*2.6. Chromosome Distribution, Gene Duplication and Synteny Analysis of NnDofs*

We investigated the evolution and expansion mechanisms of *NnDofs* genes by assessing their chromosomal location and gene duplication events. The number distribution of *NnDofs* on chromosomes was heterogeneous, with 29 *NnDofs* located on seven of the eight chromosomes and nine *NnDofs* found on Chr 1, but no *NnDof* genes located on chr 7 (Figure 6). *NnDofs* are localized in the front end of chr3 and chr4 but are separated in chr1, chr2, and chr5. Both tandem and segmental duplications contribute to gene family expansion. We evaluated duplication events in *NnDofs* and highlighted 10 segmental duplications of *NnDofs* gene pairs with red lines. Based on the research published by Holub, two pairs of *NnDofs*, NnDof3-NnDof4 and NnDof25-NnDof26, were identified as tandem duplications.

We also conducted a synteny analysis of *Dof* genes across lotus, tomato, Arabidopsis, and rice (Figure 7). There are 26 orthologous pairs between lotus and Arabidopsis, and 53 orthologous pairs between lotus and tomato. Six *NnDofs* in lotus (*NnDof11*, *NnDof12*, *NnDof15*, *NnDof17*, *NnDof19*, and *NnDof23*) have two pairs of homologous genes in Arabidopsis, and two *NnDofs* (*NnDof1* and *NnDof10*) have three pairs of homologous genes in Arabidopsis thaliana. Analysis of the number of collinear homologous genes between lotus and tomato revealed that four *NnDofs* (*NnDof12*, *NnDof14*, *NnDof18*, and *NnDof28*) have two homologous genes in tomato, three *NnDofs* (*NnDof1* and *NnDof17*) have four homologous genes in tomato genes, and three *NnDofs* (*NnDof10*, *NnDof15*, and *NnDof19*) have five homologous genes in tomato. A total of 11 *SlDofs* genes showed syntenic relationships with those in rice.

**Figure 4.** The prediction of a cis-acting element in the 2000 bp promoter upstream of the *NnDofs*. The main cis-acting elements are marked in the upper right corner.

**Figure 5.** Protein interaction network of NnDofs protein with other Arabidopsis proteins predicted using the STRING-DB. The minimum required interaction score is a moderate confidence level of 0.400. Organism set: Arabidopsis. These lines reflect the interaction of Nndofs homologs with other proteins. The darkness of the circle color indicates the strength of the data support.
