Genome-Wide Identification of the MADS-Box Gene Family during Male and Female Flower Development in Chayote (Sechium edule)

The MADS-box gene plays an important role in plant growth and development. As an important vegetable of Cucurbitaceae, chayote has great edible and medicinal value. So far, there is little molecular research on chayote, and there are no reports on the MADS-box transcription factor of chayote. In this study, the MADS-box gene family of chayote was analyzed for the first time, and a total of 70 MADS-box genes were identified, including 14 type I and 56 type II MICK MADS genes. They were randomly distributed on 13 chromosomes except for chromosome 11. The light response element, hormone response element and abiotic stress response element were found in the promoter region of 70 MADS genes, indicating that the MADS gene can regulate the growth and development of chayote, resist abiotic stress, and participate in hormone response; GO and KEGG enrichment analysis also found that SeMADS genes were mainly enriched in biological regulation and signal regulation, which further proved the important role of MADS-box gene in plant growth and development. The results of collinearity showed that segmental duplication was the main driving force of MADS gene expansion in chayote. RNA-seq showed that the expression levels of SeMADS06, SeMADS13, SeMADS26, SeMADS28, SeMADS36 and SeMADS37 gradually increased with the growth of chayote, indicating that these genes may be related to the development of root tubers of ‘Tuershao’. The gene expression patterns showed that 12 SeMADS genes were specifically expressed in the male flower in ‘Tuershao’ and chayote. In addition, SeMADS03 and SeMADS52 may be involved in regulating the maturation of male flowers of ‘Tuershao’ and chayote. SeMADS21 may be the crucial gene in the development stage of the female flower of ‘Tuershao’. This study laid a theoretical foundation for the further study of the function of the MADS gene in chayote in the future.


Introduction
MADS-box is a very important transcription factor widely existing in eukaryotes. MADS-box is an abbreviation of four genes initials: Mini chromosome maintenance 1 (MCM1) of Saccharomyces cerevisiae [1], AGAMOUS (AG) of Arabidopsis thaliana [2], DE-FICIENS (DEF) of Antirrhinum majus [3], and the serum response factor (SRF) of Homo sapiens [4]. The N-terminal of the MADS-box protein contains a conserved domain of MADS-box (M) composed of about 58 amino acids [5]. According to the classification criteria of MADS gene structure and phylogenetic analysis, the MADS gene can be divided into type I and type II [6]. Type I includes Mα, Mβ and Mγ. The type II MADS gene is also called the MICK gene family because it contains four domains: MADS-box (M), intermediate region (I), keratin-like (K), and carboxyl-terminal (C). MICK type genes can be divided into MICK c and MICK*, in which MICK c includes 13 subfamilies [7]. The type I MADS gene structure contains only 1-2 exons and a highly conserved MADS-box (M) domain, but lacks a K domain, which is the characteristic of distinguishing type I and type II MADS genes. The type II MADS gene contains 6-8 exons and is also the most studied MADS gene [8].
The MADS-box gene plays an important role in plant growth and development, especially in floral organ development. The classical ABC model proposed that the development of the floral organ was controlled by different genes. In Arabidopsis, sepal development is controlled by class A genes (AP1 and AP2), petal development is determined by class A and class B genes (AP3 and PI) together, carpel development is determined by separate class C genes (AG), and stamen development is controlled by class B and class C genes together [9]. Subsequently, the ABC model was extended to the ABCDE model [9]. In this model, class D genes (SKT) and class C genes together control ovule development, class A genes and class E genes (SEP1-4) participate in sepal development, and class C genes and class E genes participate in carpel development. Stamen development is jointly regulated by class A genes and class C genes, and petal development is jointly regulated by A, B and E genes [10]. At present, in the widely accepted ABCDE model, except AP2 in the class A gene, all other functional genes related to flower organ development are transcription factors, which contain a highly conserved DNA binding domain and belong to MADS-box family [11,12]. In addition, the MADS-box gene is also indispensable to regulate plant growth and development and resist various abiotic stresses [13]. Studies have shown that the MADS gene can regulate the development of sweet potato root tubers [14]. Overexpression of the ZmSOC1 gene can improve maize yield and grain quality [15]. In addition, a large number of studies have shown that the MADS gene also plays an important role in response to abiotic stress. The overexpression of the OsMADS25 gene in rice can improve salt tolerance and regulate auxin synthesis to promote root growth [16]. AGL16, a member of the Arabidopsis MADS-box family, negatively regulates stress response and is an important regulator to balance plant stress response and growth [17]. In addition, the MADS gene of Rhododendron also plays an important role under heat and waterlogging stresses [13].
Chayote (Sechium edule), 2n = 2x = 28, is a perennial herb climbing plant of Cucurbitaceae [18]. Chayote originated in Mexico and is widely planted in India, Brazil and Sichuan, Guizhou, Yunnan and other places in China. Its fruits, root tubers and leaves are rich in cucurbitoids, amino acids, vitamins, phenols and flavonoids, which not only make them widely used as an important vegetable, but also have the effect of treating diabetes and hypertension [19][20][21]. Chayote is mainly an edible fruit; and the chayote tuber is mainly an edible root. The fruit of chayote is developed from the expansion of female flowers after pollination. Its flowering period is from May to July and September to November every year. From May to July, only male flowers generally open. From September to November, the female flowers will bear fruit; the chayote tuber only blooms from September to November every year, and its female flower cannot normally expand into fruit [22]. Compared with Cucurbitaceae crops such as cucumber, melon, pumpkin and watermelon, the research on chayote is relatively sparse. The current research also focuses on the identification of fruit components. However, with the publication of the chayote genome, it provides great convenience for the research at the molecular level. Therefore, this study mainly uses the genome data of chayote to identify and analyze the MADS-box gene family and clarify the related mechanism of female and male floral organ development of chayote, and provide a theoretical basis for studying the molecular function of the MADS-box gene.

