*2.5. Chromosomal Locations and Gene Duplication Events of JcDof Genes*

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*, *JcDof-22*, and *JcDof-24*, are evolutionarily too close to resolve their gene duplication order (the duplication pairs are described in detail in Supplementary Table S5). The gene expansion of the *Dof* family in physic nut mainly resulted from segmental duplication, and tandem duplication also played a minor role. In total, 26 pairs of segmental duplicated *JcDof* genes (93% of all duplicated genes) and two pairs of tandem duplicated *JcDof* genes (7% of all duplicated genes) were found. For 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 higher sequence similarity than segmental duplicated genes (Table S5).

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 duplicated genes whose *Ka*/*Ks* ratio were less than one (accounting for 18% of all the duplicated gene pairs) (Table S5). This implied that most of the *Dof* duplicated gene pairs tended to be subjected to positive selection, which may play important roles in the origin of adaptive phenotypes and the possible function divergence in *JcDof* genes. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 8 of 15 duplicated genes whose *Ka*/*K s*ratio were less than one (accounting for 18% of all the duplicated gene pairs) (Table S5). This implied that most of the *Dof* duplicated gene pairs tended to be subjected to positive selection, which may play important roles in the origin of adaptive phenotypes and the possible function divergence in *JcDof* genes.

**Figure 4.** Chromosomal locations and gene duplication events of *JcDof*. Respective scaffold numbers are indicated at the top of each bar. The scale on the left is in centimorgan (cM). The *JcDof* gene pairs of segmental and tandem duplication are linked by pale blue lines and marked in dark blue rectangles, respectively. The detailed information of duplication pairs are described in Supplementary Table S5. **Figure 4.** Chromosomal locations and gene duplication events of *JcDof*. Respective scaffold numbers are indicated at the top of each bar. The scale on the left is in centimorgan (cM). The *JcDof* gene pairs of segmental and tandem duplication are linked by pale blue lines and marked in dark blue rectangles, respectively. The detailed information of duplication pairs are described in Supplementary Table S5.

**Figure 5.** The *Ka*/*Ks* value of duplicated *JcDof* gene pairs. 28 pairs of duplicated *JcDof* genes and 23 pairs duplicated genes with *Ka*/*Ks* more than one. The detailed *Ka*/*Ks* information of duplication

In order to further study the possible function divergence of *JcDof* genes, we investigated the expression level of *JcDof* genes under various abiotic stresses and hormonal treatments by using the public transcriptome data from NCBI SRA database (Supplementary Tables S6 and S7 for detailed information). We employed a heatmap to visualize a global transcription profile of the *JcDof* genes. As shown in Figure 6, *JcDof* genes showed diverse responses to various treatments, and significant differences were found in response to 6-Benzylaminopurine (BA), salt, and drought treatments (two-

*2.6. Expression Patterns of JcDof Genesunder Different Abiotic Stress and Hormone Treatments* 

pairs are described in Supplementary Table S5.

fold increases or decreases compared to controls).

Supplementary Table S5.

possible function divergence in *JcDof* genes.

**Figure 4.** Chromosomal locations and gene duplication events of *JcDof*. Respective scaffold numbers are indicated at the top of each bar. The scale on the left is in centimorgan (cM). The *JcDof* gene pairs of segmental and tandem duplication are linked by pale blue lines and marked in dark blue

duplicated genes whose *Ka*/*K s*ratio were less than one (accounting for 18% of all the duplicated gene pairs) (Table S5). This implied that most of the *Dof* duplicated gene pairs tended to be subjected to positive selection, which may play important roles in the origin of adaptive phenotypes and the

**Figure 5.** The *Ka*/*Ks* value of duplicated *JcDof* gene pairs. 28 pairs of duplicated *JcDof* genes and 23 pairs duplicated genes with *Ka*/*Ks* more than one. The detailed *Ka*/*Ks* information of duplication pairs are described in Supplementary Table S5. **Figure 5.** The *Ka*/*Ks* value of duplicated *JcDof* gene pairs. 28 pairs of duplicated *JcDof* genes and 23 pairs duplicated genes with *Ka*/*Ks* more than one. The detailed *Ka*/*Ks* information of duplication pairs are described in Supplementary Table S5.

