*2.2. Classification and Phylogenetic Analysis of Tomato MADS-Box Genes*

To study the phylogenetic relationships among MADS-box genes in tomato and Arabidopsis [6], a phylogenetic tree was drawn by the neighbor-joining (NJ) method using MEGA 5.02 (Figure S1A). Based on previous reports on Arabidopsis, the 131 tomato MADS-box genes were classified into two types: type I (81) and type II (50). Based on the phylogenetic tree, type I and type II tomato MADS-box genes were subdivided into more detailed subgroups. Then, type I could be further divided into four

groups (Mα, Mβ, Mγ, and Mδ), while Type II (MIKC) could be further divided into MIKC\* and MIKCC. The MIKCC-type genes included the *AP3*/*PI*, *SVP*, *AGL15*, *SEPALLATA* (*SEP*), *AGL6*, *AP1*, *FLOWERING LOCUS C* (*FLC*) *SOC1*, *AGAMOUS* (*AG*), *TM8,* and *DEFICIENS* (*DEF*)/*GLOBOSA* (*GLO*) subfamilies, similar to the MADS-box genes in other plants species. In addition, the phylogenetic tree of type I and type II MADS-box protein in tomato plants were constructed to identify the phylogenetic relationships of gene numbers in the two types of tomato MADS-box family more clearly (Figure S1B,C). identify the phylogenetic relationships of gene numbers in the two types of tomato MADS-box family

#### *2.3. Conserved Motif and Gene Structure Analysis of Tomato MADS-Box Genes* more clearly (Figure S1B,C).

The intron–exon organization was analyzed to investigate the structural diversity and evolution of the 131 tomato MADS-box genes. As shown in Figure 1, we obtained each gene's intron/exon arrangement by comparing their CDS with their genomic sequences using the program Gene Structure Display Server (GSDS). The number of introns in tomato MADS-box genes ranged from one to 11. Similar to Arabidopsis, the distribution of introns in tomato was different in type I and type II genes [6]. In our study, we found that the Mα, Mβ, and Mγ groups of the type I genes usually had no introns or one intron, which might be explained by the diversity of the reverse-transcribed origin or the differences in acquisition or loss introns by the ancestors of these three groups of genes [6]. Based on the genomic data, the Mδ group of type I and the type II genes contained multiple introns. Among the Mδ clade and the type II genes, 52 of 56 (92.9%) genes had more than five introns. Additionally, the gene structures of closely related genes in tomato MADS-box genes were more similar, and the differences were only in the lengths of introns and exons. However, some close gene pairs showed different intron/exon arrangements. For instance, *SlMBP61* has one exon, whereas its close homologs *SlMBP51* and *SlMBP10* both have two, although their phylogenetic relationship displayed a high bootstrap value (Figure S1B). *2.3. Conserved Motif and Gene Structure Analysis of Tomato MADS-Box Genes*  The intron–exon organization was analyzed to investigate the structural diversity and evolution of the 131 tomato MADS-box genes. As shown in Figure 1, we obtained each gene's intron/exon arrangement by comparing their CDS with their genomic sequences using the program Gene Structure Display Server (GSDS). The number of introns in tomato MADS-box genes ranged from one to 11. Similar to Arabidopsis, the distribution of introns in tomato was different in type I and type II genes [6]. In our study, we found that the Mα, Mβ, and Mγ groups of the type I genes usually had no introns or one intron, which might be explained by the diversity of the reverse-transcribed origin or the differences in acquisition or loss introns by the ancestors of these three groups of genes [6]. Based on the genomic data, the Mδ group of type I and the type II genes contained multiple introns. Among the Mδ clade and the type II genes, 52 of 56 (92.9%) genes had more than five introns. Additionally, the gene structures of closely related genes in tomato MADS-box genes were more similar, and the differences were only in the lengths of introns and exons. However, some close gene pairs showed different intron/exon arrangements. For instance, *SlMBP61* has one exon, whereas its close homologs *SlMBP51* and *SlMBP10* both have two, although their phylogenetic relationship displayed a high bootstrap value (Figure S1B).