Identification and Physicochemical Properties Analysis of the MADS-Box Gene Family of Sechium edule
A total of 70 MADS genes were identified from the genome of chayote and named SeMADS01-70 based on the conserved MADS-box model ( Table 1). The physical and chemical properties of these MADS genes were analyzed by ExPASY online software. The results showed that the 70 MADS-box genes were predicted to encode polypeptides from 63 (SeMADS11) to 674 (SeMADS69) amino acids, with a predicted molecular weight ranging from 7133.35 to 77846.80 Da. The isoelectric point of SeMADS ranged from 4.94 (SeMADS64) to 9.99 (SeMADS48), while the grand average of the hydropathicity of all MADS proteins except SeMADS48 (0.069) was negative, ranging from −0.943 (SeMADS02) to −0.275 (SeMADS14), indicating that they were hydrophilic proteins. The total number of negatively charged residues (Asp + Glu) of SeMADS was 7 to 74, and the total number of positively charged residues (Arg + Lys) was 13 to 106. Subcellular localization predicted that all SeMADS were distributed in the nucleus. Table 1. Physical and chemical properties of MADS-box genes family in S. edule. (MW: molecular weight; PI: isoelectric point; ASP + Glu: total number of negatively charged residues (ASP + Glu); Arg + Lys: total number of positively charged residues (Arg + Lys); GRAVY: Grand average of hydropathicity; SLP: subcellular localization prediction).

Phylogenetic Analysis of SeMADS-Box
A phylogenetic tree was constructed using 70 SeMADS and 108 AtMADS proteins to clarify the evolutionary relationship of MADS in chayote. All MADS were divided into 15 clades marked with different colors (Figure 1). SeMADS, except for the FLC, formed 15 subfamilies. The maximum number of SeMADS found in the AP1 branch was 11, followed by the SVP branch with 9 SeMADS, while only one SeMADS gene was found in the AGL12 and Bs branches. The remaining SeMADS genes were distributed in 11 subfamilies, with two to eight genes in each subfamily.

Chromosomal Localization of SeMADS-BOX
Chromosome localization of 70 SeMADS-box genes was performed based on the genome annotation file of chayote ( Figure 2). The results showed that 70 SeMADS genes were unevenly distributed on the other 13 chromosomes except the LG11 chromosome. The number of MADS genes on each chromosome ranged from two to ten. There were 10 MADS genes on chromosome LG09, accounting for 14.3%, and only two MADS genes on chromosome LG02, LG03 and LG12, accounting for 2.9%. There were eight genes on chromosome LG10, accounting for 11.4%, seven genes on chromosome LG01, LG05 and LG13, accounting for 10%, six genes on chromosome LG06, accounting for 8.6%, five genes on chromosome LG04, LG07 and LG14, accounting for 7.1%, and four genes on chromosome LG08, accounting for 5.7%.

Chromosomal Localization of SeMADS-BOX
Chromosome localization of 70 SeMADS-box genes was performed based on the genome annotation file of chayote ( Figure 2). The results showed that 70 SeMADS genes were unevenly distributed on the other 13 chromosomes except the LG11 chromosome. The number of MADS genes on each chromosome ranged from two to ten. There were 10 MADS genes on chromosome LG09, accounting for 14.3%, and only two MADS genes on chromosome LG02, LG03 and LG12, accounting for 2.9%. There were eight genes on chromosome LG10, accounting for 11.4%, seven genes on chromosome LG01, LG05 and LG13, accounting for 10%, six genes on chromosome LG06, accounting for 8.6%, five genes on chromosome LG04, LG07 and LG14, accounting for 7.1%, and four genes on chromosome LG08, accounting for 5.7%.

Gene Structure and Conserved Motif Analysis of the SeMADS-Box
We used the GSDS program to analyze and display the exon and intron structures of 70 SeMADS genes to understand the diversity of the MADS-box gene structure of chayote ( Figure 3A,B). The results showed that the structure of 70 SeMADS genes showed diver-

Gene Structure and Conserved Motif Analysis of the SeMADS-Box
We used the GSDS program to analyze and display the exon and intron structures of 70 SeMADS genes to understand the diversity of the MADS-box gene structure of chayote ( Figure 3A,B). The results showed that the structure of 70 SeMADS genes showed diversity, but the same subfamily showed similarity. The number of exons varies from 1 to 21, and SeMADS69 has the largest number of 21 exons. Furthermore, we found that Mα, Mβ and Mγ mostly have no intron structure, or have one to two introns. These three subfamilies belonged to type I genes. This was the same as the type I gene of MADS in Arabidopsis and tomato, which may be due to the different trend of intron acquisition or loss or the reverse-transcribed origin of type I genes ancestors [23,24].

