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Genome-Wide Identification, Characterization, and Transcriptomic Analysis of the Cyclin Gene Family in Brassica rapa

Cyclins are involved in cell division and proliferation by activating enzymes required for the cell cycle progression. Our genome-wide analysis identified 76 cyclin genes in Brassica rapa, which were divided into nine different types (A-, B-, C-, D-, H-, L-, P-, T-, and SDS-type). Cyclin genes were unevenly scattered on all chromosomes, with a maximum of 10 on A08 and a minimum of 2 on A04. The gene structure and conserved motif analysis showed that the cyclins which belonged to the same type or subgroup have a comparable intron/exon pattern or motif. A total of 14 collinear gene pairs suggested that the B. rapa cyclin genes experienced a mass of segmental duplication. The Ka/Ks analysis revealed that the Brcyclin gene family has undergone an extensive purifying pressure. By analyzing the cis-elements in the promoters, we identified 11 cis-elements and five of them are related to the hormone response. We observed 48 potential miRNAs targeting 44 Brcyclin genes, which highlighted the involvement of miRNAs in the regulation of cyclin genes. An association analysis between the leaf size and SNPs in mutants and a transcriptome analysis of two Chinese cabbage-cabbage translocation lines also showed that the Brcyclin gene family was involved in the development of the leaves. The functional characterization of the B. rapa cyclin gene family will provide the foundation for future physiological and genetic studies in the regulation of leaf growth.


Introduction
The cyclin protein family controls cell progression (division and expansion) by interacting with cyclin-dependent kinases (CDKs), which regulate the cell cycle in plants [1,2]. Cyclin, first identified in the soybean [3], plays an important role in the cell cycle. Cyclins are classified on the basis of their amino acid sequence and the point or phase in the cell cycle during which they activate their CDK partners. The phase has been divided into two categories, M-and G1-phases [4]. The former includes A-and B-type cyclins, which aid in the entry of cells into the M-phase through the nuclear division (mitosis), followed by the cytoplasmic division (cytokinesis), whereas the latter includes cyclins C-, D-, and E-type, that become active towards the end of the G1-phase and are responsible for ushering the cell into the S-phase. The G1 phase also serves as a critical checkpoint for cells, determining whether the cell will divide or remain stalled in the G1 phase of the cell cycle. Typically, cyclin contains a conserved region known as the cyclin core, which consists of two domains: the first one is cyclin N, known as the cyclin box, and is more conserved than the second domain, which is cyclin C [5]. Some cyclins only contain a cyclin-N domain but have no cyclin-C domain [6], which indicates the cyclin-C domain isn't crucial for its function. In

Identification of the Cyclin Gene Family in Brassica rapa
To identify the cyclin genes in the Brassica rapa genome, BLASTP was used on the Brassica database (http://brassicadb.cn/#/, accessed on 1 September 2022) by using the 10 Arabidopsis genes as a query sequence. As a result, we discovered 181, 196, 16,222,7,14,17,37, and 49 members in the A-, B-, C-, D-, H-, L-, T-, SDS-, and P-types, respectively. We compared all of the identified genes and found that the A-, B-, D-, and SDS-types shared the same cyclin genes (repetition of the same transcripts), but some of them were different, whereas the same scenario was found with the C-, H, L-, and T-types. However, the P-type was found to contain different genes (no repetition of the transcript IDs was found). Repeated transcripts were excluded, and the actual remaining 76 distinct cyclin genes in the entire genome were kept for further analysis. We decided that the candidates containing at least one cyclin-N domain should be considered as "true" cyclins [7]. Of the 76 unique members, 59 genes contain both N-and C-domains, and only 17 genes contain only the N-domain (Table S1). The length of the B. rapa cyclin proteins ranges from 113 to 1229 amino acids (aa), with an average of 400 aa. The smallest B. rapa cyclin protein, B. rapa CYC-P-76 (113 aa), and the biggest B. rapa cyclin protein, B. rapaCYC-C-51 (1229 aa), contain both N-and C-domains. All predicted genes and sequencing IDs, gene descriptions, chromosome locations, instability index, protein length, molecular weight, PI, and GRAVY have been presented ( Table 1).
The putative cyclin genes were localized on all chromosomes, but the general distribution was mostly unequal, with a maximum of 10 on A08 and a minimum of 2 on A04 ( Figure 1). the same cyclin genes (repetition of the same transcripts), but some of t whereas the same scenario was found with the C-, H, L-, and T-type type was found to contain different genes (no repetition of the transcr Repeated transcripts were excluded, and the actual remaining 76 dist the entire genome were kept for further analysis. We decided that the ing at least one cyclin-N domain should be considered as "true" cy unique members, 59 genes contain both N-and C-domains, and on only the N-domain (Table S1). The length of the B. rapa cyclin proteins 1229 amino acids (aa), with an average of 400 aa. The smallest B.rapa cy CYC-P-76 (113 aa), and the biggest B.rapa cyclin protein, B. rapaCYCtain both N-and C-domains. All predicted genes and sequencing IDs chromosome locations, instability index, protein length, molecula GRAVY have been presented ().
The putative cyclin genes were localized on all chromosomes, bu bution was mostly unequal, with a maximum of 10 on A08 and a mi ( Figure 1).

