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Genome-wide identification and expression pattern analysis of MIKC-Type MADS-box genes in Chionanthus retusus, an androdioecy plant



The MADS-box gene family is widely distributed in the plant kingdom, and its members typically encoding transcription factors to regulate various aspects of plant growth and development. In particular, the MIKC-type MADS-box genes play a crucial role in the determination of floral organ development and identity recognition. As a type of androdioecy plant, Chionanthus retusus have unique gender differentiation. Manifested as male individuals with only male flowers and female individuals with only bisexual flowers. However, due to the lack of reference genome information, the characteristics of MIKC-type MADS-box genes in C. retusus and its role in gender differentiation of C. retusus remain largely unknown. Therefore, it is necessary to identify and characterize the MADS-box gene family within the genome of the C. retusus.


In this study, we performed a genome-wide identification and analysis of MIKC-type MADS-box genes in C. retusus (2n = 2x = 46), utilizing the latest reference genome, and studied its expression pattern in individuals of different genders. As a result, we identified a total of 61 MIKC-type MADS-box genes in C. retusus. 61 MIKC-type MADS-box genes can be divided into 12 subfamilies and distributed on 18 chromosomes. Genome collinearity analysis revealed their conservation in evolution, while gene structure, domains and motif analysis indicated their conservation in structure. Finally, based on their expression patterns in floral organs of different sexes, we have identified that CrMADS45 and CrMADS60 may potentially be involved in the gender differentiation of C. retusus.


Our studies have provided a general understanding of the conservation and characteristics of the MIKC-type MADS-box genes family in C. retusus. And it has been demonstrated that members of the AG subfamily, CrMADS45 and CrMADS60, may play important roles in the gender differentiation of C. retusus. This provides a reference for future breeding efforts to improve flower types in C. retusus and further investigate the role of MIKC-type MADS-box genes in gender differentiation.

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The ornamental value and biodiversity of plants are increasingly appreciated in landscaping worldwide [1, 2]. Among the many plants used in urban landscapes, those that are cold-resistant and adaptable are particularly valuable [3]. This study aims to explore a plant with unique aesthetic and ecological value, namely the Chionanthus retusus. Our research not only enriches our understanding of ornamental tree species but also promotes the protection and utilization of their genetic diversity.

C. retusus (also known as Chinese Fringe tree), belonging to the Oleaceae family within the order Lamiales, is a true dicotyledonous plant [4]. It is valued for its unique landscape value and adaptive characteristics [5]. The Oleaceae family is a large family that includes multiple plant species, among which the Chionanthus genus is composed of multiple species, C. retusus, as one of its members, is particularly prominent in the eastern region of Asia [6]. This species is not only famous for its striking white flowers (Fig. 1), but also widely used in urban greening due to its strong adaptability and easy cultivation [7].

Fig. 1
figure 1

The C. retusus blooms in April

As an androdioecy plant, individuals of C. retusus are categorized into male and female based on the presence of a pistil in the flowers [8]. Male individuals produce solely male flowers with stamens, while female individuals bloom bisexual flowers featuring both stamens and pistils [6]. These two individuals coexist in the distribution area and can produce offspring through female self-pollination or hybridization [9]. This is a very rare sex system in flowering plants, which has only been clearly reported in a few species, such as Datisca glomerata [10], Tapiscia sinensis [11], and Osmanthus delavayi [12]. This sex system is considered by some studies to be a transitional state of evolution to dioecious plants [13, 14].

The C. retusus is widely distributed in China, mainly in the eastern and central southern regions. In addition, it is also distributed in Japan, South Korea and other places [4, 15]. The C. retusus exhibits rich morphological variation within the species, especially in the forms of flowers [6]. There are differences in the size, shape, and structure of petals and sepals of its flowers [8]. These morphological variations not only have adaptive significance for their natural survival, but also provide abundant materials for ornamental breeding [6, 8]. The gender differentiation mechanism and morphological variation of C. retusus may be related to the role of the MIKC-type MADS-box gene family. The MADS-box gene family plays a crucial role in regulating flower development and determining floral organ identity [16]. In C. retusus, studying this gene family not only provides a deeper understanding of the genetic basis for gender differentiation and morphological variation, but also may provide new molecular tools and strategies for ornamental cultivation, further promoting genetic improvement and variety innovation in C. retusus and other ornamental plants.

