*4.7. Validation of DEGs by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)*

Twelve differentially expressed candidate genes were assessed by qRT-PCR to measure the reliability of the RNA-seq data, as previously described [79]. The primer sequences are presented in Table S8. The SYBR qPCR Master Mix (Vazyme) was used with the CFX96 for qRT-PCR analysis (BIO-RAD). Each sample was subjected to three technical replications. The 2−∆∆CT method was utilized to determine the relative expression of target genes using *B. napus* ACTIN2 as an internal control. [80].

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23147958/s1.

**Author Contributions:** X.D. planned and supervised the research; N.A., B.S., S.I., L.K., Z.T. and X.W. performed root traits investigation and analyzed the data; N.A. wrote the manuscript; X.D. and H.W. contributed to modify the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the Major Project of Hubei Hongshan Laboratory (2021HSZD004), Agricultural Science and Technology Innovation Project (CAAS-ZDRW202109), Central Public-interest Scientific Institution Basal Research Fund (No.1610172020004), Agricultural Science and Technology Innovation Project (CAAS-ASTIP-2013-OCRI), and China Agriculture Research System of MOF and MARA (CARS-12).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated or analyzed during the present study are available from the corresponding authors on reasonable request.

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

#### **References**


**Huanhuan Qi , Feng Yu , Jiao Deng † and Pingfang Yang \***

State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Science, Hubei University, Wuhan 430062, China; qihuanhuan0911@163.com (H.Q.); yufeng@hubu.edu.cn (F.Y.); ddj613@163.com (J.D.)

**\*** Correspondence: yangpf@hubu.edu.cn; Tel.: +86-27-88661237 † Current Address: Research Center of Buckwheat Industry Technology, School of Life Sciences,

Guizhou Normal University, Guiyang 550001, China.

**Abstract:** Lotus (*Nelumbo nucifera*), under the Nelumbonaceae family, is one of the relict plants possessing important scientific research and economic values. Because of this, much attention has been paid to this species on both its biology and breeding among the scientific community. In the last decade, the genome of lotus has been sequenced, and several high-quality genome assemblies are available, which have significantly facilitated functional genomics studies in lotus. Meanwhile, re-sequencing of the natural and genetic populations along with different levels of omics studies have not only helped to classify the germplasm resources but also to identify the domestication of selected regions and genes controlling different horticultural traits. This review summarizes the latest progress of all these studies on lotus and discusses their potential application in lotus breeding.

**Keywords:** lotus; genome; variant; germplasm; breeding; omics

**Citation:** Qi, H.; Yu, F.; Deng, J.; Yang, P. Studies on Lotus Genomics and the Contribution to Its Breeding. *Int. J. Mol. Sci.* **2022**, *23*, 7270. https:// doi.org/10.3390/ijms23137270

Academic Editors: Zhiyong Li and Jian Zhang

Received: 26 May 2022 Accepted: 27 June 2022 Published: 30 June 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).