Promoter Region Analysis of SeMADS-Box
Transcription factors can bind to the promoter region to regulate the expression of target genes. To predict cis-regulatory elements in the promoters of SeMADS, we analyzed 2 kb promoter sequences in the PlantCARE database. The results showed that all SeMADS genes contained light response elements (G-box, GT1 motif, Box4, ATCT motif, GATA motif, I-box, AE-box), which accounted for 51.7% of all cis-regulated elements. Hormonal response elements such as auxin response elements (TGA-element and AuxRRcore), abscisic acid response elements (ABRE), gibberellin response elements (TATC-box, p-box and GARE motif), MeJA-response (CGTCA-motif and TGACG-motif), salicylic acid response (TCA-element), and abiotic stress response elements such as drought induction (MBS), low-temperature response (LTR), anaerobic induction (ARE), defense and stress responses (TC-rich repeats) and wound-responsive element (WUN-motif) were widely distributed in MADS genes in various subfamilies. These response elements enabled chayote to cope with various adverse environmental changes and ensure its normal growth and development ( Figure 4). The protein sequences of 70 SeMADS genes were analyzed by the meme online program. A total of 10 motifs were obtained, named motifs 1-10 ( Figure 3C). The results showed that all MADS proteins had motif 1 at the N-terminal of the sequence except for SeMADS42. This was a conserved motif that was thought to encode the MADS-box domain. We analyzed SeMADS42 with SMART software and found that it has a MADS domain. In addition, motif 2 was distributed in other subfamilies besides Mα, Mβ and Mγ. Motif 2 encoded the K domain, which was a marker to distinguish type 1 and type 2 MADS genes. Motifs 3, 4, 5 and 6 were also widely distributed in type II MADS genes. Motifs 7 and 8 were only distributed in the Mβ subfamily. Motif 9 was distributed in the protein sequences of SeMADS27, 28 and 35, and motifs 10 were only distributed in the SOC1 subfamily.

Promoter Region Analysis of SeMADS-Box
Transcription factors can bind to the promoter region to regulate the expression of target genes. To predict cis-regulatory elements in the promoters of SeMADS, we analyzed 2 kb promoter sequences in the PlantCARE database. The results showed that all SeMADS genes contained light response elements (G-box, GT1 motif, Box4, ATCT motif, GATA motif, I-box, AE-box), which accounted for 51.7% of all cis-regulated elements. Hormonal response elements such as auxin response elements (TGA-element and AuxRRcore), abscisic acid response elements (ABRE), gibberellin response elements (TATC-box, p-box and GARE motif), MeJA-response (CGTCA-motif and TGACG-motif), salicylic acid response (TCA-element), and abiotic stress response elements such as drought induction (MBS), low-temperature response (LTR), anaerobic induction (ARE), defense and stress responses (TC-rich repeats) and wound-responsive element (WUN-motif) were widely distributed in MADS genes in various subfamilies. These response elements enabled chayote to cope with various adverse environmental changes and ensure its normal growth and development ( Figure 4).

Collinearity Analyses of the SeMADS-Box within and between Species
The expansion of the gene family mainly includes five categories: whole-genome replication, scattered duplication, tandem duplication, segmental duplication, and proximal duplication [13]. We used BLASTp and MCScanX software (California, USA, http://chibba.pgml.uga.edu/mcscan2/, accessed on 12 January 2022) to analyze the replication type of SeMADS genes and conducted a collinearity analysis to explore the amplification mechanism of the chayote MADS gene family. The results showed that a total of eight SeMADS genes (11.43%) were found to form five tandem duplication gene pairs, and 50 SeMADS genes (71.43%) formed 38 segmental duplication gene pairs ( Figure 5A and Table S1). Therefore, segmental duplication was the main driving force for the expansion of the SeMADS genes family. It is worthy of note that most gene pairs were distrib-