MADS-box gene family encodes a class of transcription factors that control various aspects of growth and development, especially the development of floral organs [17]. MADS-box genes are mainly divided into two large groups, termed type I and type II [18, 19]. The type II genes are commonly referred to as MIKC-type MADS-box genes, most of these genes are described as controlling the homeotic functions of floral organ [20,21,22]. The MIKC-type proteins are composed of four typical domains, MADS-box, Intervening, Keratin-like and C-terminal domain [23]. The MADS domain is highly conserved between species and has a dual function: it binds to specific DNA sequences and promotes dimerization [24]. The intervening (I) and keratin-like (K) domains exhibit moderate conservation, which is crucial for the assembly of protein complexes [23, 25, 26]. Meanwhile, the C-terminal domain, known for its diversity, plays a crucial role in the transcriptional activation of MIKC-type MADS-box proteins [23]. In the widely accepted ABCDE model, sepal development is controlled by Class A genes, petal development is controlled by Class A and B genes, stamen development is controlled by Class B and C genes, carpel development is controlled by Class C genes, and Class E genes are involved in the formation of all floral organs [20]. All ABCDE genes belong to the MIKC-type MADS-box gene family, except for APETALA 2 [20]. The MIKC-type MADS-box genes is mainly divided into twelve main subfamilies, including AG, SVP, FLC, AP3/PI, ALG12, SOC1, SEP, AGL6, AP1, AGL17, AGL15, MIKC* [17]. AG subfamily for determining the identity of stamen and carpel, as well as the DEF and GLO subfamilies for determining the identity of stamen and petal [17]. Different combinations of MICK-type MADS-box genes control the identification of different floral organs [19]. In Arabidopsis, the functional deficiency of the SEPs gene leads to the transformation of floral organs into leaf-like organs [21]. In rice, the dual mutants of mads3-mads58 can lead to a complete loss of reproductive organ characteristics of flowers [27]. The MIKC-type MADS-box genes controls the formation of floral organs in a complex way of interaction [28].

Due to the important role of the MIKC-type MADS-box genes in floral organ development, and our understanding of its characteristics in C. retusus is still limited, therefore, in this study, we identified the MIKC-type MADS-box genes of C. retusus using the latest reference genome information (2n = 2x = 46). Syringa oblata [29] and Olea europaea [30], like the C. retusus, belong to the family Oleaceae and have well assembled genomic information, we also performed genomic collinearity analysis on their MADS-box genes. In addition, we also conducted gene structure, conserved motifs and domains, and cis-acting element analysis on the gene and protein sequences of C. retusus MIKC-type MADS-box genes. Intended to expand our understanding of the MADS-box gene in C. retusus through the above analysis. Finally, to understand the role of MADS-box genes in gender differentiation of C. retusus, transcriptome analysis was performed on the expression patterns of the MICK-type MADS-box genes at different development stages of flowers from ‘XueZaoHua’, ‘XueDengLong’, and ‘XueXuan’ varieties, and the results were validated using qRT-PCR in eight varieties of different genders. Our research findings extend our understanding of the MIKC-type MADS-box genes in woody plant C. retusus and provide a reference for future flower directed breeding of C. retusus.


Identification and phylogenetic analysis of MIKC-type CrMADS genes

By determining the MADS-box domain and K-box domain, combined with the phylogenetic tree, we obtained a total of 61 confidence MIKC-type CrMADS genes, and named CrMADS1-CrMADS61 based on its position on the chromosome (Table 1). The amino acid lengths of all MIKC-type CrMADS proteins were 164 – 586 residues, the MWs ranging from 18.97 – 65.92 kDa, the pI values varying from 5.28 – 9.77 (Table 1).

Table 1 ID and physicochemical properties of the MIKC type MADS gene

To determine the subfamily classification of 61 CrMADS, we constructed a phylogenetic tree by combining their full-length amino acid sequences with the amino acid sequences of 41 type II MADS-box genes from Arabidopsis. According to phylogenetic analysis, the CrMADSs of C. retusus were well mapped to 12 subfamilies of Arabidopsis, each subfamily containing at least one CrMADS (Fig. 2). In 10 subfamilies of all, the number of CrMADSs in C. retusus increased compared to Arabidopsis, namely AP3/PI, AG, AGL12, SOC1, SEP, AP1, AGL17, AGL15, SVP, MIKC*. In the SVP and AGL12 subfamilies, the number of C. retusus MADS-box is 3 and 2.5 times that of Arabidopsis. However, compared to Arabidopsis, the number of MADS-box genes in the two subfamilies of C. retusus is lower. In the FLC subfamily, Arabidopsis has 7 MADS-box genes, while C. retusus only has two. In the AGL6 subfamily, Arabidopsis has two members, while C. retusus has only one. This may be due to the genome duplication of Brassicales relative to Oleaceae [31]. The expansion and contraction between different subfamilies may suggest different evolutionary directions between the C. retusus and Arabidopsis.