**Figure 1.** Gene structure analysis of MADS-box genes in tomato. The Gene Structure Display Server (GSDS) database was used to perform the exon–intron structure analyses. Lengths of exons and introns of each MADS-box gene were displayed proportionally. The blue boxes represent upstream/downstream, the yellow boxes represent exons, and the black lines represent introns. **Figure 1.** Gene structure analysis of MADS-box genes in tomato. The Gene Structure Display Server (GSDS) database was used to perform the exon–intron structure analyses. Lengths of exons and introns of each MADS-box gene were displayed proportionally. The blue boxes represent upstream/downstream, the yellow boxes represent exons, and the black lines represent introns.

To better analyze conserved motifs in tomato MADS-box proteins, we constructed a conserved motif figure using the Multiple EM for Motif Elicitation (MEME) program and annotated them using

To better analyze conserved motifs in tomato MADS-box proteins, we constructed a conserved motif figure using the Multiple EM for Motif Elicitation (MEME) program and annotated them using SMART. A total of 10 conserved motifs, named 1 to 10, were identified (Figure 2). The details of the motifs are shown in Figure S2. As expected, the same types of genes tend to possess the same motifs. Motif 1—one of the most typical MADS-box domains—comprised 42 amino acids was found in the majority of tomato MADS-box proteins. Motif 3 was also conserved across most of the tomato MADS-box proteins, including type I and type II genes. Motifs 2 and 4 represent the K domain, which plays an important role in protein–protein interactions among MADS-box proteins, and they were found only in type II MADS-box proteins. Motif 2 was identified in almost all the type II proteins except for TM8/TDR8, SlMADS86, SlMADS87, and SlMADS83. In the type II proteins, a large number of proteins had motif 4, with seven exceptions (SlGLO2, SlGLO1, SlMBP11, SlMADS84, SlMADS86, SlMADS87, and SlMADS83). Motif 9 is also a MADS-box domain that is present in a small number of tomato MADS-box proteins. However, some motifs (6, 7, 8, and 10) were shown to be weakly conserved in tomato MADS-box proteins, and they were found only in type I MADS-box proteins.

**Figure 2.** Conserved motif analyses of MADS-box genes in tomato. All the conserved motifs were identified by the Multiple EM for Motif Elicitation (MEME) database using the complete amino acid sequences of tomato MADS-box genes. Ten conserved different motifs were indicated by different colors. The details of motifs refer to the Supplementary Figure S2. **Figure 2.** Conserved motif analyses of MADS-box genes in tomato. All the conserved motifs were identified by the Multiple EM for Motif Elicitation (MEME) database using the complete amino acid sequences of tomato MADS-box genes. Ten conserved different motifs were indicated by different colors. The details of motifs refer to the Supplementary Figure S2.

#### *2.4. Chromosomal Locations of Tomato MADS-Box Genes 2.4. Chromosomal Locations of Tomato MADS-Box Genes*

According to physical genome annotation files that were obtained by using genomic sequences from the SGN and NCBI databases, 131 members of the MADS-box genes were located on all 12 tomato chromosomes, implying that the MADS-box transcription factor family may have multiple functions in tomato plants. According to physical genome annotation files that were obtained by using genomic sequences from the SGN and NCBI databases, 131 members of the MADS-box genes were located on all 12 tomato chromosomes, implying that the MADS-box transcription factor family may have multiple functions in tomato plants.