Collinearity Analyses of the SeMADS-Box within and between Species
The expansion of the gene family mainly includes five categories: whole-genome replication, scattered duplication, tandem duplication, segmental duplication, and proximal duplication [13]. We used BLASTp and MCScanX software (California, USA, http: //chibba.pgml.uga.edu/mcscan2/, accessed on 12 January 2022) to analyze the replication type of SeMADS genes and conducted a collinearity analysis to explore the amplifica-tion mechanism of the chayote MADS gene family. The results showed that a total of eight SeMADS genes (11.43%) were found to form five tandem duplication gene pairs, and 50 SeMADS genes (71.43%) formed 38 segmental duplication gene pairs ( Figure 5A and Table S1). Therefore, segmental duplication was the main driving force for the expansion of the SeMADS genes family. It is worthy of note that most gene pairs were distributed in the same subfamily on the phylogenetic tree. We further calculated Ka/Ks to detect the selection pressure during the replication of 43 pairs of SeMADS genes (Table S1). The results showed that only one of the tandem duplication gene pairs (SeMADS45 and SeMADS46) had Ka/Ks > 1, indicating that it has been positively selected in the process of evolution. Other tandem duplication and all segmental duplication gene pairs had Ka/Ks < 1, suggesting that most SeMADS genes were under purification selection. followed by watermelon (84), cucumber (74) and melon (73) ( Figure 5B, Table S2). The number of collinear gene pairs between chayote and these species was not related to their own genome size. In addition, chayote and melon have no collinear gene pairs on chromosomes 1 and 5, while there were no gene pairs on chromosomes 5 and 11 between chayote and pumpkin ( Figure 5B). 15  In addition, we also found that SeMADS23 was only homologous to melon and SeMADS27, and SeMADS28 was only homologous to pumpkin. Interestingly, some SeMADS genes, such as SeMADS07, SeMADS57 and SeMADS67, formed two to five collinear gene pairs, especially three to four collinear gene pairs with four species of Cucurbitaceae, suggesting that they play an important role in the evolution of the MADS gene family of chayote (Table S2).  To further understand the evolution of the MADS gene family of chayote, we analyzed the collinear relationship between chayote and Arabidopsis, as well as some representative species of Cucurbitaceae (cucumber, melon, pumpkin and watermelon). The results showed that there were 55 pairs of homologous MADS genes between chayote and Arabidopsis ( Figure 5B), 104 pairs of homologous genes with Cucurbitaceae pumpkin, followed by watermelon (84), cucumber (74) and melon (73) ( Figure 5B, Table S2). The number of collinear gene pairs between chayote and these species was not related to their own genome size. In addition, chayote and melon have no collinear gene pairs on chromosomes 1 and 5, while there were no gene pairs on chromosomes 5 and 11 between chayote and pumpkin ( Figure 5B). 15  In addition, we also found that SeMADS23 was only homologous to melon and SeMADS27, and SeMADS28 was only homologous to pumpkin. Interestingly, some SeMADS genes, such as SeMADS07, SeMADS57 and SeMADS67, formed two to five collinear gene pairs, especially three to four collinear gene pairs with four species of Cucurbitaceae, suggesting that they play an important role in the evolution of the MADS gene family of chayote (Table S2).

GO and KEGG Analysis of SeMADS-Box
To further understand the function of SeMADS genes, 70 SeMADS genes were analyzed by GO enrichment and KEGG pathway analysis. Seventy SeMADS genes were significantly enriched in 52 biological processes (BP), four molecular function (MF) and 3 cellular components (CC) ( Figure 6A, Table S3). In the BP category, three SeMADS genes were enriched in the regulation of flower development (GO:0009909) and the regulation of shoot system development (GO:0048831). The anchored component of plasma membrane (GO:0046658) and DNA binding transcription factor activity (GO:0000981) were the main enriched items in the CC and MF categories. The results of the KEGG pathway showed that 70 SeMADS genes were mainly significantly (Q < 0.05) enriched in MAPK signaling pathway-fly (ko04013), cGMP-PKG signaling pathway (ko04022), apelin signaling pathway (ko04371), and parathyroid hormone synthesis, secretion and action (ko04928) ( Figure 6B, Table S3).

GO and KEGG Analysis of SeMADS-Box
To further understand the function of SeMADS genes, 70 SeMADS genes were analyzed by GO enrichment and KEGG pathway analysis. Seventy SeMADS genes were significantly enriched in 52 biological processes (BP), four molecular function (MF) and 3 cellular components (CC) ( Figure 6A, Table S3). In the BP category, three SeMADS genes were enriched in the regulation of flower development (GO:0009909) and the regulation of shoot system development (GO:0048831). The anchored component of plasma membrane (GO:0046658) and DNA binding transcription factor activity (GO:0000981) were the main enriched items in the CC and MF categories. The results of the KEGG pathway showed that 70 SeMADS genes were mainly significantly (Q < 0.05) enriched in MAPK signaling pathway-fly (ko04013), cGMP-PKG signaling pathway (ko04022), apelin signaling pathway (ko04371), and parathyroid hormone synthesis, secretion and action (ko04928) ( Figure 6B, Table S3). (A)

Protein-Protein Interaction Network Analysis of the SeMADS-Box
Almost all life activities in cells depend on protein-protein interaction [25]. We predicted the protein interaction of SeMADS based on the homologous MADS gene in Arabidopsis using the STRING online database. As shown in Figure 7, FUL (ortholog of SeMADS02), AP1 (ortholog of SeMADS36) and AG (ortholog of SeMADS51) could co-regulate the development of flower meristem. SVP (SeMADS53 orthologous), AGL24 (ortholog of SeMADS10) and AGL6 (ortholog of SeMADS09) could participate in flowering regulation. SHP1 (ortholog of SeMADS01) and SHP2 (ortholog of SeMADS22) regulate ovule development. AGL14 (ortholog of SeMADS07), AGL21 (ortholog of SeMADS52) and AGL44 (ortholog of SeMADS54) co-regulate root development.

Protein-Protein Interaction Network Analysis of the SeMADS-Box
Almost all life activities in cells depend on protein-protein interaction [25]. We predicted the protein interaction of SeMADS based on the homologous MADS gene in Arabidopsis using the STRING online database. As shown in Figure 7, FUL (ortholog of SeMADS02), AP1 (ortholog of SeMADS36) and AG (ortholog of SeMADS51) could coregulate the development of flower meristem. SVP (SeMADS53 orthologous), AGL24 (ortholog of SeMADS10) and AGL6 (ortholog of SeMADS09) could participate in flowering regulation. SHP1 (ortholog of SeMADS01) and SHP2 (ortholog of SeMADS22) regulate ovule development. AGL14 (ortholog of SeMADS07), AGL21 (ortholog of SeMADS52) and AGL44 (ortholog of SeMADS54) co-regulate root development.

