*2.4. JcDof Gene Structures and Conserved Motifs in JcDof Proteins*

Introns and exons are the backbones of genes. Their numbers and distribution patterns are an evolutionary mark for a gene family. We, therefore, compared the intron-exon structure of each *JcDof* gene. The results revealed that the gene structure pattern was consistent with the phylogenetic analysis. Based on the exon-intron structures, the number of introns varied from one to three in *J. curcas* (Figure 3b). There are

ten *JcDof* genes with one intron (41.7%), 12 *JcDof* genes with two introns (50%), and two *JcDof* genes with three introns (8.3%). All of the *JcDof* genes in subfamily A possessed two introns, while the number of introns of the *JcDof* gene in subfamily C varied from one to three.

Our classification of *Dof* genes was also verified by the conserved motif analysis. All of the Dof protein sequences were loaded into the MEME analysis tool to identify the conserved motifs. As a result, a total of ten conserved motifs were observed, which were statistically-significant with *<sup>E</sup>*-values less than 1<sup>×</sup> <sup>10</sup>−<sup>40</sup> (Figure 3a, described in detail in Supplementary Figure S1 and Table S4). The motifs of Dof proteins identified by MEME were between 13–43 amino acids in length. Among them, Motif-1 is a common motif in all Dof proteins, corresponding to the CX2CX21CX2C single zinc-finger structure in the Dof domain, which was the highly-homologous core region of *Dof* family (Figure 3c). While all of the Group B proteins and many of the Group C1 and C2 proteins only contain Motif-1, some Dof proteins have extra specific motifs, which may be relevant to different functions. The Dof proteins from Group A had the most complicated motif patterns, and Motif-2, Motif-4, Motif-5, and Motif-9 were specific for them. While Group C members have relatively simple motif patterns compared with Group A, they also had group-specific motifs, such as Motif-6, Motif-8, and Motif-10, but not all the group members have these specific motifs. For further elucidation of the potential roles of the Group A specific motifs, we checked the GO annotations of the Group A genes in *Arabidopsis*. Interestingly, we found that comparing with the *Arabidopsis Dof* genes in other groups, most of the genes in Group A (5 out of 7) have some flower-development-related annotations, such as "flower development", "negative regulation of long-day photo periodism", "flowering", "negative regulation of short-day photo periodism", "regulation of timing of transition from vegetative to reproductive phase", and "vegetative to reproductive phase transition of meristem", which implied the possible function divergence of the *Dof* genes in group A (see Supplementary Table S3 for detailed information).

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 7 of 15

**Figure 3.** *JcDof* gene structures and conserved motifs in JcDof proteins. (**a**) The distribution of 10conserved motifs in Dof proteins; (**b**) Gene structures of *JcDof* genes. CDS, UTR and introns were depicted by filled red boxes, blue boxes, and single black lines; and (**c**) Motif-1, corresponding to theCX2CX21CX2C single zinc-finger structure. The detailed motif's sequences are shown in Figure S1 and Table S4. *2.5. Chromosomal Locations and Gene Duplication Events ofJcDofGenes*  **Figure 3.** *JcDof* gene structures and conserved motifs in JcDof proteins. (**a**) The distribution of 10 conserved motifs in Dof proteins; (**b**) Gene structures of *JcDof* genes. CDS, UTR and introns were depicted by filled red boxes, blue boxes, and single black lines; and (**c**) Motif-1, corresponding to theCX2CX21CX2C single zinc-finger structure. The detailed motif's sequences are shown in Figure S1 and Table S4.

In order to explore the mechanism of evolution and amplification of *JcDof* gene, the chromosomal locations and gene duplication events of *JcDof* genes were further analyzed. The chromosomal distribution of *JcDof* genes was plotted using Map Inspect software (Figure 4). The duplication events of *JcDof* genes were also examined, and *Dof* gene-pairs arising from segmental and tandem duplication were marked with light blue line and dark blue rectangles, respectively. From Figure 4 we can find that some *Dof* genes, such as *JcDof-19*, have been duplicated several times to form more than one duplicated gene-pair with other genes; and some *JcDof* genes, such as *JcDof-15*,

most of the duplicated gene pairs (22 out of 28), the pairwise *JcDof* genes often came from the same phylogenetic group, with very high sequence similarities. Specifically, tandem duplicated genes have

To further understand the evolutionary constraints acting on all of the duplicated *JcDof* genes, we calculated the non-synonymous substitution rate (*Ka*), synonymous substitution rate (*Ks*) and *Ka*/*Ks* for all of the 28 pairs duplicated genes (Figure 5 and Table S5). We found 23 pairs duplicated genes whose *Ka*/*Ks* were more than one (accounting for 82% of all the duplicated genes) and five pairs

higher sequence similarity than segmental duplicated genes (Table S5).