As shown in Figure 3, the tomato MADS-box genes are distributed unevenly on each chromosome. Chromosome 1 contains the most MADS-box genes (24), while chromosome 8 and 9 contain the fewest (two). Gene duplication events have a significant function in amplifying gene family numbers and genome complexity in eukaryotes [73,74]. The tandem amplification or segmental duplication of chromosomal regions can increase gene families. In this study, the results showed that chromosomes 1, 3, 4, 10, and 12 contain gene clusters or gene hotspots; in particular, chromosome 1 contains eight MADS-box genes within a short region. Additionally, we confirmed that internal chromosome duplication (tandem duplication) occurred in these genes. As shown in Figure 3, the tomato MADS-box genes are distributed unevenly on each chromosome. Chromosome 1 contains the most MADS-box genes (24), while chromosome 8 and 9 contain the fewest (two). Gene duplication events have a significant function in amplifying gene family numbers and genome complexity in eukaryotes [73,74]. The tandem amplification or segmental duplication of chromosomal regions can increase gene families. In this study, the results showed that chromosomes 1, 3, 4, 10, and 12 contain gene clusters or gene hotspots; in particular, chromosome 1 contains eight MADS-box genes within a short region. Additionally, we confirmed that internal chromosome duplication (tandem duplication) occurred in these genes.

**Figure 3.** Chromosomal locations of tomato MADS-box genes. A total of 12 chromosomes of tomato were labeled with their names, chromosomes 1 to 12, which are indicated at the top of each bar. The position of tomato MADS-box genes on the chromosome was based on the Sol Genomics Network (SGN) and National Center for Biotechnology Information (NCBI) database and the Tomato-EXPEN 2000 was used to draw the physical map of the tomato MADS-box genes. **Figure 3.** Chromosomal locations of tomato MADS-box genes. A total of 12 chromosomes of tomato were labeled with their names, chromosomes 1 to 12, which are indicated at the top of each bar. The position of tomato MADS-box genes on the chromosome was based on the Sol Genomics Network (SGN) and National Center for Biotechnology Information (NCBI) database and the Tomato-EXPEN 2000 was used to draw the physical map of the tomato MADS-box genes.

#### *2.5. Predictions of Expression Profiles of Tomato MADS-Box Genes in Different Organs 2.5. Predictions of Expression Profiles of Tomato MADS-Box Genes in Di*ff*erent Organs*

To investigate the tomato MADS-box genes expression patterns in different tissues of tomato plants, we analyzed tomato transcript expression (RNA-seq) data in nine different tomato tissues at different developmental stages. This included the expression in the whole root (RT), young leaf (YL), mature leaves (ML), young flower buds (YFB), fully open flowers (F), and at the immature green (IMG), mature green (MG), break (B), and mature (MF) stages of fruit development and ripening. These datasets were searched using the locus/gene names in SGN of 124 tomato MADS-box gene To investigate the tomato MADS-box genes expression patterns in different tissues of tomato plants, we analyzed tomato transcript expression (RNA-seq) data in nine different tomato tissues at different developmental stages. This included the expression in the whole root (RT), young leaf (YL), mature leaves (ML), young flower buds (YFB), fully open flowers (F), and at the immature green (IMG), mature green (MG), break (B), and mature (MF) stages of fruit development and ripening. These datasets were searched using the locus/gene names in SGN of 124 tomato MADS-box gene sequences, except for

sequences, except for *SlMADS4*, *SlMADS11*, *SlMADS37*, *SlMADS44*, *SlMADS46*, *SlMADS56*,

*SlMADS4*, *SlMADS11*, *SlMADS37*, *SlMADS44*, *SlMADS46*, *SlMADS56*, *SlMADS68*, *SlMADS70* and *SlMADS89*, which were not accurately found in TFGD. Then, we constructed a hierarchical clustering heat map using these datasets (Figure 4). *SlMADS68*, *SlMADS70* and *SlMADS89*, which were not accurately found in TFGD. Then, we constructed a hierarchical clustering heat map using these datasets (Figure 4).

**Figure 4.** Heat map representation of tomato MADS-box genes in various tissues. The tissues included the whole root (Rt), young leaf (YL), mature leaves (ML), young flower buds (YFB), and fully open flowers (F), which were at the immature green (IMG), mature green (MG), break (B), and mature (MF) stages of fruit development and ripening. The bar at the bottom of the heat map represents relative gene expression values. **Figure 4.** Heat map representation of tomato MADS-box genes in various tissues. The tissues included the whole root (Rt), young leaf (YL), mature leaves (ML), young flower buds (YFB), and fully open flowers (F), which were at the immature green (IMG), mature green (MG), break (B), and mature (MF) stages of fruit development and ripening. The bar at the bottom of the heat map represents relative gene expression values.