Cyclin Gene Structure and the Conserved Domain and Motif Analysis
A gene's structure (exon/intron) distribution is strongly linked to its function. The B. rapa evolutionary tree showed that the maximum cyclin members within a group contained the same gene structure (Figure 2a,b). Both cyclin domains N-and C-were present in all members of the A-, B-, and D-types (plant-specific) of the B. rapa cyclin genes (Table S1). Using the MEME algorithm, 10 types of motifs and their distribution in the B. rapa cyclins were predicted to understand the distribution of the motifs in cyclin proteins and their function. The study revealed that cyclins belonging to the same group have comparable motifs. Furthermore, all probable B. rapa cyclins were grouped into five major groups (Groups (I) red, (II) purple, (III) blue, (IV) yellow, and (V) green). Group (I) contained 16 Brcyclin members; out of them, four members contained similar conserved motifs, 11 members contained two similar conserved motifs, and only one member contained three similar motifs. Group (II) contained varying numbers of motifs; with 15 Brcyclin members, six have three motifs, one has two, and eight have four conserved motifs and one variable motif. Group (III) consisted of 12 Brcyclin members; 11 have four conserved motifs, and one has three conserved motifs. Group (IV) consisted of 14 Brcyclin members; six contained eight conserved motifs, seven members contained nine conserved motifs, and one member has three conserved motifs. Group (V) contained 19 members; all have nine conserved motifs ( Figure 2b).

Comparative Phylogenetic Analysis and Classification of the B. rapa Cyclin Gene
A phylogenetic study was performed using the maximum likelihood tree approach to understand the evolutionary relationship of the B. rapa cyclins with the Brassicaceae species: B. napus (Bna), B. oleracea (Bol), B. juncea (Bju), B. nigra (Bni), and A. thaliana (At). The tree was built using 76, 27, 25, 34, 38, and 49 cyclin genes from Brassica rapa, Brassica napus, Brassica oleracea, Brassica juncea, Brassica nigra, and Arabidopsis, respectively. The tree was divided into six clusters (I-VI) ( Figure 2). According to our results, Group I comprised 58 cyclin members, Group II comprised 37, Group III comprised 38, Group IV comprised 47, Group V comprised 30, and Group VI comprised 50 cyclin members ( Figure 3). In addition, it is established that the B. rapa cyclins evolved closely with other Brassicaceae species. Furthermore, CYC-A was one of the major types in Brassica rapa, with 15 described genes, followed by CYC-D with seven defined and five uncharacterized genes, and the CYC-B type cyclin with nine genes.

