*2.2. Phylogenetic Analysis of the UGlcAE Genes in Tomato and Other Species*

To evaluate the classification of the *UGlcAE* genes in *S. lycopersicum*, we analyzed the sequence features in 10 different species, including *S. lycopersicum*, *C. sativus*, *C. annuum*, *S. tuberosum*, *A. thaliana*, *N. tabacum*, *P. trichocarpa*, *S. pennelli*, *Z. mays*, and *A. lyrata* subsp. *lyrata*, and we constructed a unrooted phylogenetic tree of the *UGlcAE* genes (Figure 1) using the N-J methods. The orthologous relationships were evident. Only the tree topology is shown, and the branch lengths do not represent the estimated numbers of amino acid replacements [28].



**Table 1.** Information about the nine isoforms of the tomato SlUGlcAE gene family.

**Figure 1.** Phylogenetic analysis of the *UGlcAE* gene family based amino acids in tomato and other nine species. The unrooted neighbor-joining phylogenetic tree is generated by MEGA 5. The sequence names included three parts: the source numbers from NCBI, the abbreviation of species names, and their respective subfamilies. Red dots highlight the tomato *UGlcAE* genes.

Combing with the sequence similarity of the mature proteins, the employed *UGlcAE* genes are distributed into seven groups (Figure 1). In addition to *UGlcAE2* being divided into two clusters, the other five subfamilies are clustered separately.

Interestingly, the *UGlcAE4* subfamily is specifically present in *A. thaliana* and *A. lyrata* subsp. *lyrata*, whereas it is absent from other species in this study. This implies that *UGlcAE4* may be associated with distinctive functions. It is noteworthy that *UGlcAE2* genes were classified into two different groups based on their evolutionary relationship. This finding indicates that *UGlcAE2* genes may evolve into new features, which have not been known until today. Moreover, phylogenetic analyses showed that

1

other five subfamilies are clustered separately.

the *UGlcAE3* gene in *A. thaliana* was clustered together with *UGlcAE2* genes of *A. thaliana* and *A. lyrata* subsp. *lyrata*, suggesting that there may be some gene fusion among them. analyses showed that the *UGlcAE3* gene in *A. thaliana* was clustered together with *UGlcAE2* genes of *A. thaliana* and *A. lyrata* subsp. *lyrata*, suggesting that there may be some gene fusion among them.

may evolve into new features, which have not been known until today. Moreover, phylogenetic

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 5 of 21

Combing with the sequence similarity of the mature proteins, the employed *UGlcAE* genes are distributed into seven groups (Figure 1). In addition to *UGlcAE2* being divided into two clusters, the

Interestingly, the *UGlcAE4* subfamily is specifically present in *A. thaliana* and *A. lyrata* subsp. *lyrata*, whereas it is absent from other species in this study. This implies that *UGlcAE4* may be

#### *2.3. Structures of the UGlcAE Genes in the Tomato and Arabidopsis Thaliana 2.3. Structures of the UGlcAE Genes in the Tomato and Arabidopsis Thaliana*

Introns, especially UTR introns, in *UGlcAE* genes may influence the expression level [29]. To analyze the structural characteristics of the *UGlcAE* genes in tomato and *Arabidopsis thaliana*, their gene structures were mapped according to the genome sequences and corresponding coding sequences of *SlUGlcAE* and *AtUGlcAE* genes (Figure 2). We found that all of the *SlUGlcAE* and *AtUGlcAE* genes do not contain intron in their genomic sequences. In other words, nine *UGlcAE* genes in the tomato and six *UGlcAE* genes in *Arabidopsis thaliana* are single exon structures. Introns, especially UTR introns, in *UGlcAE* genes may influence the expression level [29]. To analyze the structural characteristics of the *UGlcAE* genes in tomato and *Arabidopsis thaliana*, their gene structures were mapped according to the genome sequences and corresponding coding sequences of *SlUGlcAE* and *AtUGlcAE* genes (Figure 2). We found that all of the *SlUGlcAE* and *AtUGlcAE* genes do not contain intron in their genomic sequences. In other words, nine *UGlcAE* genes in the tomato and six *UGlcAE* genes in *Arabidopsis thaliana* are single exon structures.

**Figure 2.** Exon-intron structures of nine tomato *UGlcAE* genes and six *Arabidopsis thaliana UGlcAE* genes. The yellow sections represent the exons, and the blue parts indicate upstream/downstream regions. **Figure 2.** Exon-intron structures of nine tomato *UGlcAE* genes and six *Arabidopsis thaliana UGlcAE* genes. The yellow sections represent the exons, and the blue parts indicate upstream/downstream regions.
