*2.2. Gene Structure, Motif, and Phylogenetic Analysis of the RcSWEET Genes*

The gene structures, including untranslated regions (UTRs), exons, and introns, of each *RcSWEET* gene were predicted based on gDNA and CDS sequences (Table S2). The majority of the *RcSWEET* genes (*RcSWEET1*, *RcSWEET2a*, *RcSWEET2b*, *RcSWEET3*, *Rc-SWEET4a*, *RcSWEET5a*, *RcSWEET5c*, *RcSWEET5d*, *RcSWEET9*, *RcSWEET10a*, *RcSWEET10b*, *RcSWEET10c*, *RcSWEET11a*, *RcSWEET12*, *RcSWEET15a*, *RcSWEET15b*, *RcSWEET17a*, and *RcSWEET17b*) contained six exons. Two *RcSWEET* genes (*RcSWEET4b* and *RcSWEET11b*) had five exons, whereas five *RcSWEET* genes (*RcSWEET5b*, *RcSWEET5e*, *RcSWEET5f*, *Rc-SWEET5g*, and *RcSWEET5h*) had only one exon and no intron (Figure 1B). The conserved motifs were investigated based on the protein sequences of the *RcSWEET* genes. Ten consensus motifs were detected in the *RcSWEET* genes (Figure 1C). Each of the 25 members contained two MtN3\_slv domains that were composed of three transmembrane helical domains. To investigate the phylogenetic relationship of the *SWEET* genes in *R. chinensis*, the identified *SWEET* family members of rose were constructed alone and combined with the *SWEET* family members of rice and Arabidopsis. The results showed that 25 *RcSWEETs* were divided into four clades, including 4 members in Clade I, 10 members in Clade II, 9 members in Clade III, and 2 members in Clade IV (Figures 1A and 2).

## *2.3. Chromosomal Location and Gene Duplication Analysis of the RcSWEET Genes*

The analysis of chromosomal location showed that 25 *RcSWEET* genes were distributed on six chromosomes of *R. chinensis* 'Old Blush'. Chr6 contained the greatest number of six *RcSWEET* genes, including *RcSWEET1*, *RcSWEET9*, *RcSWEET10b*, *RcSWEET10c*, *Rc-SWEET11b*, and *RcSWEET11b*. Both Chr1 and Chr7 contained five genes, which were *RcSWEET3, RcSWEET5f, RcSWEET5g, RcSWEET5h*, and *RcSWEET17a* on Chr1 and *Rc-SWEET4b, RcSWEET5a, RcSWEET5d, RcSWEET10a*, and *RcSWEET12* on Chr7. There were four genes, including *RcSWEET5e, RcSWEET15a, RcSWEET15b*, and *RcSWEET17b*, on Chr4. Three genes, including *RcSWEET2a, RcSWEET2b*, and *RcSWEET5b*, are located on Chr5. Only two genes, *RcSWEET4a* and *RcSWEET5c*, are distributed on Chr2. However, there was no *RcSWEET* gene on Chr3.

Gene duplication events usually play an indispensable role in gene expansion and the evolution of gene families. A pair of tandemly duplicated genes (*RcSWEET10a* and *RcSWEET12*) located on Chr7, and another cluster of five tandemly duplicated genes (*RcSWEET9*, *RcSWEET10b*, *RcSWEET10c*, *RcSWEET11a*, and *RcSWEET11b*) located on Chr6 (Figure 3A). In addition, two segmental duplication events, including *RcSWEET4a*/ *RcSWEET5a* and *RcSWEET12*/*RcSWEET15a*, were detected on three different chromosomes (Figure 3B). The selection pressure was further estimated by the Ka (nonsynonymous) and Ks (synonymous) substitution ratios [46]. Only one pair of segmentally duplicated *RcSWEETs*, namely *RcSWEET4a* and *RcSWEET5a*, had a Ka/Ks ratio of 0.15, which was significant, and indicated a synonymous change that has been selected during plant genome evolution (Table S4).

**Figure 1.** Phylogenetic relationship, gene structure, and conserved motif analysis of the *RcSWEET* genes. (**A**) Phylogenetic tree of 25 RcSWEET proteins. The unrooted neighbor-joining (NJ) phylogenetic tree was constructed with MEGA7 using amino acid sequences of 25 RcSWEET proteins, and the bootstrap test replicate was set at 1000 times. (**B**) Gene structure of *RcSWEET* genes. Yellow boxes represent CDS, and black lines represent introns. The upstream/downstream regions of the *RcSWEET* genes are indicated in blue boxes. (**C**) Distributions of conserved motifs in *RcSWEET* genes. Ten putative motifs are indicated in different colored boxes. The MtN3\_slv domains were marked in two red frames.

**Figure 2.** Phylogenetic tree of SWEET proteins of Arabidopsis, rice, and rose. The phylogenetic tree was constructed using the NJ (neighbor joining) method with 1000 bootstrap replications. The 4 subfamilies were distinguished by different colors.

**Figure 3.** Chromosomal location and gene duplication of the *RcSWEET* genes. (**A**) Tandem duplication of *RcSWEET* genes. The tandemly duplicated genes are marked by laurel-green rectangles. (**B**) Segmental duplication of the *RcSWEET* genes. The segmentally duplicated genes of the *R. chinensis* genome are connected by gray lines, and the segmentally duplicated genes of the *RcSWEET* family are highlighted.