Duplication Event in the Brassica rapa Cyclins and Synteny Analysis
The tandem and segmental duplication advance the development and progression of plant genomes by promoting new gene family members. Through polyploidy, segmental duplications result in gene duplication, whereas tandem duplications result from the crossing over of shorter fragments [23][24][25]. We used BlastP searches to comprehend the tandem and segmental gene duplication in cyclins. Out of 76 Brcyclin genes, we found 14 collinear gene pairs, indicating that all pairs were segmentally duplicated, and no tandem duplicated pairs were found ( Figure 4a). These findings show that segmental duplication played an important role in the expansion of the Brcyclin family genes.
A synteny analysis showed that the B. rapa cyclins displayed the most collinearity with B. napus, B. juncea, and B. carinata, followed by B. oleracea, B. nigra, and A. thaliana (Figure 4b). A total of 75 B. rapa cyclin genes were found to syntenic with B. napus, B. juncea, and 69 with B. carinata, 67 with B. oleracea, and B. nigra, while 62 with A. thaliana. The study revealed that B. rapa and the other six Brassicaceae species had strong orthologs of the cyclin genes. Several homologs of other Brassicaceae crops maintained a syntenic relationship with the B. rapa cyclin genes, implying that whole genome duplication, along with segmental repetition, played a crucial role in the Brcyclin gene family evolution. Furthermore, we determined the nonsynonymous (Ka) and synonymous (Ks) ratios for each pair of duplicated Brcyclins under a selective sweep (

Comparative Phylogenetic Analysis and Classification of the B.rapa Cyclin Gene
A phylogenetic study was performed using the maximum likelihood tree approach to understand the evolutionary relationship of the B. rapa cyclins with the Brassicaceae species: B. napus (Bna), B. oleracea (Bol), B. juncea (Bju), B. nigra (Bni), and A. thaliana (At). The tree was built using 76, 27, 25, 34, 38, and 49 cyclin genes from Brassica rapa, Brassica napus, Brassica oleracea, Brassica juncea, Brassica nigra, and Arabidopsis, respectively. The tree was divided into six clusters (I-VI) ( Figure 2). According to our results, Group I comprised 58 cyclin members, Group II comprised 37, Group III comprised 38, Group IV comprised 47, Group V comprised 30, and Group VI comprised 50 cyclin members ( Figure 3). In addition, it is established that the B. rapa cyclins evolved closely with other Brassicaceae species. Furthermore, CYC-A was one of the major types in Brassica rapa, with 15 described genes, followed by CYC-D with seven defined and five uncharacterized genes, and the CYC-B type cyclin with nine genes.

Duplication Event in the Brassica rapa Cyclins and Synteny Analysis
The tandem and segmental duplication advance the development and progression of plant genomes by promoting new gene family members. Through polyploidy, segmental duplications result in gene duplication, whereas tandem duplications result from the crossing over of shorter fragments [23,24,25]. We used BlastP searches to comprehend the tandem and segmental gene duplication in cyclins. Out of 76 Brcyclin genes, we found 14

Cis-Elements in the Promoters of the Brassica rapa Cyclin Genes
The 2.0 kb upstream sequences from the translation start sites of the cyclin genes were submitted to the PlantCARE database to detect the cis-elements in the promoters of the Brcyclin genes, to study the potential regulatory mechanisms in the biological processes, particularly in plant hormones, meristem development, cell cycle regulation, and pathogen infection. Results showed 11 cis-elements (Table S2), among them five were hormone-related response elements, i.e., abscisic acid (ABA), auxin, methyl jasmonate (MeJA), gibberellin (GA), and salicylic acid (SA), implying that these genes may respond to phytohormones (Figure 5a and Table S2). Brcyclin genes CYCA2;3, CYCA2;4, CYCD3, CYCD3;1, and some uncharacterized cyclin genes, are hormone-correlated responsive elements that play a significant role in plant growth and development (Table 1). Furthermore, the stress responsive cis-elements were also found, which include drought, low temperature-responsive elements, defense, stress, light, and circadian (Figure 5a,b). The maximum elements were found for the light response and the auxin hormone response, and the minimum elements were found for the circadian responsive elements in all of the promoters of Brcyclin genes (Figure 5b), implying that Brcyclin genes may respond to stress stimuli as well and have a wide range of functions during plant development.

