**3. Discussion**

The Euphorbiaceae family includes some of the most efficient biomass accumulators, such as physic nut, castor bean, cassava, and rubber tree [9,31]. Crop improvement in Euphorbiaceae for sustainable industrial raw materials and food production requires more extensive genome-wide studies on these species. Notably, physic nut has become an ideal model organism in Euphorbiaceae for further functional genomics analysis due to its sequenced genome, genetic linkage map, and abundance of high-throughput transcriptome data. Studies on physic nut will provide insights into the investigation of other Euphorbiaceae organisms.

Genome-wide gene family analysis is abasic and a key step to understanding the gene structure, function, and evolution [32]. The *Dof* gene family has been shown to play crucial roles in the regulatory network of plant defense, including responses to diverse biotic and abiotic stresses [22,23,33,34]. Until now, the *Dof* genes have been identified and characterized in different plant species, but not in the promising energy plant physic nut yet. Therefore, we conducted a comprehensive analysis of the *JcDof* family in physic nut, along with their homologs in *R. communis* and *A. thaliana*, to study their phylogenetic relationships and potential functions.

In total, we identified 24 *JcDof* genes in the physic nut genome. Compared with the number of *Dof* genes in *A. thaliana* (36 genes from TAIR), the size of physic nut *Dof* gene family is much smaller [35], although the assembled genome size of physic nut is approximately three times larger than the *A. thaliana* genome (320.5 Mbp vs. 125 Mbp) [9,36]. Correspondingly, we had discovered that the members from Group B, one of the major groups in the phylogenetic tree, all pertained to *AtDof* genes. In addition, Subgroup C1 contained 13 *AtDof* genes; while only nine *JcDof* genes were noted. Subgroup C2 had 12 *AtDof* genes and 10 *JcDof* genes. These results suggested that *JcDof* and *AtDof* genes should arise through different duplication events, and might have undergone species/lineage-specific gene gain or loss.

Both tandem duplication and segmental duplication contributed to the variation in gene family number and distribution [37,38]. In total, 26 gene-pairs from segmental duplication and two from tandem duplication were found in physic nut. We calculated the *Ka*/*Ks* ratios for these duplicated *JcDof* paralog genes, and found most of the duplicated genes pairs had *Ka*/*Ks* ratios over 1, implying that positive selection played an important role in the evolution of *JcDof* genes, and high-throughput expression data analysis further confirmed the functional diversity of *JcDof* genes. *JcDof* genes showed diverse responses to various treatments, and might participate in different stress/hormone-responding regulatory processes. This work provides valuable information for understanding the evolution of *JcDof* genes and lays a foundation for future functional analysis of *Dof* genes in the process of growth, development, and Dof-mediated regulation in physic nut.