Fig. 2
figure 2

Phylogenetic Tree of MADS genes in C. retusus and Arabidopsis. The blue font represents the MADS genes of C. retusus, the black font represents the MADS genes of Arabidopsis, and different colored backgrounds represent different subfamilies

Chromosomal location, genome synteny, and gene duplication of CrMADSs

The C. retusus genome consists of 23 chromosomes, with 61 CrMADS genes located on 18 of them (Fig. 3). The chromosome 13 have the highest number of CrMADS genes, followed by chromosome 1, 11, and 14, which have 6 CrMADS genes each. In order to further understand the duplication and evolution of the C. retusus MADS genes, we conducted a genomic collinearity analysis of C. retusus. Based on the collinearity analysis results, there are extensive collinear segments between the chromosomes of C. retusus (Fig. 3). For example, there are collinear segments between chromosome 14 and 16, as well as chromosome 15 and 17. This indicates that the C. retusus genome has undergone duplication event. To further investigate the impact of chromosome duplication on the number of CrMADS genes, we mapped them to the collinear segments of the chromosome. A total of 21 CrMADS gene pairs were identified in the collinearity segments. Some of these genes share collinearity with multiple genes, so these 21 collinear gene pairs involve a total of 34 CrMADS genes. This quantity accounts for half of the total MADS genes in the C. retusus (Fig. 3). This indicates that after genome duplication, these 34 genes underwent retention and evolution, and these genes were preserved within the collinear segments. In addition, we also identified tandem duplication CrMADS genes in the C. retusus genome. There are a total of 4 tandem duplication genes, namely CrMADS22 and CrMADS23, as well as CrMADS34 and CrMADS35 (Fig. 3). Therefore, segmental duplication and tandem duplication both contribute to the amplification and evolution of the CrMADS genes, increasing the diversity of the C. retusus MADS gene family. At the same time, this also explains to some extent the quantitative expansion of some subfamilies relative to Arabidopsis. For example, the CrMADS9 and CrMADS60 genes in the AG subfamily are located on chromosome 3 and 23, respectively. The corresponding segments of these two chromosomes have a clear collinearity relationship, and these two genes are located within them. This indicates that these two genes may have been fixed into two by the same gene after undergoing possible genome duplication. The same examples also include CrMADS12 and CrMADS31 in the AP3/PI subfamily, as well as CrMADS13 and CrMADS59 in the AGL12 subfamily. In addition, the tandem repeats between CrMADS22 and CrMADS23, as well as the tandem repeats between CrMADS34 and CrMADS35, to some extent explain the increase in the number of members in the SVP and AGL17 subfamilies.

Fig. 3
figure 3

Chromosome distribution and duplication event analysis of CrMADSs. Red font represents tandem repeat genes

Conducting inter-species comparative analysis of the C. retusus genome, along with the genomes of S. oblata and O. europaea, would be beneficial for a better understanding of the evolution of the MADS-box gene family. S. oblata, O. europaea, and C. retusus all have the same number of chromosomes, with each species having 23 chromosomes. Based on the collinearity analysis results, it can be observed that there is a strong collinearity relationship between the corresponding chromosomes across these species (Fig. S1). However, there have been certain chromosome segment rearrangements that have occurred between these species. We have identified MADS-box genes located within these collinear regions, and we have found a total of 79 pairs of MADS-box genes located within the collinear regions between C. retusus and O. europaea (Fig. S1, Table 2). Between C. retusus and S. oblata, this number is 64 pairs. It is suggested that C. retusus has a closer genetic relationship with O. europaea. Subsequently, the Ka/Ks values were calculated for all collinear MADS-box gene pairs, and the results showed that the Ka/Ks values for all MADS-box gene pairs were less than 1 (Table 3). Therefore, after the divergence of C. retusus and these two species, the MADS-box genes have undergone purifying selection. The above results indicate that MADS-box genes in the Oleaceae family are highly conserved.

Table 2 The MADS-box genes sharing relationship between C. retusus, S. oblata, and O. europaea
Table 3 The Ka, Ks, and Ka/Ks values between collinear MADS gene pairs

In order to determine the phylogenetic relationship between the MADS-box genes of these three species in the Oleaceae family, we constructed a phylogenetic tree by combining the MADS-box genes of O. europaea and S. oblata with those of C. retusus. Interestingly, even within the same family, the MADS-box gene undergoes a certain degree of differentiation (Fig. S3). For example, in the AGL17 subfamily, the MADS-box gene of S. oblata is almost in a different evolutionary branch from that of C. retusus and O. europaea, while in the AGL15 subfamily, both C. retusus and O. europaea have two members, while S. oblata only has one. However, overall, the corresponding relationship between MADS-box genes in the C. retusus, O. europaea, and S. oblata is still very good.