3D Protein Structure of the Cyclin Genes in Brassica rapa
Proteins are important molecules that play a significant role in different biological processes. The function of a protein is largely dependent on its 3D structure, so protein structure prediction or modeling is very significant. The 3D structure of a protein offers insight into how it functions and how it might be utilized, to control or modify it. It also enables the understanding of several biological interactions, and the prediction of which

3D Protein Structure of the Cyclin Genes in Brassica rapa
Proteins are important molecules that play a significant role in different biological processes. The function of a protein is largely dependent on its 3D structure, so protein structure prediction or modeling is very significant. The 3D structure of a protein offers insight into how it functions and how it might be utilized, to control or modify it. It also enables the understanding of several biological interactions, and the prediction of which molecules will bind to that protein. In this study, 3D protein structures were predicted to understand the attributes of proteins and facilitate comparative studies of them. We inferred that all 76 Brcyclin genes have a conserved 3-dimensional structure with alpha (α)_ helix and beta (β) sheets after constructing the 3D protein structures (Figure 6), because proteins with similar structures often have similar functions. The protein structures were modelled via the c2khoA template at a confidence level of 100%. On the basis of the protein structure guide design, binding ligands can be determined, that are essential to bind with the external or internal residues. In addition, the cyclin protein family's tertiary structure predicts the physical connections, different functional roles, independent divergence sequences, and conserved biological features.

Genome-wide Analysis of the miRNA Targeting Cyclin Genes in Brassica rapa
miRNAs play a critical role in plant growth and development, including the signaling pathways [26] and organ morphology [27], by silencing and upregulating their target genes. The variability of the DNA sequences at the miRNA target sites alters the complementation between miRNAs and their target genes, which influences the target gene expression levels and the traits relevant to these genes (Table S3) [28]. "MicroRNA-targeting a specific gene family analysis" conveys the positional information needed to generate the developmental patterns of a specific genetic regulator [29]. We observed 48 potential miR-NAs targeting 44 Brcyclin genes, which highlighted the involvement of miRNAs in the regulation of the cyclin genes (Figure 7). B. rapaCYC-17 was targeted by a maximum of 14

Genome-Wide Analysis of the miRNA Targeting Cyclin Genes in Brassica rapa
miRNAs play a critical role in plant growth and development, including the signaling pathways [26] and organ morphology [27], by silencing and upregulating their target genes. The variability of the DNA sequences at the miRNA target sites alters the complementation between miRNAs and their target genes, which influences the target gene expression levels and the traits relevant to these genes (Table S3) [28]. "MicroRNA-targeting a specific gene family analysis" conveys the positional information needed to generate the developmental patterns of a specific genetic regulator [29]. We observed 48 potential miRNAs targeting 44 Brcyclin genes, which highlighted the involvement of miRNAs in the regulation of the cyclin genes (Figure 7). B. rapaCYC-17 was targeted by a maximum of 14 miRNAs, followed by B. rapaCYC-10, which was targeted by 12 miRNAs, B. rapaCYC-11, and B. rapaCYC-18, were targeted by 10 miRNAs (Table S3 and Figure 7).

Functional Annotation Analysis of the Cyclin Genes in Brassica rapa
The GO annotation and enrichment analysis were carried out to understand the function of the Brcyclin genes in biological process (BP), molecular function (MF), and cellular component (CC). The GO annotation was only detected in the P-type cyclin, and the annotations were only found in BP and MF (Table S4). In the GO-MF, only one highly enriched term (GO:0000079) was found, which demonstrated its involvement in protein kinase binding activity, cell growth and proliferation, as well as the initiation and modulation of the immunological responses. Similarly, in the GO-BP, only one highly enriched term was anticipated (GO:0019901) which was found to be involved in the control of the cyclin-dependent protein serine/threonine kinase activity. These GO enrichment results suggested that the Brcyclin genes play a pivotal role in growth and development.

The Functional Characterization of the Brassica rapa Cyclin Genes by the SNP Analysis
Out of the 76 cyclin genes, 74 genes showed 523 SNPs in the CCEMD, which was created by our research team (Table S5), and 61 cyclin genes had 138 non-synonymous SNPs in the body region of the gene (Table S6) [30]. These 138 non-synonymous mutations were attained from 106 mutant plants, and among them, 21 mutant plants showed altered leaf size, as compared to the wild-type plants (Table S7).

