*5.6. Stress Treatments and Quantitative Real-Time PCR Analysis*

*Nelumbo nucifera* cv. 'Taikonghongqi', a typical cultivated variety, was used throughout the study. The variety was cultivated in a greenhouse at Fuyang Normal University, Fuyang City, Anhui Province, China, from June 2021 to July 2021. 'Taikonghongqi' seeds were surface sterilized in 75% ethanol for 2 min before being peeled open and fully soaked in water. They were then provided an artificial environment with 16 h of light and 8 h of darkness at a temperature of 24 ◦C for four weeks. Lotus seedlings with similar development status were selected and randomly divided into two groups and cultured under the same culture conditions for 72 h. For the salt-stress treatment group, the roots of the lotus seedlings were exposed to high salt stress (200 mM NaCl) for 24 h. For all treatments, plant materials from three biological replicates were harvested immediately, frozen in liquid nitrogen, and then stored at −80 ◦C until RNA isolation.

Total RNA was extracted with a plant RNA extraction kit (Huayueyang, Beijing, China) and treated with RNase-free DNase I to remove potential genomic DNA contamination. Qualified RNA was selected as the template to generate first-strand cDNA by gel electrophoresis and A260/A280 ratio determination. Complementary cDNA was generated with reverse transcriptase (TransGen Biotech, Beijing, China). Specific NnDof gene primers were designed using Primer Premier 5, and the expression levels of different sampling cycles were normalized with NnActin gene as a reference gene. Supplementary Table S3

provides all primer information. qRT-PCR was performed using 2×SYBR Green qPCR Mix (with ROX) (Sparkjade, Qingdao, China) and amplified using 96-well plates and a CFX96 TouchTM RT-PCR system (Biorad, Los Angeles, CA, USA). Each reaction was performed in biological triplicate. Data from qRT-PCR amplifications were analyzed using the 2−ΔΔCt method.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants11152057/s1, Table S1: List of the 29 NnDof genes identified in this study; Table S2: RNA-seq data of 29 Nndof genes that were used in this study; Table S3: Primers of NnDof genes used in RT-qPCR; Table S4: The lotus Dof gene sequence identified in this study.

**Author Contributions:** L.R., W.W. and X.C. conceived and designed the experiments; D.Y. and H.M. performed the experiments; W.W. analyzed the data; X.D., H.Y. and X.C. contributed reagents/materials/analysis tools; X.C. and W.W. wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant from the Natural Science Key Foundations of the Anhui Bureau of Education (KJ2021A0677), the research funds for postdoctoral researchers in Anhui Province (2020B434), the science and technology special project of Fuyang Normal University-Fuyang City Municipal School Cooperation (SXHZ202105) and Fuyang Normal University's major scientific and technological achievements incubator fund project (kjfh201703).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
