*2.7. Expression Patterns of NnDofs in Lotus*

RNA-seq data were used to discover how *NnDof* was expressed in different tissues of the lotus (Figure 8). We investigated the expression patterns of *NnDofs* since their distinct physiological roles are interrelated with their expression patterns in different tissues and at different developmental stages. The expression patterns of *Dof* genes in different tissues and organs may be grouped into three groups based on clustering results, and members of the same group have comparable expression profiles. In group 1, *NnDofs* expression was low in most tissues; in group 2, certain *NnDofs* were dominantly expressed in some tissues; and in group 3, *NnDofs* expression was high in most tissues. In general, genes have similar expression patterns. For example, some genes, such as NnDof19 and *NnDof28*, are highly expressed in all tissues, while *NnDof24* and *NnDof27* are lowly expressed in all tissues, and *NnDof2*, *NnDof16*, *NnDof22*, and *NnDof28* are predominantly expressed in the seed coat. Furthermore, 26 (90%) *NnDofs* were expressed in at least one tissue and developmental period (log2 (FPKM) > 0); six (21%) *NnDofs* were expressed in all samples tested, which suggests that these *NnDofs* act at multiple developmental stages; and only one (3%) *NnDof* was lowly expressed in all samples tested and may be specifically expressed in other tissues and developmental periods.

**Figure 6.** Chromosomal locations and segmental duplication events of NnDofs. Red lines represent segmental duplication events.

**Figure 7.** Synthetic analysis of *NnDofs* in lotus, rice, tomato, and Arabidopsis genomes. The red lines represent homologous gene pairs between two adjacent species.

**Figure 8.** Expression pattern of *NnDofs* in different tissues of lotus. The expression pattern was generated based on FPKM plus 1 after log2 transformation and analyzed by heatmap hierarchical clustering.

Whole plant leaves were used when extracting total RNA to prepare for the qRT-PCR analysis, which was validated to discover how the NnDofs genes were expressed under salt stress (Figure 9). Under salt stress, the expression of *NnDofs* was rapidly upregulated at 1 h, except for *NnDof15*, *NnDof16*, *NnDof19*, and *NnDof20*. Some *NnDofs* genes were downregulated at 4 h, 12 h, and 24 h, but *NnDof3*, *NnDof11*, *NnDof12*, *NnDof17*, *NnDof18*, *NnDof22*, *NnDof25*, and *NnDof26* were upregulated at 24 h.

**Figure 9.** Expression profiles of *NnDofs* genes responding to salt stress. Blue indicates decreased expression levels and red indicates increased expression levels. Heatmaps were generated with TBtools software.

## **3. Discussion**

The ornamental lotus is a traditional Chinese flower and an important aquatic plant, which is loved and widely cultivated for its brilliant color, rich flower shape, and fragrant smell. It has been reported that common lotus species are sensitive to salt stress, and increasing soil salt concentration can significantly affect their growth and development processes, further affecting lotus cultivation and production. Therefore, mining and analyzing the genes associated with salt stress resistance in lotus is important to improve the resistance of lotus. *Dofs* have been reported to play an important role in salt stress resistance in rice and tomato. In this study, we performed a genome-wide search for *NnDofs* with a focus on salt stress resistance. This will help us learn more about how *Dof* genes work and how to improve the genetics of lotus.