#### *2.6. Expression Patterns of JcDof Genesunder Different Abiotic Stress and Hormone Treatments 2.6. Expression Patterns of JcDof Genesunder Different Abiotic Stress and Hormone Treatments*

In order to further study the possible function divergence of *JcDof* genes, we investigated the expression level of *JcDof* genes under various abiotic stresses and hormonal treatments by using the public transcriptome data from NCBI SRA database (Supplementary Tables S6 and S7 for detailed information). We employed a heatmap to visualize a global transcription profile of the *JcDof* genes. As shown in Figure 6, *JcDof* genes showed diverse responses to various treatments, and significant differences were found in response to 6-Benzylaminopurine (BA), salt, and drought treatments (twofold increases or decreases compared to controls). In order to further study the possible function divergence of *JcDof* genes, we investigated the expression level of *JcDof* genes under various abiotic stresses and hormonal treatments by using the public transcriptome data from NCBI SRA database (Supplementary Tables S6 and S7 for detailed information). We employed a heatmap to visualize a global transcription profile of the *JcDof* genes. As shown in Figure 6, *JcDof* genes showed diverse responses to various treatments, and significant differences were found in response to 6-Benzylaminopurine (BA), salt, and drought treatments (two-fold increases or decreases compared to controls).

In the BA treatment experiments (gene expression data collected from roots), compared with the negative control (mock), three genes (*JcDof-1*, *JcDof-8*, and *JcDof-10*) exhibited significant responses. Among them, *JcDof-1* and *JcDof-10* showed reduced expression when responding to BA treatment, with more than two-fold (*JcDof-1*) and nearly four-fold (*JcDof-10*) decreasing, respectively. Meanwhile, *JcDof-8* showed a significantly up-regulated expression with more than four-fold increase. We further checked the GO annotations of their Arabidopsis orthologs, and found they were annotated as "seed coat development" (*JcDof-1*, *AT1G29160.1*), "guard cell differentiation, positive regulation of transcription, regulation of cell wall pectin metabolic process, stomatal movement" (*JcDof-8*, *AT5G65590.1*), and "flower development" (*JcDof-10*, *AT5G39660.1*) respectively, which may imply the possible roles of these three genes (Supplementary Table S3 for detailed information).

We further analyzed the expression patterns of the *JcDof* genes in salt- and drought-stressed roots and leaves at different times: 2 h, 2 days, and 7 days (salt-stressed); 13 days, 49 days, and 52 days (drought-stressed). The fold changes of gene expression were calculated between abiotic stress treatments and controls. Many *JcDof* genes exhibited significant responses, and some of them showed significant up- or down-regulation in both roots and leaves, such as *JcDof-8*, *JcDof-17*, and *JcDof-20* in salt-stressed treatments, and *JcDof-6*, *JcDof-8*, *JcDof-10*, *JcDof-14*, *JcDof-17*, and *JcDof-21* in drought-stressed treatments. Most of these significantly up- or down-regulated genes (seven out of nine) tended to show similar expression changes (up- or down-regulation) in both roots and leaves. The only two exceptions were *JcDof-20* and *JcDof-14*. *JcDof-20* showed significantly reduced expression in leaves (from 2 h to 7 days) when responding to salt treatment, while *JcDof-20* expression

in salt-treated roots first decreased (at 2 h), and then increased significantly (two days and seven days). Another gene, *JcDof-14*, showed significantly reduced expression in leaves (in 49 days) when responding to drought treatment, while *JcDof-14* expression in drought-treated roots first increased (in 13 days), and then decreased significantly (49 days and 52 days). leaves (from 2 h to 7 days) when responding to salt treatment, while *JcDof-20* expression in salttreated roots first decreased (at 2 h), and then increased significantly (two days and seven days). Another gene, *JcDof-14*, showed significantly reduced expression in leaves (in 49 days) when responding to drought treatment, while *JcDof-14* expression in drought-treated roots first increased (in 13 days), and then decreased significantly (49 days and 52 days).

