*2.6. The Critical Tomato MADS-Box Genes Involved in Floral Organ Development*

Based on the phylogenetic analysis of the MADS-box genes that participated in floral organ development in petunia, which has been reported previously [32,77] (Figure S3), 15 tomato MADS-box genes that are possibly involved in floral organ development were screened out, including tow class A genes (*MADS-MC* and *SlMBP20*), four class B genes (*TAP3*, *TM6*, *SlMBP1,* and *SlMBP2*, two class C genes (*TAG1* and *TAGL1*), two class D genes (*SlMBP3* and *SlMBP22*), and five class E genes (*TAGL2*, *TM5*, *SlMADS1*, *SlMBP21,* and *SlMBP6*), as shown in Table S2.

According to the expression profile predictions shown in Figure 4, among the 15 MADS-box genes, nine genes (*MADS-MC*, *TAP3*, *TM6*, *SlMBP1*, *SlMBP2*, *TAG1*, *SlMBP22, SlMBP21,* and *SlMBP6*) were extremely highly expressed in flowers. However, *TAGL1*, *SlMBP3*, *TAGL2*, *TM5*, and *SlMADS1* were mainly expressed during the stages of fruit development and ripening, and the expression of *SlMBP20* was particularly high in leaves.

To further investigate the potential role of these MADS-box genes in floral organ development, a comparison of the expression patterns of 15 tomato MADS-box genes in four whorls of floral organs (sepal, petal, stamen, and carpel) were analyzed by qPCR. As shown in Figure 5A,B, the class A genes *MADS-MC* and *SlMBP20* were highly expressed in sepals. The expression levels of class B genes (*TAP3*, *SlMBP2,* and *SlMBP1)* were notably high in the petal and stamen (Figure 5C–E), whereas the transcription of *TM6* was found to be markedly higher in carpel and stamen compared with other floral organs (Figure 5F). Two class C genes, *TAG1* and *TAGL1*, were found to be mainly expressed in stamens and carpels (Figure 5G,H). Moreover, the class D genes, *SlMBP3* and *SlMBP22,* showed organ-specific expression patterns that were exclusively expressed in carpel (Figure 5I,J). The expression patterns of class E genes indicated that the expression of *TAGL2* and TM5 were significantly higher expressed in the petal, stamen, and carpel than in the sepal (Figure 5K,L), and *SlMBP6* was highly expressed in petals and carpels (Figure 5M). In comparison, the other class E genes (*SlMADS1* and *SlMBP21*) were shown to exhibit higher expression levels in the sepal and carpel (Figure 5N,O).

In addition, the interaction networks of these 15 tomato MADS-box proteins were predicted by STRING software (Figure S4). The results showed that they established interactions with other proteins, directly or indirectly. The TAGL2, SlMBP3, TAGL1, and SlMBP22 proteins can interact directly with each other. Apart from that, the TAGL1 protein showed complex interactions with several other proteins, including the SlMBP3 and TAP3 proteins. The TM5 protein was also shown to directly interact with the TAP3 and TM6 proteins.

#### *2.7. Di*ff*erential Expression Analysis of Tomato MADS-Box Genes at Di*ff*erent Stages of Fruit Development and Ripening* to directly interact with the TAP3 and TM6 proteins. *2.7. Differential Expression Analysis of Tomato MADS-Box Genes at Different Stages of Fruit Development*

In addition, the interaction networks of these 15 tomato MADS-box proteins were predicted by STRING software (Figure S4). The results showed that they established interactions with other

several other proteins, including the SlMBP3 and TAP3 proteins. The TM5 protein was also shown

