into three subfamilies (CMF, COL, and PRR) and highlighted in distinct colors. CMF subfamily is shown in blue, COL subfamily in red, and PRR subfamily in green. The various icons represent the CCT genes in diverse species, with red stars representing B. rapa, blue circles representing A. thaliana, and green squares representing O. sativa.
Horticulturae 11 00848 g007
Horticulturae 11 00848 g008
Figure 8. Expression analysis in different tissues and transcriptome analysis of BrCMF genes. (a) Expression profiles of BrCMF genes in different tissues. Heat map showing the expression profiles of 28 BrCMF genes in various tissues, including root, stem, leaf, flower, bud, and pod. The samples are grouped based on tissue type. High expression levels are shown in dark orange, and low expression levels are shown in light orange. (b) Transcriptome analysis of BrCMF genes. Heat map illustrating the expression profiles of BrCMF genes in ‘FT’ and ebm5 under LD condition. High expression levels are shown in dark orange, and low expression levels are shown in light orange.
Figure 8. Expression analysis in different tissues and transcriptome analysis of BrCMF genes. (a) Expression profiles of BrCMF genes in different tissues. Heat map showing the expression profiles of 28 BrCMF genes in various tissues, including root, stem, leaf, flower, bud, and pod. The samples are grouped based on tissue type. High expression levels are shown in dark orange, and low expression levels are shown in light orange. (b) Transcriptome analysis of BrCMF genes. Heat map illustrating the expression profiles of BrCMF genes in ‘FT’ and ebm5 under LD condition. High expression levels are shown in dark orange, and low expression levels are shown in light orange.
Horticulturae 11 00848 g008
Horticulturae 11 00848 g009aHorticulturae 11 00848 g009b
Figure 9. Expression analyses of BrCMF genes in ‘FT’ and ebm5 under LD and SD conditions. RT-qPCR analyses of BrCMF genes in ‘FT’ and ebm5 under LD and SD conditions. * p < 0.05, ** p < 0.01, ns indicates no significant difference.
Figure 9. Expression analyses of BrCMF genes in ‘FT’ and ebm5 under LD and SD conditions. RT-qPCR analyses of BrCMF genes in ‘FT’ and ebm5 under LD and SD conditions. * p < 0.05, ** p < 0.01, ns indicates no significant difference.
Horticulturae 11 00848 g009aHorticulturae 11 00848 g009b
Table 1. Physiological characteristics of the CCT gene family in Brassica rapa.
Table 1. Physiological characteristics of the CCT gene family in Brassica rapa.
Gene NameGene IDChromosomeProtein (aa)pIMW (Da)
BrCMF1BraA01g015270.3.5CA013106.0533,558.27
BrCMF2BraA01g016770.3.5CA013826.3742,602.83
BrCOL1BraA01g033110.3.5CA013865.5443,028.5
BrCMF3BraA01g033700.3.5CA012906.2631,727.19
BrPRR1BraA02g000660.3.5CA027608.2382,882.73
BrCMF4BraA02g005440.3.5CA023188.4935,569.73
BrCOL2BraA02g006280.3.5CA023157.1435,249.63
BrCMF5BraA02g010650.3.5CA0212810.514,454.51
BrCMF6BraA02g010730.3.5CA022416.4126,671.66
BrCOL3BraA02g012450.3.5CA023425.4937,592.09
BrCMF7BraA02g012780.3.5CA023947.7343,853.06
BrCMF8BraA02g015010.3.5CA022985.3533,624.66
BrCOL4BraA02g019020.3.5CA024015.3945,585.3
BrCOL5BraA02g041160.3.5CA023515.4838,961.19
BrPRR2BraA02g043910.3.5CA025097.6357,088.59
BrCMF9BraA03g006340.3.5CA033019.3433,962.85
BrCMF10BraA03g014440.3.5CA032694.7730,419.14