Expression Profiles of the SeMADS-Box
The root tubers of chayote have important economic value. In order to further investigate the potential role of the MADS gene in root tubers, we conducted RNA-seq on the root tubers of 'Tuershao' at three different developmental stages. The results showed that most MADS genes were differentially expressed at different developmental stages (Figure 8). In the T1 period, the gene expression levels of SeMADS01, SeMADS18, SeMADS27, SeMADS33, SeMADS55, SeMADS70, SeMADS08 etc. were relatively high. SeMADS53, SeMADS15, SeMADS38, SeMADS01, SeMADS60, SeMADS58, SeMADS45 etc. genes were highly expressed in the T2 period. The expression levels of SeMADS43 and SeMADS57 were higher in the T3 period. It is worth noting that the expression levels of SeMADS36, SeMADS37, SeMADS13, SeMADS28, SeMADS06 and SeMADS26 gradually increased with the growth of 'Tuershao' time, indicating that these genes may be related to the root tuber development of 'Tuershao'.

Expression Profiles of the SeMADS-Box
The root tubers of chayote have important economic value. In order to further investigate the potential role of the MADS gene in root tubers, we conducted RNA-seq on the root tubers of 'Tuershao' at three different developmental stages. The results showed that most MADS genes were differentially expressed at different developmental stages ( Figure  8). In the T1 period, the gene expression levels of SeMADS01, SeMADS18, SeMADS27, SeMADS33, SeMADS55, SeMADS70, SeMADS08 etc. were relatively high. SeMADS53, SeMADS15, SeMADS38, SeMADS01, SeMADS60, SeMADS58, SeMADS45 etc. genes were highly expressed in the T2 period. The expression levels of SeMADS43 and SeMADS57 were higher in the T3 period. It is worth noting that the expression levels of SeMADS36, SeMADS37, SeMADS13, SeMADS28, SeMADS06 and SeMADS26 gradually increased with the growth of 'Tuershao' time, indicating that these genes may be related to the root tuber development of 'Tuershao'.

Expression Analysis of SeMADS-Box in Different Tissue and Stage
To investigate the chayote MADS-box genes expression of different tissues, we selected 12 MADS genes for quantitative analysis in roots, stems, leaves, tendrils, male flowers, and female flowers (Figure 9). Most MADS-box genes were highly expressed in the male flowers of 'Tuershao', including SeMADS03, SeMADS12, SeMADS15, SeMADS19,

Expression Analysis of SeMADS-Box in Different Tissue and Stage
To investigate the chayote MADS-box genes expression of different tissues, we selected 12 MADS genes for quantitative analysis in roots, stems, leaves, tendrils, male flowers, and female flowers (Figure 9). Most MADS-box genes were highly expressed in the male flowers of 'Tuershao', including SeMADS03, SeMADS12, SeMADS15, SeMADS19, SeMADS21, SeMADS42, SeMADS47, SeMADS49, SeMADS56, and SeMADS57, while Se-MADS03, SeMADS15, SeMADS21, SeMADS42, SeMADS47, SeMADS49 and SeMADS57 were highly expressed in the male flowers of chayote. SeMADS12 and SeMADS52 had the highest relative expression in the leaf of chayote and in root of 'Tuershao'. SeMADS54 was expressed in all organs in 'Tuershao' and chayote. These results indicated that SeMADS genes had tissue specificity. To further verify the functions of SeMADS in the process of floral organ development, we analysed the 12 SeMADS genes expression from five developmental stages of male and female flowers in 'Tuershao' and chayote by qRT-PCR (Figures 10 and 11). In To further verify the functions of SeMADS in the process of floral organ development, we analysed the 12 SeMADS genes expression from five developmental stages of male and female flowers in 'Tuershao' and chayote by qRT-PCR (Figures 10 and 11). In the process of male flower development, SeMADS03, SeMADS12, SeMADS15, SeMADS47 and SeMADS52 were highly expressed at S4 in 'Tuershao', while SeMADS42 and SeMADS56 were expressed to a lower degree; the expression of SeMADS03, SeMADS19, SeMADS52, SeMADS54 and SeMADS57 was highest at S5 in chayote ( Figure 10); it is speculated that these genes are related to the maturation of male flowers in 'Tuershao' and chayote. In the process of female flower development, most SeMADS genes showed a single peak trend in S2 or S3 (Figure 11), which indicated that these SeMADS genes were important to 'Tuershao' and chayote in the early and middle stage of female flower development. Across S1-S5, the expression of SeMADS21 in the female flower of 'Tuershao' gradually increased ( Figure 11