Gene structure, conserved protein motifs and domain analysis

The gene structure of CrMADS genes, as shown in the Fig. S2, is characterized by multiple exons and long introns, similar to other species in this regard [32,33,34]. Most C. retusus MADS-box genes have 7 or 8 exons, accounting for 25/61 and 26/61, respectively. At this point, C. retusus is similar to other species. Additionally, CrMADS22 has the highest numbers of exons, with a total of 12. On the other hand, CrMADS32 and CrMADS48 have the fewest number of exons, with a total of 6. Overall, the average number of exons of 61 CrMADS genes is 7.8. The structure of MADS-box genes varies significantly among different subfamilies but remains relatively conserved within each subfamily. In the AP3/PI subfamilies, CrMADS genes commonly have shorter introns, while in the AGL17 and FLC subfamilies, CrMADS genes generally have longer introns (Fig. S2).

Furthermore, we identified the conserved domains and motifs of 61 CrMADS protein sequences. The results revealed that all CrMADS proteins possess the MADS-box domain, corresponding to motif-1 (Fig. 4). Additionally, the K-box domain is highly conserved in non-MIKC* subfamilies, corresponding to motif-4, motif-5 and motif-6. However, typical K-box domains and their corresponding motifs were not identified in the MIKC* subfamily. This could be due to the rearrangement of exons in ancient MIKC* genes [18, 35]. In addition to highly conserved motifs, there are also some motifs that only exist in specific subfamilies. For example, the AP1 subfamily possesses a unique motif, motif-7. Similarly, the AG subfamily has motif-14 and motif-15 as its distinctive motifs. These subfamily-specific motifs are often found at the C-terminus of CrMADS protein sequences, indicating that the C-terminus of CrMADS proteins possesses a relatively higher structural diversity. The conserved MADS-box and K-box domains, along with the diverse C-terminal domains, confirm the previous view that the CrMADS family proteins have undergone functional differentiation while retaining the conserved MADS-box genes shared functions [36,37,38]. Considering that tassels, as perennial woody plants, may have differences in the distribution of motifs compared to Arabidopsis.

Fig. 4
figure 4

Phylogenetic relationships, domains, and motifs composition of CrMADSs

Identification and enrichment analysis of cis-acting elements in promoters of CrMADSs

In gene regulation, promoter cis-acting elements play crucial roles in controlling gene expression [39]. The promoters of 61 CrMADS genes were analyzed using PlantCARE tool [40]. The identified cis-acting elements were than classified based on their potential regulatory functions. In our analysis, we identified a total of 2760 number of specific cis-acting elements in the 2000 bp upstream sequence of the 61 CrMADS genes (Fig. 5). According to their functional annotations, they are divided into 5 categories, namely growth-related elements, hormone-responsive elements, light-responsive elements, stress-responsive elements, and transcription factor binding sites. Among them, the largest quantity is the light-responsive elements (879), followed by transcription factor binding sites (784) and stress-responsive elements (607). The least abundant are hormone-responsive elements and growth-related elements (288 and 202, respectively). This suggests that the CrMADS genes may respond to light and stress stimuli, and they may be regulated by various transcription factors. Among the 61 CrMADS genes, CrMADS1 possesses the highest number of cis-acting elements. A total of 136 elements were identified in its promoter sequence, which is significantly higher than average count of 45.2 (Fig. 5). These include 22 MYB elements, 14 G-box elements, and 12 GT1-motif. The significantly higher number of cis-acting elements in CrMADS1 compared to the average indicates that it may be plays an important role in floral organ development.

Fig. 5
figure 5

Enrichment and distribution of cis-acting elements in the promoter of CrMADSs. The color and size of the point indicate the number of elements, while red and large dimensions indicate more element

The differential expression patterns of the C. retusus MADS-box genes in androdioecy flowers

The C. retusus, as an androdioecy plant, exists in nature only as hermaphroditic flower individuals and male flower individuals. To investigate the expression patterns of CrMADS genes during the development of C. retusus flowers in different sexes, we selected floral organs from three varieties: ‘XZH’ (hermaphroditic flowers), ‘XDL’ (male flowers), and ‘XX’ (male flowers). We measured their transcriptomes during four development stages: bud stage (B), initial flowering stage (I), full flowering stage (Full), and final flowering stage (Final). The heatmap (Fig. 6) displays the expression patterns of 61 CrMADS genes. The 61 CrMADS genes exhibit different expression patterns during the flower development process in different varieties. It should be noted that CrMADS genes from the same subfamily often exhibit similar expression patterns. For example, within the MIKC* subfamily, MADS-box genes such as CrMADS19, CrMADS5, CrMADS27, and CrMADS29; and within the SEP subfamily, CrMADS1, CrMADS24, and CrMADS18 exhibit similar expression patterns (Fig. 6).