Expression Profiles of the Brassica rapa Cyclin Genes by a Transcriptome Analysis
According to our transcriptome results between the normal and small-leaf plants, out of 76 Brcyclin genes, a total of 35 expressed genes were identified in the Chinese cabbagecabbage 16-1 translocation line. There were 13 genes with log2FC| ≥ 1, FDR ≤ 0.01 and all were downregulated in 16-1S plants, compared with 16-1N plants (Figure 8 and Table S8), including six CYCA genes, two CYCB genes, two CYCD genes, and three uncharacterized genes. (Figure 8, Table S8). It suggests that the CYCA genes could play a significant role in regulating the size of 16-1 plant leaves. Similarly, in the qdh-263 Chinese cabbagecabbage translocation line, 31 expressed genes were identified out of 76 Brcyclin genes, with an FPKM value ≥ 3.17, of them 17 had |log2FC| ≥ 1 and FDR ≤ 0.01 (Figure 9 and Table S8). In our transcriptomic results, CYCA2;3 (BraA09g058650.3C) and CYCA3;1 were downregulated in the 16-1S plants. The downregulation of these genes may be the reason for the small leaf size of the 16-1S plants because CYCA2;3 acts as a major regulator of the ploidy levels in Arabidopsis endoreduplication by negatively regulating the endocycles [11]. CYCA2;3 alters the ploidy levels and endocycles, which affect leaf development. Corneillie, De Storme et al. 2019 have reported the morphometric analysis of leaves in which the polyploidy influences the epidermal pavement cells, resulting in larger cell sizes and fewer cells per leaf blade, as the ploidy rises [31]. Further studies with reference to the ploidy level and its effects on leaf size can bring new perspectives to leaf development. The genes CYCB2;3 (BraA08g027760.3C) and CYCD3;1 (BraA08g16120.3C, BraA01g004130.3C) play an important role in the cell cycle and cell division, respectively [32]. The downregulation of these genes affects the growth and development of plant organs, which is consistent with our transcriptomic results.
Furthermore, there are a few uncharacterized genes in the cyclin gene family, i.e., BraA07g016670.3C, BraA07g027470.3C, and BraA07g039870.3C (core cell cycle genes), according to the Brassica database http://brassicadb.cn/#/Annotations/, accessed on 1 September 2022 (Table 3 and Table S8), which need to be studied and are probably involved in the regulation of leaf size. A similar cyclin gene family behavior was observed in the qdh-263 translocation line. The expression of all plant-specific cyclin types A-, B-, and D-was downregulated (Figure 9). These results imply that in our accessions, the small leaf size might be due to the poor functioning of the cell cycle genes. It also suggests that in both translocation lines, the pathway of the Brcyclin genes in the regulation of the leaf size is same.  (Tables 3 and S8), which need to be studied and are probably involved in the regulation of leaf size. A similar cyclin gene family behavior was observed in the qdh-263 translocation line. The expression of all plant-specific cyclin types A-, B-, and D-was downregulated (Figure 9). These results imply that in our accessions, the small leaf size might be due to the poor functioning of the cell cycle genes. It also suggests that in both translocation lines, the pathway of the Brcyclin genes in the regulation of the leaf size is same.     Table 3. Gene annotation of the differentially expressed cyclin genes.

Brassica rapa Gene ID
Arabidopsis Gene ID Gene Name Gene Annotation

Discussion
In plants, the cyclin-dependent kinases (CDKs) regulate cell division, plant growth, and development, including leaf development and branch formation. The CDK activity is regulated by the kinase's interaction with a regulatory cyclin component, which regulates the timing of CDK activation, the complex's subcellular location, and its substrate specificity. Cyclins are involved in virtually every step of the mitotic process by interacting with CDKs and other proteins [33].

Identification of the Cyclin Gene Family and Its Structure in Brassica rapa
In plant genomes, the number of cyclin genes varies; we found 76 cyclin genes in the Brassica rapa genome of nine different types (A-, B-, C-, D-, H-, L-, P-, T-, and SDS-types), and the A-type was the largest one. The rice genome has been found to include 49 cyclins, which are divided into nine types: A-, B-, D-, F-, H-, L-, P-, T-, and SDS-types [34]. The maize genome contains 59 cyclins that can only be divided into six types: A-, B-, D-, F-, T-, and SDS-type cyclins [15]. The poplar genome contains 45 cyclins that can be categorized into seven types: A-, B-, C-, D-, Q-, T-, and Z-types [35]. There are 50 cyclins in the Arabidopsis genome, which are divided into ten types: A-, B-, C-, D-, H-, L-, U-, T-, J18-, and SDS-types [7]. 52 cyclin genes in the tomato genome have been identified, which are also classified into 10 types: A-, B-, C-, D-, H-, L-, P-, T-, J18-, and SDS-types, and legumes share the same categories as Arabidopsis. Previously, it was discovered that cyclin types A-, B-, D-, and U-were further divided into 3, 3, 7, and 4 subgroups, respectively, while other types C-, H-, L-, SDS-, and T-were not divided into any other subgroups [20]. All plant genomes have A-, D-, and T-type cyclin genes, so these three types are more conserved. The structural investigation of the B. rapa cyclins demonstrates their remarkable conservation and distinguishing characteristics (Figure 2a).