According to the expression profiles predictions, we selected five tomato MADS-box genes (*SlMBP3*, *MADS-RIN*, *TAGL1*, *TM4*, and *SlMBP7*) that may take part in fruit development and ripening. We analyzed their expression patterns by qPCR at five different stages of fruit development and ripening, including the immature green (IMG), mature green (MG), and break (B) stages, as well as at four days after break (B+4) and seven days after break (B+7). *SlMBP3* exhibited a strikingly high expression level at the IMG stage, and showed extremely low expression at the other stages (Figure 6A). The expression levels of *MADS-RIN* and *TAGL1* exhibited an increasing tendency from the MG to the B+4 stage, and then decreased at B+7 (Figure 6B,C). *TM4* expression increased continuously during the process of fruit development and exhibited its maximum expression level at the B+7 stage (Figure 6D). Compared with the other three stages, the expression of *SlMBP7* was slightly higher at B (Figure 6E). These results indicated that the qPCR data were consistent with the predictions of expression profiles and that our predictions are suitable for investigating the expression patterns of tomato MADS-box genes. *and Ripening*  According to the expression profiles predictions, we selected five tomato MADS-box genes (*SlMBP3*, *MADS-RIN*, *TAGL1*, *TM4*, and *SlMBP7*) that may take part in fruit development and ripening. We analyzed their expression patterns by qPCR at five different stages of fruit development and ripening, including the immature green (IMG), mature green (MG), and break (B) stages, as well as at four days after break (B+4) and seven days after break (B+7). *SlMBP3* exhibited a strikingly high expression level at the IMG stage, and showed extremely low expression at the other stages (Figure 6A). The expression levels of *MADS-RIN* and *TAGL1* exhibited an increasing tendency from the MG to the B+4 stage, and then decreased at B+7 (Figure 6B,C). *TM4* expression increased continuously during the process of fruit development and exhibited its maximum expression level at the B+7 stage (Figure 6D). Compared with the other three stages, the expression of *SlMBP7* was slightly higher at B (Figure 6E). These results indicated that the qPCR data were consistent with the predictions of expression profiles and that our predictions are suitable for investigating the expression patterns of tomato MADS-box genes.

**Figure 6.** Relative expression of tomato MADS-box genes in difference stages of fruit development and ripening by qPCR analysis. Expression patterns of *SlMBP3* (**A**), *MADS-RIN* (**B**), *TAGL1* (**C**), *TM4*  (**D**), and *SlMBP7*(**E**) in different organs, including mature green (MG), break (B), four days after break (B+4), and seven days after break (B+7). Each value represents the mean ± SE of three technical replicates of a single biological sample. The *SlCAC* gene of tomato was used as the internal standard. **Figure 6.** Relative expression of tomato MADS-box genes in difference stages of fruit development and ripening by qPCR analysis. Expression patterns of *SlMBP3* (**A**), *MADS-RIN* (**B**), *TAGL1* (**C**), *TM4* (**D**), and *SlMBP7*(**E**) in different organs, including mature green (MG), break (B), four days after break (B+4), and seven days after break (B+7). Each value represents the mean ± SE of three technical replicates of a single biological sample. The *SlCAC* gene of tomato was used as the internal standard.

#### **3. Discussion 3. Discussion**

#### *3.1. Characterization of MADS-Box Genes in Tomato 3.1. Characterization of MADS-Box Genes in Tomato*

The MADS-box genes control diverse biological processes in plants, including vegetative growth and reproductive development. They mainly play key roles in the developmental processes of The MADS-box genes control diverse biological processes in plants, including vegetative growth and reproductive development. They mainly play key roles in the developmental processes of inflorescences, flowers, and fruits [78,79]. 24 MIKCC-type MADS-box transcription factors have been identified, and their functions and evolutions in tomatoes were thoroughly studied in 2006 [35]. However, the MIKCC-type MADS-box members are only part of the MADS-box transcription factor family, and to date, there has been no comparative report on the tomato MADS-box genes. It is well known that genome-wide analysis of gene families is a major and necessary approach to analyze the structure, evolution, and function of genes. In this study, 131 tomato MADS-box proteins were identified, and 96 new tomato MADS-box proteins with unknown functions were systemically named (Table 1). This study is the first comparative analysis of the tomato MADS-box gene family, and we

believe that the resolving confusion in naming the genes will facilitate further functional analysis of the tomato MADS-box genes.