Fig. 6
figure 6

Expression and differential analysis of CrMADSs. a Heatmap of the expression of 61 CrMADSs in flower organs at four stages of three varieties. The expression heatmap of 61 MADS in flower organs of three varieties at four stages. B represents the Bud stage, I represents the Initial flowering stage, Full represents the Full flowering stage, and Final represents the Final flowering stage. b Genes differentially expressed in the bud stage between ‘XZH’ and two other varieties. c Wayne diagram of differentially expressed genes

Compared to the ‘XX’ and ‘XDL’ varieties, ‘XZH’ exhibits specific upregulation of certain genes during the bud stage, initiation flowering, and full flowering stage. For instance, genes such as CrMADS60 and CrMADS48 show higher expression levels in ‘XZH’ than in ‘XX’ and ‘XDL’. ‘XX’ and ‘XDL’ are male individuals, while ‘XZH’ is female individuals. In ‘XZH’, there are both complete pistils and stamens, while in ‘XX’ and ‘XDL’, only stamens exist and the pistils are completely absent. Therefore, we assume that MADS-box genes related to carpel development, such as class C genes, will exhibit differential expression between ‘XDL’ and the other two varieties. In order to identify genes in the MADS-box family that may be involved in gender differentiation of C. retusus flower, we screened for differentially upregulated CrMADS genes during the bud stage in ‘XZH’ compared to ‘XX’ or ‘XDL’ (Fig. 6). We then took the intersection of these genes, resulting in a total of six differential genes identified as gender-related CrMADS genes (Fig. 6). These six differential genes primarily belong to the AP3/PI, SVP, AG, and AGL15 subfamilies. Subsequently, to validate the expression patterns of these six genes in flowers of different genders, we selected an additional eight varieties, including four male flower individuals (‘XL’, ‘BM’, ‘H19’, ‘XRQ’) and four hermaphrodite flower individuals (‘XI’, ‘NY’, ‘T-8’, ‘MZ’) (Fig. 7). The qRT-PCR was performed to test the expression patterns of these six candidate genes in the bud flowers of these eight varieties. The results indicated that out of the six candidate genes, only CrMADS45 and CrMADS60 exhibited significant gender-specific expression patterns (Fig. 7). Among them, CrMADS45 showed expression levels ranging from 77.8 to 141.1 times higher in hermaphrodite flowers compared to male flowers. The expression fold of CrMADS60 is 23.9 to 62.7 times. Importantly, it is noteworthy that both CrMADS45 and CrMADS60 both belong to the AG subfamily, whose members in Arabidopsis have been shown to play critical roles in carpel development [41, 42].

Fig. 7
figure 7

The floral organs of eight varieties and the expression patterns of CrMADSs in floral organs. a Vertical sectional views of floral organs during the bud stage. Among them, BM, XRQ, XL, H19 are male flower individuals, XI, T-8, MZ, NY are bisexual flower individuals. Both individuals have stamens, while only bisexual individuals have pistils. b The relative expression levels of differentially expressed CrMADSs in flower organs of 8 varieties. c The relative expression levels of CrMADSs in the AG subfamily in flower organs of 8 varieties

In addition to CrMADS45 and CrMADS60, there are three other members in the AG subfamily of C. retusus, namely CrMADS9, CrMADS55, and CrMADS61 (Fig. 2). To determine whether the three were also involved in the gender differentiation of C. retusus, we also measured their expression levels in eight varieties. However, the results showed that CrMADS9, CrMADS55, and CrMADS61 did not exhibit the same expression patterns as CrMADS45 and CrMADS60 (Fig. 7). Hence, we conclude that only CrMADS45 and CrMADS60 play important roles in the differentiation of male flowers and hermaphrodite flowers in C. retusus.