Discussion
With the completion of genome sequencing, many MADS-box whole gene family analyses have been reported, such as Arabidopsis [23], tomato [24], and wheat [26]. In this study, we analyzed the genome of Chayote and found 70 MADS-box genes. Compared with other species of Cucurbitaceae, including watermelon (39) [27], cucumber (43) [28] and melon (62) [29], chayote has relatively more MADS-box genes. On the one hand, this phenomenon may be related to the genome size. The genome of chayote was 606.42Mb, which was larger than those of these three plants [18]; On the other hand, during the evolution of Cucurbitaceae, 184 gene families in the genome of chayote contracted and 200 expanded, while 138 in watermelon contracted and 109 expanded, 193 in Cucumber contracted and 91 expanded, and 116 in melon contracted and 162 expanded [18]. More expansion genes in chayote may lead to more MADS-box genes [18]. In addition, the number of MADS-box genes in the Cucurbitaceae species was less than that in Arabidopsis (108) [23], wheat (201) [26] and tomato (131) [24]. It seems that Cucurbitaceae species have lost some MADS-box genes in evolution [27]. The MADS-box gene was usually divided into type I and type II. Type I included Mα, Mβ, and Mγ. Type II was further divided into MICK c and MiCK*. According to the classification method of the MADS-box gene in Arabidopsis, the phylogenetic tree of the MADS gene family in Chayote was constructed. Seventy SeMADS genes were divided into 14 subfamilies, type I Mα (7) (2), SEP (4), and AP1 (11). Some 80% of them belong to type II MADS, which is similar to that reported in the MADS gene family, indicating that type II MADS genes were more conservative than type I MADS genes [13,14,30]. The FLC gene was

Discussion
With the completion of genome sequencing, many MADS-box whole gene family analyses have been reported, such as Arabidopsis [23], tomato [24], and wheat [26]. In this study, we analyzed the genome of Chayote and found 70 MADS-box genes. Compared with other species of Cucurbitaceae, including watermelon (39) [27], cucumber (43) [28] and melon (62) [29], chayote has relatively more MADS-box genes. On the one hand, this phenomenon may be related to the genome size. The genome of chayote was 606.42 Mb, which was larger than those of these three plants [18]; On the other hand, during the evolution of Cucurbitaceae, 184 gene families in the genome of chayote contracted and 200 expanded, while 138 in watermelon contracted and 109 expanded, 193 in Cucumber contracted and 91 expanded, and 116 in melon contracted and 162 expanded [18]. More expansion genes in chayote may lead to more MADS-box genes [18]. In addition, the number of MADS-box genes in the Cucurbitaceae species was less than that in Arabidopsis (108) [23], wheat (201) [26] and tomato (131) [24]. It seems that Cucurbitaceae species have lost some MADS-box genes in evolution [27]. The MADS-box gene was usually divided into type I and type II. Type I included Mα, Mβ, and Mγ. Type II was further divided into MICK c and MiCK*. According to the classification method of the MADS-box gene in Arabidopsis, the phylogenetic tree of the MADS gene family in Chayote was constructed. Seventy SeMADS genes were divided into 14 subfamilies, type I Mα (7) (2), SEP (4), and AP1 (11). Some 80% of them belong to type II MADS, which is similar to that reported in the MADS gene family, indicating that type II MADS genes were more conservative than type I MADS genes [13,14,30]. The FLC gene was reported to regulate flowering with the spring flower pathway [14,27]. The FLC gene was not found in type II MICK c , indicating that chayote can blossom without going through the vernalization process, or the vernalization process was not inhibited by the FLC gene. The deletion of the FLC subfamily genes was also found in the watermelon and cucumber of Cucurbitaceae [27,28]. BS subfamily genes were involved in seed pigmentation and endothelial development [30]. We found a Bs gene SeMADS49, but no Bs gene was found in cucumbers, watermelon and melon in Cucurbitaceae [27][28][29]. SeMADS49 was likely to be related to the process of seed and fruit development of chayote. In addition, we found an AGL12 subfamily gene, SeMADS55. The study showed that AGL12 was related to root development [31]. An AGL12 gene was also identified in watermelon [27], indicating that the root development of chayote was likely to be regulated by SeMADS55.
Moreover, we noticed that 70 SeMADS genes were distributed on 13 chromosomes, except chromosome LG11. The number of MADS genes distributed on each chromosome was not positively correlated with chromosome length, and showed a random distribution on each chromosome. In addition, the physical and chemical properties of 70 SeMADS proteins showed that the subcellular localization of all proteins was in the nucleus, suggesting that the MADS gene of chayote may play a transcriptional regulatory role in the nucleus. Gene structure and conserved motif analysis showed that there were differences in the MADS gene of chayote. We found that the gene length of the type II gene was longer and contained more exons than that of the type I gene, which was also an important feature in distinguishing type I from type II. Different subfamilies contain different numbers and types of conserved motifs [30]. We found that motif 1 was the most conserved motif, which was related to the SRF domain. Motif 2, motif 3, motif 4 and motif 5 were mainly distributed in the type II MADS gene, which was related to the semi conserved k-domain and the low conserved I-domain and C-domain.