First, we presented the phylogenetic relationships of 131 tomato MADS-box proteins with Arabidopsis MADS-box proteins to classify the tomato MADS-box proteins into five subfamilies (MIKC, Mα, Mβ, Mγ, and Mδ), as shown Figure S1A. Compared with Arabidopsis, a larger number of MADS-box proteins were found in tomato. In total, 81 tomato MADS-box genes were determined to be type I genes, including the Mα, Mβ, Mγ, and Mδ groups, which is more than that in Arabidopsis. We speculate that tomato type I MADS-box genes may have a higher duplication rate and/or a lower gene loss rate after duplication. Nevertheless, 50 tomato MADS-box genes were classified as type II genes, including MIKC<sup>c</sup> and MIKC\*, which is comparable to that in Arabidopsis. These results indicate that the genes' retention duplication have been different in various species, leading to different numbers of MAD-box genes among different species, with different evolutionary constraints [80]. Then, in order to investigate the phylogenetic relationships of MADS-box genes in tomato, a phylogenetic tree for two types of tomato MADS-box genes was constructed (Figure S1B,C). This showed that tomato MADS-box genes are conservative in subfamilies.

To obtain insight into the structural diversity of the tomato MADS-box genes, the intron–exon organization was analyzed (Figure 1). Previous studies have postulated that an intron-rich gene would lose multiple introns simultaneously by retrotransposition, thereby producing intron-less ancestral genes. In this study, we found that the Mα, Mβ and Mγ groups of the type I genes usually have no introns or one intron, which may experience the loss of multiple introns during MADS-box gene family diversification. In addition, the distribution of introns in tomato type I and type II genes were different, and the Mδ group of the type I and type II genes had more introns than the Mα, Mβ, and Mγ groups genes. Similar cases have also been detected in Arabidopsis and rice [7,13], suggesting the evolutionary conservation among plants. However, some close gene pairs showed different intron/exon arrangements, indicating that a more complicated gene structural evolution may exist in tomato MADS-box genes. The conserved motif analysis indicated that the same group contained most conserved motifs (Figure 2). The results suggested that these conserved motifs play important roles in group-specific functions. However, high structural divergence was found between the different groups. An analysis of the gene structures and conserved motifs could provide more clues about the evolutionary relationships of the MADS-box family in tomato.

Gene duplication (segmental duplication and tandem duplication) as well as transposition events were prevalent forces that result in the expansion of family members and genome complexity in eukaryotes [74]. The duplication of more than two genes located on one chromosome is confirmed as a tandem duplication event, whereas gene duplication that occurs on different chromosomes is identified as segmental duplication [73,81]. Both tandem and segmental duplication can play crucial roles in MADS-box gene expansion the tomato genome. In our study, a chromosomal location analysis of the tomato MADS-box genes showed that the MADS-box genes are distributed on 12 chromosomes (Figure 3). The tomato MADS-box genes had a high-density distribution on chromosome 1, which had 24 genes, suggesting that they might be caused by tandem duplications. The closely related tomato MADS-box genes formed tandem arrays on chromosomes 1, 3, 4, 10, and 12, which may help the tomato evolve distinct characterizations from other plants.

Since gene expression profiles can provide significant clues about gene function, the expression of tomato MADS-box genes in whole root (Rt), young leaf (YL), mature leaves (ML), young flower buds (YFB), fully open flowers (F), and five different fruit tissues were examined by transcription expression (RNA-seq) data. All the tested 124 tomato MAD-box genes that were expected to contain *SlMADS4*, *SlMADS11*, *SlMADS37*, *SlMADS44*, *SlMADS46*, *SlMADS56*, *SlMADS68*, *SlMADS70*, and *SlMADS89* showed distinct expression patterns (Figure 4). This finding may supply insight for future studies on the functions of MADS-box genes in tomato plant growth and development. For example, we found that the *SlMADS23* gene was only expressed in the root, so we speculate that the *SlMADS23* gene may play a key role in root growth and development. The roots of plants determined the

capacity of plants to acquire and distribute nutrients and water, as well as provide a means to suit the environmental conditions. Thus, the root architecture is extremely important for plant development and breeding. In the future, we will verify whether *SlMADS23* is related to root growth by constructing a *SlMADS23* overexpression vector and generating transgenic overexpression tomato plants to study the regulation of *SlMADS23* gene on root growth and development. In addition, performing *SlMADS23* gene mutagenesis with the CRISPR/Cas9 system transformation method may also prove to be a helpful strategy.