As a garden ornamental plant, the cultivation of C. retusus focuses on the diversity of flower morphology [6]. The morphology of plant flowers is mainly determined by the arrangement and quantity of floral organs [35]. In Arabidopsis, the MADS-box gene family consists of 108 members and is divided into two main group: type I and type II [17]. Among them, the type II genes, specifically the MIKC-type MADS-box genes, are primarily associated with flower organ development, there are a total of 45 members in the MIKC-type MADS-box gene family of Arabidopsis [16]. The number of MADS-box genes varies significantly among different species. Here, we identified a total of 61 MIKC-type MADS-box genes in the genome of C. retusus. This number is higher compared to several other dicotyledonous plants, such as Arabidopsis with 45 MIKC-type MADS-box genes, sacred lotus with 28 genes [43], and sesame with 33 genes [34]. However, the number of MIKC-type MADS-box genes in C. retusus is significantly lower than that in the monocotyledonous plant wheat, which has 201 MIKC-type MADS-box genes [44]. Within the Oleaceae family, such as S. oblata, O. europaea, and C. retusus, the number of MADS-box genes is generally similar (Fig. S3). This indicates that MIKC-type MADS-box genes have undergone complex expansions and losses during the evolution of different species.

In previous studies, MIKC-type MADS-box genes have been classified into 12 major subfamilies based on their functional characteristics and sequence similarity [17]. Here, 61 MIKC-type MADS-box genes in C. retusus have been successfully mapped to the 12 subfamilies, with each subfamily containing at least one CrMADS genes. However, compared to Arabidopsis, C. retusus MADS-box genes have undergone significant doubled in several subfamilies, such as the AGL12 and SVP subfamilies. In contrast, in some other subfamilies, such as the FLC, the number of MADS-box gens in C. retusus has significantly halved. Both the FLC and SVP subfamilies have been shown to be involved in the transition to flowering, while AGL12 is considered to be associated with root development and the transition to flowering [45,46,47]. Therefore, the expansions and contractions of C. retusus different MADS-box subfamilies reflect its potential adaptations in various aspects of growth and development.

C. retusus, as an androdiecious plant, exists in nature as only two types of individuals: male flower individuals that produce only male flowers and hermaphroditic flower individuals that produce only hermaphrodite flowers [6, 9]. In our study, by analyzing the transcriptomes of different gendered varieties of C. retusus flowers, we identified 6 genes that showed differential expression during the floral bud stage, include members of the AP3/PI, AG, SVP, and AGL15 subfamilies. Previous studies have shown that AP3/PI predominantly functions in determining the identity of petals and stamens, known as the B-class function [48, 49]. AG primarily acts in determining the identity of carpels and stamens, referred to as the C-class function [27]. SVP and AGL15 play significant roles in the transition to flowering, and AGL15 also plays a role in embryogenesis [45, 50, 51]. In the process of flower development, there may be differences between ‘XZH’ and ‘XX’ /’XDL’ in these aspects. However, in the subsequent qRT-PCR results of eight varieties, only CrMADS45 and CrMADS60 showed significant differential expression in male flowers and hermaphroditic flowers. Interestingly, both of these two genes belong to the AG subfamily. Previous studies have shown that members of the AG subfamily mainly play a role in C-class functions, and some members also play D-class functions. C-class genes are associated with the formation of both stamens and pistils [52, 53]. The genes of the AG subfamily should also regulate the development of pistils in C. retusus through this very complex mechanism, and thus affect their gender differentiation. Just as, subsequent measurements of the expression levels of the other three members of the AG subfamily did not show the same expression pattern, indicating that not all AG subfamily genes are involved in the gender differentiation of C. retusus, or play different roles in this process. By reviewing our results on the cis-acting elements of promoters and conserved motifs of amino acid sequences, we hope to find relevant information on why these genes in the AG subfamily exhibit different performances. Unfortunately, we have not found any useful clues. However, future research on the population of C. retusus may provide an answer.

In summary, our study involved the sequence analysis and the expression analysis of the MADS-box genes in a recently sequenced species called C. retusus (Chinese Fringe tree), and identified two MADS-box genes that may be involved in sex differentiation of C. retusus. This research provides a valuable reference for future studies on gene function and breeding strategies in C. retusus.


In this study, we identified 61 MIKC-type MADS-box genes within the C. retusus genome. Through phylogenetic analysis, these genes were classified into 12 distinct subfamilies. The analysis of genomic collinearity reveals the duplication events experienced by CrMADS genes and the purification selection accompanying the diversification process of plants in the Oleaceae family. Conservation domain and motif analyses demonstrated their structural conservation. The determination of gene expression levels in flower organs of different genders showed that CrMADS45 and CrMADS60 exhibited significant high expression in bisexual flowers, indicating that they may play a key role in the sex determination process of C. retusus. In summary, our efforts not only increase the existing knowledge of the MADS-box gene family within C. retusus, but also provide a foundation for further exploration of the flower evolution and gender determination mechanisms of this species.


