*5.2. Conserved Domain, Conserved Structure, and Phylogenetic Analysis of NnDofs*

Multiple protein sequence alignments of Dof domains were constructed using ClustalW, and the results were submitted to GeneDoc software for optimization. After analyzing the full-length sequence, the gene structure pattern distribution of NnDofs was drawn using Tbtools software. The conserved motifs of NnDof sequences in lotus were scanned using

MEME (https://meme-suite.org/meme/tools/meme/, accessed on 3 May 2021), with a maximum number of motifs of 10 and the remaining parameters set to default values. The Dof-deduced sequences in Arabidopsis and lotus were aligned using MUSCLE with default settings. Phylogenetic trees were constructed using MEGA 7.0 (http://www.megasoftware.net/, accessed on 5 May 2021) with the neighbor-joining (NJ) method and 1000 bootstraps.

#### *5.3. Gene Duplication and Syntenic Analysis of NnDofs*

All NnDofs genes were mapped to lotus chromosomes based on physical location information from the database of lotus genome using Circos. The multiple collinear scanning toolkit (MCScanX, http://chibba.pgml.uga.edu/mcscan2/, accessed on 7 May 2021) was used to analyze gene duplication events in lotus, and for Arabidopsis, tomato, soybean, rice, maize, and lotus protein sequences, a collinearity analysis was performed between them. Finally, gene duplication and collinearity were shown using Circos software and Tbtool software with default parameters [64].

## *5.4. Cis-Acting Element Analysis and Protein Interaction Network Prediction of NnDofs*

Genomic DNA sequences 2000 bp upstream of each NnDofs transcription start site were extracted and submitted to the PlantCARE database (http://bioinformatics.psb.ugent. be/webtools/plantcare/html/, accessed on 9 May 2021) to predict the cis-acting elements in the NnDofs promoter region.

All putative NnDof protein sequences were uploaded to the webserver STRING (version 11.5; http://string-db.org, accessed on 11 May 2021) to build an interaction network, and the NnDof protein interaction network was mapped using Cytoscape.

#### *5.5. Expression Patterns Analysis of NnDofs by RNA-Seq Data*

A study by Zhang describes information from transcriptomic data from various tissues of the lotus [63]. To analyze the expression patterns of NnDofs, we generated 20 groups of tissue-specific transcript abundances at different developmental stages based on the data obtained. The 20 groups of tissues are from the seed coat, cotyledon, root, rhizome (mixed), leaf, petiole, receptacle, stamen, petal, carpel, rhizome internode, rhizome elongation zone, and rhizome apical meristem. The seed coat data were taken at 18 days after pollination, and the cotyledon data were taken at 15 days after pollination. Triplicate data were averaged, data were transformed by log2 (FPKM+1), and heatmaps were created using a plugin within the Tbtools software. All the detailed data are shown in Table S2.