Gene replication plays an important role in the amplification and evolution of transcription factors. In this study, we found 43 MADS gene replication events, and 84% of SeMADS genes were generated through segmental replication, indicating that segmental replication plays an important role in the amplification of the chayote MADS gene. The same results were found in Cyclocarya paliurus [32], Rhododendron hainanense Merr. [13], and Fagopyrum tataricum [33]. In addition, we found that one of the five tandem replication gene pairs (SeMADS45 and SeMADS46) underwent purifying selection by calculating Ka/Ks. This pair of genes belong to the AP1 subfamily and have a similar gene structure, which is consistent with previous reports, suggesting that tandem replication genes may produce genes with similar functions after purifying selection [34].
Through collinearity analysis among different species, it was found that 55, 73, 74, 104 and 84 pairs of homologous gene pairs were identified between chayote and Arabidopsis and Cucurbitaceae plants (melon, cucumber, pumpkin and watermelon), suggesting that Cucurbitaceae species may have an expansion after divergence. Studies have shown that Arabidopsis and Cucurbitaceae plants have a WGD event of about 101-156 Mya [18], resulting in more homologous gene pairs in Cucurbitaceae plants than in Arabidopsis. In addition, the second WGD event occurred in Cucurbitaceae at 27-51Mya [18]. The phylogenetic tree analysis showed that pumpkin was relatively distant from melon, cucumber and watermelon, suggesting that there were more homologous gene pairs between chayote and pumpkin. Cis-acting elements such as the light response element, hormone response element, and abiotic stress response element were found in the SeMADS genes promoter region. The research shows that chayote has strong growth ability and stress resistance [21]. These elements may be the key ones related to the growth, development and stress resistance of chayote fruit and root tubers. In addition, a GO and KEGG enrichment analysis also found that the SeMADS genes are mainly enriched in biological regulation, the MAPK signaling pathway-fly (ko04013), the cGMP PKG signaling pathway (ko04022), the Apelin signaling pathway (ko04371), and parathyroid hormone synthesis, secret and action (ko04928), further indicating that the SeMADS genes can not only regulate the growth and development of chayote, but also regulate the genes related to the active components of chayote. A protein interaction analysis showed that the interaction of SeMADS genes jointly affected the growth and development of chayote. It is worth noting that the gene expression of SeMADS06, SeMADS13, SeMADS26, SeMADS28, SeMADS36 and SeMADS37 gradually increased in three different periods of root tuber growth of 'Tuershao'. They may be the key genes regulating the root tuber development of 'Tuershao'.
Floral organ development is an important turning point in the process of plant growth and development [35]. The sequencing of the chayote genome has promoted the breeding values [18]. 'Tuershao' is a kind of chayote variety with a high yield of root tuber, but the female flower cannot expand normally to form fruit. The correct formation of the female/male flower could theoretically form normal fruit. MADS-box genes play an important role in floral organ development of angiosperms. In recent years, many studies have reported that the MADS-box gene is involved in the regulation of floral organ development.
LsMADS55 plays an important role in the development of floral organs of lettuce [36]. JmMADS58 and JmMADS29 play a positive regulatory role in the development of male and female flowers of Juglans mandshurica [35]. AcANR1b and AcAGL11 are essential to the development of floral organs of pineapple, regulating the development of male and female flowers respectively [37]. AcMADS04 is an important candidate gene for male and the female flower differentiation of kiwifruit [38]. In addition, more than 30 MADS-box genes were found to be highly expressed in flowers in Dactylis glomerata, indicating that they play a critical role in flower development [39]. However, the role of MADS-box gene in the floral organ development of chayote is still unclear. In this study, we selected 12 SeMADS genes for tissue specificity and flower development expression analysis. All SeMADS genes were highly expressed in the male or female flowers of 'Tuershao' and chayote. Most genes are highly expressed in male flowers at the S4 or S5 stage, which indicates that these genes may regulate the development of male flowers in 'Tuershao' and chayote. SeMADS19 and SeMADS54 are homologous with the AGL15 and SVP genes of Arabidopsis. AGL15 and SVP are flowering repressors in Arabidopsis [40], while in 'Tuershao' and chayote, SeMADS19 and SeMADS54 are highly expressed in male and female flowers, and the same phenomenon is also observed in Cunninghamia lanceolata [41], which suggests that similar inhibitors may exist in chayote to limit the development of male and female flowers. Furthermore, the differential expression of SeMADS19 and SeMADS54 genes in different stages of female and male flower development in 'Tuershao' and chayote may be the reason for their different flowering time. Further functional verification is necessary. The SEP gene of Arabidopsis thaliana is involved in the development of male flowers [23]. SeMADS03 and SeMADS47 are homologous genes of SEP, which are highly expressed in the male flowers of 'Tuershao' and chayote. SeMADS03 and SeMADS47 may be the crucial genes regulating the development of male flowers in the late stage of 'Tuershao' and chayote. SeMADS21, which is homologous with PI, is involved in the development of female flowers in Arabidopsis [23], and is highly expressed in the S1-S5 stage of female flower in 'Tuershao', suggesting that it may be the key gene in the regulation of the development of female flowers. These SeMADS genes could regulate the floral organ development of chayote, which needs to be further verified by systematic experiments.