Plant materials

The experimental materials used for transcriptome sequencing were obtained from three C. retusus varieties (‘XueZaoHua’ or ‘XZH’, ‘XueDengLong’ or ‘XDL’, and ‘XueXuan’ or XX) grown at the experimental field of Shandong Agricultural University (36°10′ N, 117°9′ W). In mid-April 2023, flower organs were collected at four different development stages, including the floral bud stage (B), initial flowering stage (I), full flowering stage (Full), and final flowering stage (Final). Sample each variety three times for each period to obtain three replicates. These samples were rapidly frozen in liquid nitrogen and stored at -80℃ for subsequent transcriptome sequencing.

Materials from an additional eight C. retusus varieties (‘XueLuo’ or ‘XL’, 'BaoMa' or ‘BM’, 'H-19–61' or ‘H19’, 'XueRongQiu' or ‘XRQ’, 'XueLi' or ‘XI’, 'NiuYe' or ‘NY’, 'T-8', and 'MuZhu' or ‘MZ’) were also collected from the same location and during the same period mentioned earlier. The entire flower organs at the initial flowering stage of these varieties were chosen as the experimental materials. A portion of the collected materials was rapidly frozen in liquid nitrogen and stored at -80℃ for subsequent qRT-PCR experiments. Another portion was subjected to vertical sectioning using a razor blade and observed under a stereomicroscope to document the growth conditions of the pistils and stamens.

Among the aforementioned 11 C. retusus varieties, XZH, XI, NY, T-8, and MZ are hermaphroditic flower varieties, meaning they only have the hermaphroditic flowers. On the other hand, XDL, XX, XL, BM, H19, and XRQ are male flower varieties, indicating that they produce only male flowers. These 11 varieties are all clones independently selected by our laboratory, and all variety names and abbreviations only represent our numbering of clones. These clones are all derived from germplasm resources in different regions of China, such as Shandong Province, Henan Province, and Beijing City, and propagated through grafting. These clones only represent individuals of different genders and have been planted in the germplasm resource garden of our forestry station located at Shandong Agricultural University (36°10′ N, 117°9′ W) for scientific research and teaching purposes for a long time. Therefore, they have not been submitted to the herbarium. We regularly maintain these plants to ensure their health and stability, and to ensure reliable experimental materials can be provided.

Identification of the MIKC-type MADS-box gene in C. retusus

The reference genome information of the C. retusus is based on the latest sequencing and assembly of the ‘XZH’ variety genome in our past study (unpublished, if you need it, you can contact the corresponding author). Hidden Markov Model (HMM) profile for the MADS-box (SRF-TF: PF00319) and K-box (K-box: PF01486) domains were downloaded from the Pfam database ( [54]. Hmmersearch was conducted using the graphical interactive software SPDE v2.0 to search for protein sequences containing the MADS-box and K-box domains in the C. retusus genome [55]. The obtained protein sequences, along with the 108 MADS-box protein sequences from Arabidopsis, were used to construct a phylogenetic tree using MEGA X software [56]. Based on the classification of type I and type II MADS-box proteins in Arabidopsis, it is possible to distinguish between type I and type II MADS-box proteins in the C. retusus. In this case, all the type II MADS-box proteins can be retained for subsequent analysis. All corresponding MADS-box genes are named based on their location on the chromosome. Additionally, the physicochemical properties of MADS-box proteins, including molecular weight (MW), theoretical isoelectric point (pI), instability index (II), and grand average of hydropathicity (GRAVY), be calculated using the ExPasy serve (

Phylogenetic and genetic structure analysis

The ClustalW algorithm was used to align the full-length sequences of 61 MIKC-type MADS-box proteins from the C. retusus and 45 MIKC-type MADS-box proteins from Arabidopsis, all parameters were set to default values. Subsequently, a maximum-likelihood phylogenetic analysis was performed under the Jones-Taylor-Thornton amino acid substitution model in MEGA X with 1000 bootstrap replicates, and the resulting tree was visualized with Evolview ( [57].

Extract the annotation information of all 61 MIKC-type MADS-box genes from the C. retusus genome annotation file, and visualize the exon–intron distribution of all genes using the Tbtools [58].

Protein conservation domain and motif analysis of CrMADSs

Protein conservation domain analysis can be performed using the CD-Search tool of NCBI ( All CrMADS protein sequences be submitted to the CD-Search website to obtain information about the location of conservation domains [59].