Segmental Duplication Event of the Brassica rapa Cyclins
Gene duplication is a major driving force in the evolution of genomes and genetic systems [36]. The tandem and segmental duplications cause the substantial variations in the family size, as observed in most gene families [25]. Studies on the Arabidopsis genome have featured a high number of large segmental duplications that came from ongoing polyploidy events and have been scrambled by chromosomal rearrangements [34]. The co-linearity of the cyclin genes among the A, B, and C genomes indicates that these genomes are the progenitors of a common ancestor and that, over the course of evolution, these genes have remained conserved, due to their utility in the cell cycle progression. Moreover, the collinearity events of the B. rapa cyclin genes with all other Brassica crops, showed their similar evolutionary progenitor. Throughout evolution, it seems that cyclin gene duplication and subsequent expansion have occurred frequently. These discoveries shed light on Brassica rapa's genome evolution, which suggests that all segmental duplicated cyclin genes have comparable or overlapping activities, despite their striking variances in amino acid sequences.

MicroRNAs Targeting the Cyclin Genes in Brassica rapa
MicroRNAs (miRNAs), a family of single-stranded, non-coding RNAs, have been discovered to have a role in post-transcriptional gene regulation [37,38]. Numerous miRNAs have been identified in Chinese cabbage through genome-wide studies in recent years [39]. In our study, 48 putative miRNAs targeting 44 Brcyclin genes have been identified (Figure 7). These findings suggest that Br-miRNAs may play a critical role in protecting the plant from a variety of stresses, by changing the transcript level of the cyclin genes in Brassica rapa.

Expression Profiles of the Brassica rapa Cyclin Genes by Analyzing the Transcriptomic Data
Out of 76 Brcyclin genes, a total of 13 differentially expressed genes were identified in 16-1S and 16-1N, and 17 differentially expressed genes were identified in qdh-263S and qdh-263N. All of them were downregulated in 16-1S and qdh-263S, compared with 16-1N and qdh-263N.
In plants, A-type cyclins are classified into three subtypes, namely, CYCA1, CYCA2, and CYCA3. These genes showed different expression levels in 16-1N and 16-1S accessions ( Figure 8). The protein CYCA1;1 is a regulatory subunit and CDC2OS-3 is a catalytic subunit of the cyclin-dependent protein kinases (CDKs), which are central to the cell cycle regulation. Both genes are regulated by gibberellic acid in a coordinated manner, with transcripts accumulating in the G2 phase, prior to the B2-type mitotic cyclins [40], suggesting a distinct role in regulating the G2/M phase progression. CYCA3;2 is a distinct member of the G1 cyclin family that plays an important role in the meristematic tissues.
The gene CYCB2;3 (BraA08g027760.3C) is the cyclin-dependent protein kinase regulator activity, which is involved in the regulation of the cell cycle. The downregulation of this gene resulted in decreased leaf size [32], which is consistent with our transcriptomic results. Therefore, we speculated that CYCB2;3 might be involved in the regulation of the leaf size in our accessions.
CYCD3;1 (BraA08g16120.3C, BraA01g004130.3C) plays a key role in integrating cell division in the leaf and lateral organ development [41]. The CYCD3;1 expression is increased by the cytokinin, which regulates a variety of developmental programs, including the shoot regeneration, leaf development, greening, and cell cycle stimulation. A mutation in CYCD3 decreases the shoot meristem activity and leads to a reduced cytokinin response [18]. It showed that the CYCD3 activity is critical for the cell quantity in growing lateral organs and mediates the cytokinin effects in the apical growth and development. These findings suggest that the small leaf size in our accessions may be due to a cell cycle malfunction; the dysfunction of the cell cycle may be caused by the cyclin genes' aberrant behavior. In the future, more research will be required to map these regulators in the bigger network of leaf growth regulation.