Construction of MADS-Box Phylogenetic Tree
The MADS-box protein sequences of Arabidopsis were downloaded from the Arabidopsis database (https://www.arabidopsis.org/, accessed on 19 January 2022). There were multiple comparisons of MADS proteins in Arabidopsis and chayote using ClustalW. The phylogenetic tree was constructed by neighbor-joining (NJ) using MEGA-X software (version 7.0.26, California, USA). The specific parameters were as follows: Poisson model, same (homogeneous), pairwise deletion, and bootstrap method with 1000 replicates [42]. Based on Arabidopsis AtMADS genes, chayote MADS genes were divided into different subfamilies, and finally the phylogenetic tree was beautified with the iTOL tool (https://itol.embl.de/, accessed on 25 January 2022).

MADS-Box Family Genes Chromosomal Localization
Based on the annotation file of chayote, the chromosome length information was obtained. The 70 MADS genes of chayote were then located on different chromosomes using the MG2C online tool (http://mg2c.iask.in/mg2c_v2.1/, accessed on 25 January 2022) [32].

Gene Structure and Conserved Motif Analysis of MADS-Box
The gene structure of the MADS gene in chayote was analyzed by GSDS (http://gsds. gao-lab.org/, accessed on 27 January 2022), including UTL (untranslated region), CDS (coding sequence), and intron. Seventy MADS protein sequences were submitted to the MEME online tool (https://meme-suite.org/, accessed on 27 January 2022) to analyze the conserved motifs. The number of motifs was set to 10, the minimum width of motifs was set to 6, and the maximum was set to 50. Other parameters were set by default [43,44]. We then downloaded the result meme.xml file and used the visualize meme/mast motif pattern tool of TBtools software_0987663 (Guangzhou, China) to visualize the results [45].

Cis-Element Analysis of MADS-Box
The sequences of 2000bp upstream of the start codon of 70 chayote MADS genes were extracted, and the sequences were then submitted to the PlantCare online website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 27 January 2022) to query the cis-acting elements of each MADS gene [46]. We downloaded and simplified the PlantCare results, and finally visualized results using TBtools software_0987663 (Guangzhou, China) [45].

Collinearity Analyses of MADS-Box within and between Species
BLASTp and MCScanX software (California, USA, http://chibba.pgml.uga.edu/ mcscan2/, accessed on 12 January 2022) were used to analyze the replication types of MADS-box genes in chayote [30], Arabidopsis and some representative species of Cucurbitaceae (cucumber, ChineseLong_v3; melon, DHL92; pumpkin, Cmoschata_v1; and watermelon, 97103_v2.5), and to analyze the collinearity within and between genomes. The genome data and annotation files of Arabidopsis and Cucurbitaceae were downloaded from the Arabidopsis database and the Cucurbitaceae website described above. The KaKs_Calculator2.0 was used to calculate Ka and Ks [32]. Finally, the Circos of TBtools was used to visualize the results [45].

GO and KEGG Analysis of MADS-Box
GO enrichment and KEGG pathway analysis were used to understand the function of MADS genes. The function of MADS genes were annotated using the eggNOG-mapper database (http://eggnog-mapper.embl.de/, accessed on 29 January 2022), and the results were then visualized using TBtools software_0987663 (Guangzhou, China) [45].

MADS-Box Protein Interaction Network Analysis
The protein-protein interaction network of MADS proteins homologous to Arabidopsis was used to further analyze the interaction relationship between MADS proteins of chayote. The protein-protein interaction network was displayed using the online website of STRING (https://string-db.org/, accessed on 29 January 2022).

MADS-Box Gene Expression Pattern
The expression data of 'Tuershao' root tubers of three different developmental stages (T1: early, T2: middle, and T3: mature stages) were obtained from our previous transcriptome data [22]. Briefly, the FPKM value of the MADS gene in transcriptome data was log2 standardized and displayed by heat map [47].

Plant Material, RNA Extraction and qRT-PCR Analysis
Chayote (Sechium edule) and 'Tuershao' (a high tuber-yield chayote cultivar) were cultivated in Chongzhou, Chengdu, China (30 • 63 N, 103 • 67 E). 'Tuershao' is a new variety selected by our research group many years ago. The female flower of 'Tuershao' cannot expand into fruit normally, as with the chayote. The edible root tuber in the underground is its main product, and the root tuber yield is much higher than that of chayote [22]. For the tissue-specific expression analysis, we collected the roots, stems, leaves, tendrils, and open male and female flowers of 'Tuershao' and chayote; for floral organ development expression analysis, the male and female flower samples of 'Tuershao' and chayote with different diameters were collected and named S1 to S5, then immediately frozen in liquid nitrogen and stored at −80 • C. Each sample contained three biological replicates. The total RNA of each sample was isolated by a Plant RNA Kit (OMEGA, Beijing, China), and the first strand cDNA was synthesized by a Script™ RT reagent Kit (TaKaRa, Beijing, China). The qRT-PCR was performed on a Bio-Rad CFX96 Real-Time System with 2X SYBR Green Abstart PCR Mix (Sangon Biotech, Beijing, China). The chayote actin gene was used as the internal reference and the relative expression levels of each MADS gene was calculated using the 2 −∆∆Ct method [22]. All of the primers used for qRT-PCR are shown in Table S4.

Conclusions
In this study, we identified 70 MADS-box genes from the genome of chayote, including 14 type I and 56 type II MADS genes. A phylogenetic analysis showed that 70 SeMADS genes were divided into 14 subfamilies. There are differences in gene structure, conserved motifs and cis-acting elements among different subfamilies. The results of a collinearity analysis speculated that segmental duplication was the main reason for the MADS-gene expansion of chayote. A GO and KEGG enrichment analysis also showed that the MADS gene played a key role in the development of chayote. SeMADS06, SeMADS13, SeMADS26, SeMADS28, SeMADS36 and SeMADS37 may be the key genes regulating the root tubers development of 'Tuershao'. SeMADS03 and SeMADS52 may play an important role in the maturation process of male flowers of 'Tuershao' and chayote. SeMADS21 may be an important regulatory gene in the development stage of female flowers of 'Tuershao'. In conclusion, our results provide a basis for the further study of the MADS gene in chayote. Funding: This study was financially supported by the Second Tibetan Plateau Scientific Expedition and Research (2019QZKK0303) and the Ya'an Science and Technology Bureau.