The conserved motif in the proteins were analyzed using the MEME ( databased [60]. The CrMADS sequences are submitted, and the following parameters are used for the analysis: classic mode, allowing zero or one occurrence per sequence, and a total number of motifs set to 20. Finally, visualize the distribution of all structural domains and motifs using Python.

Analysis of promoter cis-acting element of CrMADSs

The 2000 bp upstream of the start codon of 61 CrMADS genes was extracted, using the SPDE v2.0 software. Subsequently, all the sequences were submitted to the PlantCARE ( database for prediction of cis-acting elements [40]. Based on the functional annotation of the elements, the specific elements were classified into five categories: growth-related elements, hormone-responsive elements, light-responsive elements, stress- responsive elements, and transcription factor binding sites. Finally, the number of specific elements was counted for visualization, and the distribution of elements was visualized using Tbtools.

Genomic collinearity analysis of CrMADS genes

To perform intra-species genome collinearity analysis of the C. retusus, MCScanX was used to identify all collinear blocks within the C. retusus genome [61]. The downstream analysis software of MCScanX, “detect_collinearity_within_gene_families”, was utilized to further analyze the collinear pairs of CrMADS genes that have a collinear relationship. Finally, Circos was used to display the collinearity information.

To analyze the collinearity relationship of MADS-box genes between the C. retusus and two other Oleaceae plants, Olea europaea and Syringa oblata, the reference genomes of O. europaea and S. oblata were downloaded [29, 30]. The same method as mentioned above was used to identify the genomic collinearity information and collinear MADS-box gene pairs. Finally, the Dual Systeny Plot for MCScanX feature in Tbtools was employed for visualization. The Ka/Ks values between all collinear CrMADS gene pairs were calculated based on the CDS sequence and amino acid sequence, and were calculated by Simple Ka/Ks Calculator of TBtools.

Expression level analysis

Fragments Per Kilobase Million (FPKM) values of 61 CrMADS genes were extracted from the previous transcriptome data (unpublished), and a heatmap was generated to display the data, the data used to construct a heatmap is obtained by taking the average of three replicates. The data in the heatmap were normalized by row using the ZeroToOne method, and row clustering was performed on the normalized data.

For qRT-PCR, total RNA from the materials at the initial flowering stage of the eight mentioned varieties was extracted using the Trizol method, and then quality-checked on a NanoDrop One UV spectrophotometer (Thermo Scientific, USA). Subsequently, the first-strand cDNA was synthesized using the Evo M-MLV Plus 1st Strand cDNA Synthesis Kit AG11615 (AG). The qRT-PCR was performed on the CFX-96 real-time PCR detection system (Bio-Rad, USA). Each experiment consisted of three independent biological replicates, with three technical replicates per sample. The UBIQUITIN CARRIER PROTEIN2 (UBC2) gene (Table S1) of C. retusus was used as an internal control. Relative expression levels of each target gene were analyzed the 2−∆∆CT method [62]. The primers used are shown in Table S1.

Availability of data and materials

The genomic information of the Chionanthus retusus mentioned in the article is available from the corresponding author upon reasonable request. This assembly used HiC and PacBio methods, with a scaffold quantity of 134. The Hi-C and ONT data, as well was the assemblies have been deposited to National Genomics Data Center with Bioproject ID of CRA011999. Accession "CRA011999" was publicly disclosed on June 20, 2024.







Base pair


Fragments per Kilobase Million


Molecular weight


Isoelectric point


Instability index


Grand average of hydropathicity


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This work was supported by the Subject of Key R & D Plan of Shandong Province (Major Scientific and Technological Innovation Project) Mining and Accurate Identification of Forest Tree Germplasm Resources (No. 2021LZGC023); and Agricultural science and Technology Fund Project of Shandong province (No. 2019LY001-4).

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This work was conceived by Jihong Li and Maotong Sun, Maotong Sun and Dongyue Wang wrote the manuscript and performed main bioinformatics analysis, Ying Li performed RNA isolation and qRT-PCR experiment. Muge Niu and Cuishuang Liu were responsible for collecting C. retusus samples, Laishuo Liu took the photos. Jihong Li and Jinnan Wang supervised this whole process and revised this manuscript. All authors have reviewed the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jinnan Wang or Jihong Li.

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Sun, M., Wang, D., Li, Y. et al. Genome-wide identification and expression pattern analysis of MIKC-Type MADS-box genes in Chionanthus retusus, an androdioecy plant. BMC Genomics 25, 662 (2024).

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