Identification and Characterization of the Cyclin Gene Family
A total of 76 cyclin genes (http://brassicadb.cn/#/BLASTP/, accessed on 1 September 2022) were retrieved from the genome assembly v3.0, on the basis of the e-value < 0.01, by using the 10 Arabidopsis cyclin genes (At1g44110, At4g37490, At5g48640, At1g70210, At5g27620, At2g26430, At1g35440, At1g14750, At3g21870, and At2g01820) as a query sequence, which were taken from the A. thaliana genome (https://www.arabidopsis.org/ about/citingtair.jsp, accessed on 1 September 2022) database. Further screening was done on the basis of the PF value (PF00134, cyclin, N-terminal, PF02984, cyclin, C-terminal) (http://pfam.xfam.org/, accessed on 1 September 2022) B. rapa cyclin proteins were further characterized by determining the molecular weight, the number of amino acids, the isoelectric point, and GRAVY through the ProtParm tool (ExPASy-ProtParam tool). The protein sequences were uploaded to the online database of Wolf PSORT for the prediction of the subcellular localization (https://wolfpsort.hgc.jp/, accessed on 1 September 2022) [42]. To visualize the chromosomal location of all obtained cyclin genes, the "Show Genes on Chromosome" feature of the TB-tools software was used.

Comparative Phylogenetic Analysis and Classification of the Brassica rapa Cyclin Gene
The protein sequences of the cyclin genes in Arabidopsis, B. nigra, B. junacea, B. olearcea, B. carinata, and B. napus were used to generate the phylogenetic tree, using MEGA (V.11 https://www.megasoftware.net/, accessed on 1 September 2022) software. The sequences were multiple aligned and employed the neighbor-joining tree method with 1000 bootstrap replicates [43].

Tandem Duplication Events of the Brassica rapa Cyclins and the Synteny Analysis
The duplicated genes were retrieved, using the Blast, MCScanX, and Advance Circos features of TB-tools (https://github.com/CJ-Chen/TBtools, accessed on 1 September 2022). For the purity selection pressure of the duplicated genes, the Ka/Ks values were calculated, using the Ka/Ks Calculator in TB-tools, and the time million years ago (TMYA) was calculated with the help of previous studies in Brassica rapa [44,45]. The synteny relationships of B. rapa with B. oleracea, B. napus, B. junacaea, B. carinata, B. nigra, and A. thaliana were developed by using MCScanX to get the collinearity files, which were then used to create the dual synteny graphics through TB-tools (https://github.com/CJ-Chen/ TBtools, accessed on 1 September 2022) [46].

Cis-Elements in the Promoters of the Brassica rapa Cyclin Genes
The putative cis-elements of all 76 Brcyclin were classified by downloading the 2000 bp upstream of the start codon from the BRAD database. For further classification, the Plant-CARE web-based tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 1 September 2022) was employed, and the findings were presented using TB-tools.

3D Protein Structure of the Cyclin Gene in Brassica rapa
The 3-dimensional structure of the Brcyclin proteins was digitally constructed, using the available web-based software PHYRE2 (PHYRE2 Protein Fold Recognition Server) (ic.ac.uk, accessed on 1 September 2022).

Cyclin Gene Structure, Conserved Domain, and the Motif Analysis
The protein conserved domain was identified using the NCBI conserved domain online server (www.ncbi.nlm.nih.gov, accessed on 1 September 2022), and the motif analysis was performed, using the MEME Suite (meme-suite.org, accessed on 1 September 2022). The gene structure (exon and intron) was predicted by the Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/, accessed on 1 September 2022), using the CDS and genomic sequences of 76 Brcyclin genes.

Genome-Wide Analysis of the miRNA Targeting the Cyclin Genes in Brassica rapa
The CDS sequences of Brcyclin were utilized to find the microRNAs, targeting the Brcyclin genes through the psRNATarget database (http://plantgrn.noble.org/psRNATarget, accessed on 1 September 2022), and the interaction was further predicted by a graphical illustration.