The expression profiles revealed that 117 genes were expressed in at least one tomato plant organ, while the other seven genes (*SlMADS24*, *SlMADS25*, *SlMADS26*, *SlMADS33*, *SlMADS45*, *SlMADS61*, and *SlMADS74*) were expressed at levels that were too low to be identified, or they had temporal and spatial specific expression patterns that showed no expression in the organs tested. Most tomato MADS-box genes displayed a broad expression range across all the organs and developmental stages, which is consistent with previous reports that the MADS-box genes may play multiple roles in plant growth and development [75,76]. However, some genes exhibited tissuespecific expression. For example, the expression of *SlMADS12*, *SlMADS20*, *SlMADS21*, *SlMADS22,*  and *SlMADS23* were restricted in whole root, and the *SlMADS16, SlMADS17,* and *SlMADS132*  transcripts were observed only during flower development. These results illustrate that these genes may be involved in the regulation of some biological process of the tomato root or in flower growth and development. Eight genes (*SlMBP2*, *SlMBP6*, *SlMBP10*, *SlMBP21*, *TAP3*, *SlMADS78*, *SlMADS92*, and *SlMADS98*) showed especially high expression in young flower buds (YFB) and fully open flowers (F), indicating that these genes may play important roles in floral organ development. We further discovered that most type II genes (*SlMBP3*, *SlMBP7*, *SlMBP11*, *SlMBP15*, *MADS-RIN*, *MADS-MC*, *TAGL1*, and *LeAP1*) were highly expressed during flower or fruit development; especially, the expression values of *MADS-RIN* and *MADS-MC* in fruits were more than 1000, suggesting that these genes may be associated with the reproductive growth of tomato. However, the The expression profiles revealed that 117 genes were expressed in at least one tomato plant organ, while the other seven genes (*SlMADS24*, *SlMADS25*, *SlMADS26*, *SlMADS33*, *SlMADS45*, *SlMADS61*, and *SlMADS74*) were expressed at levels that were too low to be identified, or they had temporal and spatial specific expression patterns that showed no expression in the organs tested. Most tomato MADS-box genes displayed a broad expression range across all the organs and developmental stages, which is consistent with previous reports that the MADS-box genes may play multiple roles in plant growth and development [75,76]. However, some genes exhibited tissue-specific expression. For example, the expression of *SlMADS12*, *SlMADS20*, *SlMADS21*, *SlMADS22,* and *SlMADS23* were restricted in whole root, and the *SlMADS16, SlMADS17,* and *SlMADS132* transcripts were observed only during flower development. These results illustrate that these genes may be involved in the regulation of some biological process of the tomato root or in flower growth and development. Eight genes (*SlMBP2*, *SlMBP6*, *SlMBP10*, *SlMBP21*, *TAP3*, *SlMADS78*, *SlMADS92*, and *SlMADS98*) showed especially high expression in young flower buds (YFB) and fully open flowers (F), indicating that these genes may play important roles in floral organ development. We further discovered that most type II genes (*SlMBP3*, *SlMBP7*, *SlMBP11*, *SlMBP15*, *MADS-RIN*, *MADS-MC*, *TAGL1*, and *LeAP1*) were highly expressed during flower or fruit development; especially, the expression values of *MADS-RIN* and *MADS-MC* in fruits were more than 1000, suggesting that these genes may be associated with the reproductive growth of tomato. However, the expression of most type II genes showed no significant difference among tissues.

expression of most type II genes showed no significant difference among tissues. In short, these results indicate that the MADS-box genes had different expression levels in various tomato organs, and the predictions of the organ expression profiles of the tomato MADS-box In short, these results indicate that the MADS-box genes had different expression levels in various tomato organs, and the predictions of the organ expression profiles of the tomato MADS-box gene

gene family may provide insight for future studies on the functions of MADS-box genes in tomato

plant growth and development.

family may provide insight for future studies on the functions of MADS-box genes in tomato plant growth and development.
