Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets

Xin Liu; Huirui Guan; Min Song; Yanping Fu; Xiaomin Han; Meng Lei; Jingyu Ren; Bin Guo; Wei He; Yahui Wei

doi:10.7717/peerj.4535

Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets

PeerJ. 2018 Apr 3:6:e4535. doi: 10.7717/peerj.4535. eCollection 2018.

Authors

Xin Liu^{1

2}, Huirui Guan^{1

2}, Min Song^{1

2}, Yanping Fu^{1

2}, Xiaomin Han^{1

2}, Meng Lei^{1

2}, Jingyu Ren^{1

2}, Bin Guo^{1

2}, Wei He^{1

2}, Yahui Wei^{1

2}

Affiliations

¹ College of Life Science, Northwest University, Xi'an, Shaanxi, China.
² Key Laboratory of Resource Biology and Biotechnology in Western China, Northwest University, Xi'an, Shaanxi, China.

Abstract

Background: Stellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported.

Method: In this study, 10 candidate RGs namely, 18S, 60S, CYP, GAPCP1, GAPDH2, EF1B, MDH, SAND, TUA1, and TUA6, were singled out from the transcriptome database of S. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper.

Result: Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND, TUA1 and CYP, GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2 and GI) further verified that the RGs that we selected were suitable for gene expression normalization.

Discussion: This work is the first attempt to comprehensively estimate the stability of RGs in S. chamaejasme. Our results provide suitable RGs for high-precision normalization in qRT-PCR analysis, thereby making it more convenient to analyze gene expression under these experimental conditions.

Keywords: Abiotic stresses; Hormone treatments; Reference gene; Stellera chamaejasme; Transcriptome; qRT-PCR.

Grants and funding

This work were supported by the Special Fund for Agro-scientific Research in the Public Interest (No. 201203062), the Scientific research plan projects of the Education Department of Shaanxi Provincial Government (No. 2010JS091), the Special Fund for Scientific Research from the Shaanxi Provincial Department of Education (No. 16JK1756), the Natural Science Foundation of China (No. 31702159; No. 81303159), the Support Plan for Young Talents of Science and Technology Association of Colleges and Universities in Shaanxi Province (No. 20150105), and the Bureau of Science and Technology in Shaanxi Province, Xi’an City, Beilin District (No. GX1702). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.