Functional Annotation Analysis of the Brcyclin genes
To investigate the functional properties, Gene Ontology (GO) annotation was used by accessing the Brassica database (http://brassicadb.cn, accessed on 1 September 2022). The transcript IDs of the Brcyclin genes were used to find the annotation of every gene.  [47]. Heterotriploids (genome AAC, 2n = 29) were produced by crossing 4XL and 11-1, then the monosomic alien addition lines of the Chinese cabbage-cabbage (AA + 1C, 2n = 20 + 1 = 21) were obtained through selfing after backcrossing AAC and a Chinese cabbage (85-1, AA, 2n = 20), the translocation lines were developed after the isolated microspore culture ( Figure 10) [48]. In our study, two phenotypes, including normal (16-1N, qdh263N) and small (16-1S, qdh263S) leaves, were present in the self-breeding offspring of 16-1 and qdh-263 ( Figure 11). For the transcriptomic analysis, the seeds were grown in a greenhouse at Hebei Agricultural University for six weeks. The fifth leaves were sampled from at least three plants with similar characteristics and phenotypes. Following the sampling, the tissues were quickly frozen in liquid nitrogen and stored at −70 • C until the RNA isolation. Three technical replicates were taken from each morphotype. crospore culture (Figure 10) [48]. In our study, two phenotypes, including normal (1 qdh263N) and small (16-1S, qdh263S) leaves, were present in the self-breeding offs of 16-1 and qdh-263 ( Figure 11). For the transcriptomic analysis, the seeds were grow a greenhouse at Hebei Agricultural University for six weeks. The fifth leaves were pled from at least three plants with similar characteristics and phenotypes. Followin sampling, the tissues were quickly frozen in liquid nitrogen and stored at −70 °C unt RNA isolation. Three technical replicates were taken from each morphotype.  qdh263N) and small (16-1S, qdh263S) leaves, were present in the self-breeding offspring of 16-1 and qdh-263 ( Figure 11). For the transcriptomic analysis, the seeds were grown in a greenhouse at Hebei Agricultural University for six weeks. The fifth leaves were sampled from at least three plants with similar characteristics and phenotypes. Following the sampling, the tissues were quickly frozen in liquid nitrogen and stored at −70 °C until the RNA isolation. Three technical replicates were taken from each morphotype.

RNA-Seq Library Construction and Sequencing
The RNA sample preparations employed a total of 3 g RNA per sample, as the input material. A NEBNext ® Ultra™ RNA Library Prep Kit for Illumina ® was used to create our RNA-Seq libraries (NEB, Ipswich, MA, USA). Using poly-T oligo-attached magnetic beads, the mRNA was extracted from the total RNA. The NEB proprietary fragmentation buffer was used to fragment the divalent cations at a high temperature (5X). On an Illumina HiSeq 4000 platform, the RNA-Seq libraries were sequenced to obtain the 150 bp paired-end reads.

Differential Expression and Cluster Analysis
The DESeq (2012) R package was used to perform the differential expression analysis. Differentially expressed genes (DEGs) were discovered using the hierarchical cluster analy-sis. In each pairwise comparison, the DEGs with a false discovery rate (FDR) ≤ 0.01, |log2 fold change| ≥ 1 were filtered.

Single Nucleotide Polymorphisms (SNP) Analysis
To determine the function of the cyclin genes, we collected SNPs of the Brcyclin genes in lines from the Chinese cabbage EMS-induced Mutant Database (CCEMD) (http: //www.bioinformaticslab.cn/EMSmutation/home/, accessed on 1 September 2022) [30] and analyzed the relationship between the phenotypic changes and the SNPs in these genes.

Conclusions
In conclusion, a total of 76 cyclin genes were identified in the Brassica rapa genome. All Brcyclin genes were found to possess similar gene structures and conserved motifs, which showed a close evolutionary and syntenic relationship with Arabidopsis and all other Brassica crops. All Brcyclin genes were highly responsive to hormones and abiotic stress. The segmental duplication occurred frequently through the evolution of the Brassica genome. The functional analysis discovered that the Brcyclin gene family plays an important role in plant growth and leaf development by regulating the cell cycle. These discoveries will lay the basis for studying the roles of cyclin genes in the Brassica rapa developmental processes by using different functional confirmation methods, such as overexpression, RNAi, and genome editing.