only two exceptions were *JcDof-20* and *JcDof-14*. *JcDof-20* showed significantly reduced expression in

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 9 of 15

In the BA treatment experiments (gene expression data collected from roots), compared with the negative control (mock), three genes (*JcDof-1*, *JcDof-8*, and *JcDof-10*) exhibited significant responses. Among them, *JcDof-1* and *JcDof-10* showed reduced expression when responding to BA treatment, with more than two-fold (*JcDof-1*) and nearly four-fold (*JcDof-10*) decreasing, respectively. Meanwhile, *JcDof-8* showed a significantly up-regulated expression with more than four-fold increase. We further checked the GO annotations of their Arabidopsis orthologs, and found they were annotated as "seed coat development" (*JcDof-1*, *AT1G29160.1*), "guard cell differentiation, positive regulation of transcription, regulation of cell wall pectin metabolic process, stomatal movement" (*JcDof-8*, *AT5G65590.1*), and "flower development" (*JcDof-10*, *AT5G39660.1*) respectively, which may imply the possible roles of these three genes (Supplementary Table S3 for detailed information).

We further analyzed the expression patterns of the *JcDof* genes in salt- and drought-stressed roots and leaves at different times: 2 h, 2 days, and 7 days (salt-stressed); 13 days, 49 days, and 52 days (drought-stressed). The fold changes of gene expression were calculated between abiotic stress treatments and controls. Many *JcDof* genes exhibited significant responses, and some of them showed significant up- or down-regulation in both roots and leaves, such as *JcDof-8*, *JcDof-17*, and *JcDof-20* in salt-stressed treatments, and *JcDof-6*, *JcDof-8*, *JcDof-10*, *JcDof-14*, *JcDof-17*, and *JcDof-21* in droughtstressed treatments. Most of these significantly up- or down-regulated genes (seven out of nine)

We have also checked the differential expression patterns of the duplicated *JcDof* gene pairs, and found that if *JcDof* genes differentially expressed in some stress treatments, and their duplicated counterparts were more likely not to show differential expression (27 pairs vs. 20 pairs, Supplementary Table S8 for detailed information). We think these results are consistent with our *Ka*/*Ks* results, that most of the duplicated *JcDof* genes tended to be subjected to positive selection, and implied the possible function divergence in *JcDof* genes. We have also checked the differential expression patterns of the duplicated *JcDof* gene pairs, and found that if *JcDof* genes differentially expressed in some stress treatments, and their duplicated counterparts were more likely not to show differential expression (27 pairs vs. 20 pairs, Supplementary Table S8 for detailed information). We think these results are consistent with our *Ka*/*Ks* results, that most of the duplicated *JcDof* genes tended to be subjected to positive selection, and implied the possible function divergence in *JcDof* genes.

**Figure 6.** Expression patterns of *JcDof* genes under different treatments. The heatmap was generated by HemI software using the expression data of the *JcDof* genes, and normalized log2 transformed values were used with hierarchical clustering represented by the color scale (0–10). Blue indicates low expression, and red indicates high expression. The samples were: roots and leaves (salt- and droughtstressed at different times), and roots (BA treatment). The detailed information of expression data are described in Supplementary Tables S6 and S7. **Figure 6.** Expression patterns of *JcDof* genes under different treatments. The heatmap was generated by HemI software using the expression data of the *JcDof* genes, and normalized log<sup>2</sup> transformed values were used with hierarchical clustering represented by the color scale (0–10). Blue indicates low expression, and red indicates high expression. The samples were: roots and leaves (salt- and drought-stressed at different times), and roots (BA treatment). The detailed information of expression data are described in Supplementary Tables S6 and S7.
