, Volume 33, Issue 6, pp 1591–1601 | Cite as

Selection and verification of candidate reference genes for gene expression by quantitative RT-PCR in Hibiscus hamabo Zucc.

  • Longjie Ni
  • Zhiquan Wang
  • Liangqin Liu
  • Jinbo Guo
  • Huogen LiEmail author
  • Chunsun GuEmail author
Original Article
Part of the following topical collections:
  1. Functional Genomics


Key message

Ten candidate reference genes were examined in Hibiscus hamabo Sieb. et Zucc. ACT and SKIP are proposed as good reference genes for gene studies in Hibiscus hamabo Sieb. et Zucc.


Because of its sensitivity and rapidness, quantitative real-time PCR (qRT-PCR) is currently extensively used to analyze gene-expression patterns. Selecting suitable reference genes to normalize qRT-PCR results is essential. Hibiscus hamabo Sieb. et Zucc. (H. hamabo) is a semi-mangrove plant that is widely used for the ecological restoration of saline-alkali land and coastal afforestation owing to its excellent salt tolerance. However, suitable reference genes used for the normalization of H. hamabo qRT-PCR data have not been selected or verified. Here, we tested the expression stabilities of ten candidate reference genes in different H. hamabo tissues under a set of abiotic stresses (salt, drought, high temperature, and low temperature) and hormonal treatments (methyl jasmonate, abscisic acid, and salicylic acid) using three statistical algorithms, i.e., NormFinder, geNorm, and BestKeeper. Actin (ACT) and ski-interacting protein (SKIP) can be regarded as good choices as reference genes in studying gene expression of H. hamabo. In addition, the qRT-PCR analysis of the NAC (NAM/ATAF1/2/CUC2) target gene’s expression pattern under NaCl-treated conditions confirmed the suitability of selected reference genes. Here, we used qRT-PCR technology to provide a stable reference gene list for H. hamabo gene-expression studies.


Reference gene Hibiscus hamabo Sieb. et Zucc. Quantitative real-time PCR NormFinder GeNorm BestKeeper 



The study was supported by Six Talent Peaks Project of Jiangsu Province (NY-042) and the 333 Talents Project of Jiangsu Province (BRA2017498).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this paper.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.College of Forest SciencesNanjing Forestry UniversityNanjingChina
  2. 2.Jiangsu Provincial Platform for Conservation and Utilization of Agricultural GermplasmInstitute of Botany, Jiangsu Province and Chinese Academy of SciencesNanjingChina

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