*2.3. RsAP2 Protein Motifs and Gene Structure Analysis*

Fifteen conserved motifs of RsAP2 proteins were identified with Multiple EM for Motif Elicitation (MEME) software. These motifs were composed of 8–60 amino acids as shown in Figure 2. The types and distribution of motifs were different in RsAP2 genes, but several combined patterns of motifs, such as motif 11-1-8 and motif 3-2, appear rather frequently. Gene structures of introns and exons were acquired from alignment of genome sequence data. Among the RsAP2 genes, 79 genes had no introns, 17 genes had 1 intron and 24 genes had 2 or more introns. To intuitively compare the features of the RsAP2 genes, Figure 3 was constructed by using a phylogenetic tree, the distribution of motifs and the gene structure of RsAP2 genes.


**Figure 2.** Fifteen main conserved motifs of RsAP2 genes.

**Figure 3.** Chromosomal distribution and gene duplication analysis of RsAP2 genes.

*2.4. Chromosome Location and Gene Duplication of RsAP2 Genes*

The 120 RsAP2 genes were distributed on the thirteen chromosomes unevenly, with the chromosome 7 containing fifteen of the RsAP2 genes and the chromosome 11 contained only three RsAP2 genes, which were the largest and smallest numbers, respectively (Figure 4).

**Figure 4.** Chromosomal distribution and location of RsAP2.

The evolution of genomes and genetic systems is considered to be driven by gene duplications and could be distinguished as segmental duplication and tandem duplication [29]. A tandem duplication event is described as a chromosomal region within 200 kb containing two or more genes, according to previous research [30]. As shown in Figure 5, thirty RsAP2 genes (RsAP2-5/6, RsAP2-27/28/29, RsAP2-50/51/52, RsAP2-61/62/63, RsAP2-65/66, RsAP2-68/69, RsAP2-70/71/72, RsAP2-78/79, RsAP2-80/81/82, RsAP2- 83/84, RsAP2-90/91/92 and RsAP2-94/95) were clustered into twelve tandem duplication event regions on *Rhododendron* chromosomes 1, 5, 7, 8, 9 and 10. The most frequent AP2/ERF gene tandem duplication event occurred on chromosome 8 with four clusters. Fifty segmental duplication events involving seventy-one RsAP2 genes were identified with Multiple Collinearity Scan toolkit X version software (MCScanX). The tandem and segmental duplication contributed greatly to the expansion and evolution process of RsAP2, especially for the latter pattern.

**Figure 5.** Synteny analysis and chromosomal relationships of *Rhododendron* RsAP2 genes. Red lines indicate duplicated RsAP2 gene pairs between different chromosomals, green lines indicate duplicated RsAP2 gene pairs in the same chromosomal and gray lines indicate all synteny blocks in the *Rhododendron* genome.