BrCOL6BraA03g033820.3.5CA033515.4738,638.54
BrCMF11BraA03g035990.3.5CA032445.928,167.87
BrPRR3BraA03g044790.3.5CA035766.6764,811.77
BrCMF12BraA03g052730.3.5CA032615.9728,333.46
BrCMF13BraA03g053790.3.5CA033836.2242,959.09
BrCMF14BraA04g014840.3.5CA043085.3334,785.22
BrCOL7BraA04g019450.3.5CA042898.2731,501.53
BrCMF15BraA04g025130.3.5CA043904.4143,197.44
BrCOL8BraA05g000130.3.5CA053828.743,594.07
BrPRR4BraA05g001060.3.5CA054126.0945,915.79
BrCMF16BraA05g011500.3.5CA053704.741,413.53
BrCOL9BraA05g020480.3.5CA053146.0737,089.29
BrCMF17BraA05g026820.3.5CA053016.3133,252.02
BrCMF18BraA05g035260.3.5CA052445.0928,037.53
BrCOL10BraA05g039120.3.5CA053685.5840,383.3
BrPRR5BraA06g033140.3.5CA065636.9862,530.66
BrCOL11BraA06g033600.3.5CA063515.5637,943.14
BrCOL12BraA06g037050.3.5CA063585.8539,397.8
BrCOL13BraA07g012300.3.5CA073085.834,026.11
BrCOL14BraA07g013220.3.5CA074085.5146,106.73
BrCOL15BraA07g031770.3.5CA074155.4847,062.04
BrCMF19BraA08g002590.3.5CA083435.4937,283.24
BrCMF20BraA08g019720.3.5CA082525.0628,599.34
BrCOL16BraA08g025070.3.5CA084185.6746,245.53
BrCOL17BraA08g025990.3.5CA084075.5845,398.2
BrCMF21BraA08g034360.3.5CA083254.8237,077.56
BrCMF22BraA08g035370.3.5CA083404.8737,905.93
BrCOL18BraA09g006160.3.5CA093455.8937,321.68
BrPRR6BraA09g006290.3.5CA095288.459,627.47
BrPRR7BraA09g007060.3.5CA095107.6957,852.13
BrCOL19BraA09g038260.3.5CA094128.4545,914.97
BrCOL20BraA09g039540.3.5CA094145.0846,495.03
BrCMF23BraA10g002920.3.5CA103864.4843,148.13
BrCMF24BraA10g005660.3.5CA102274.9726,904.79
BrCMF25BraA10g012030.3.5CA103475.0838,537.5
BrCMF26BraA10g015500.3.5CA103979.1444,406.02
BrCOL21BraA10g015920.3.5CA103566.0839,055.67
BrCMF27BraA10g017860.3.5CA102406.326,425.3
BrPRR8BraA10g017910.3.5CA104845.8153,046.54
BrCOL22BraA10g023820.3.5CA103376.2337,950.39
BrCOL23BraA10g023830.3.5CA103457.1939,033.48
BrCMF28BraA10g024780.3.5CA103529.7240,132.2
BrPRR9BraA10g032970.3.5CA107227.6278,528.65
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Fu, W.; Jia, X.; Li, S.; Zhou, Y.; Zhang, X.; Jiang, L.; Hao, L. Genome-Wide Identification and Analysis of the CCT Gene Family Contributing to Photoperiodic Flowering in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Horticulturae 2025, 11, 848. https://doi.org/10.3390/horticulturae11070848

AMA Style

Fu W, Jia X, Li S, Zhou Y, Zhang X, Jiang L, Hao L. Genome-Wide Identification and Analysis of the CCT Gene Family Contributing to Photoperiodic Flowering in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Horticulturae. 2025; 11(7):848. https://doi.org/10.3390/horticulturae11070848

Chicago/Turabian Style

Fu, Wei, Xinyu Jia, Shanyu Li, Yang Zhou, Xinjie Zhang, Lisi Jiang, and Lin Hao. 2025. "Genome-Wide Identification and Analysis of the CCT Gene Family Contributing to Photoperiodic Flowering in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)" Horticulturae 11, no. 7: 848. https://doi.org/10.3390/horticulturae11070848

APA Style

Fu, W., Jia, X., Li, S., Zhou, Y., Zhang, X., Jiang, L., & Hao, L. (2025). Genome-Wide Identification and Analysis of the CCT Gene Family Contributing to Photoperiodic Flowering in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Horticulturae, 11(7), 848. https://doi.org/10.3390/horticulturae11070848

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop