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Systematic assessment of reference genes for RT-qPCR across plant species under salt stress and drought stress

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Abstract

Suitable reference gene (RGs) is the prerequisite for accurate normalization of real-time quantitative PCR (RT-qPCR) data. However, previous results are diverse in various researches that focused on selecting stable RGs. This study aims at systematically assessing various RGs in plants under salt stress or drought stress by collection of geNorm rankings of genes, data transformation and statistic analysis. Although none of the analyzed genes can guarantee universally stable expressions in plant species under salt stress or drought stress, we found that 18S (18S ribosomal RNA) was generally the least stable gene under salt and drought stress. This gene should not be used as the RG in RT-qPCR. On the contrary, it is least risk to use EF1 for salt stress and TIP41 for drought treatment experiments. We compared the effects of salt and drought stresses on 7 frequently used RGs through paired-samples T test. The expression of Ubiquitin gene under drought stress is much more unstable than that under salt stress. The tested genes belonging to multi-gene family and having different stability could be one reason of variations in the published studies, which was supported by the analysis of expression profile of Salicornia europaea transcriptome. This is the first systematic assessment quantifying global stability of RGs across plant species under salt stress and drought stress, which will improve our understanding of RGs and facilitate the future work on RGs selection.

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Abbreviations

RG:

Reference gene

RT-qPCR:

Real-time quantitative PCR

EF1 :

Elongation factor 1

EIF :

Eukaryotic initiation factor

PP2A :

Protein phosphatase 2A

GAPDH :

Glyceraldehyde-3-phosphate dehydrogenase

RPL :

Ribosomal protein L

TIP41 :

Tonoplast intrinsic proteins-41

His :

Histone

APRT :

Adenine phosphoribosyl transferase

UBQ :

Ubiquitin

UBC :

Ubiquitin-conjugating enzyme

CAC :

Clathrin adaptor complexes medium subunit

CYP :

Cyclophilin

ACT :

Actin

TUA :

α-Tubulin

TUB :

β-Tubulin

18S :

18S ribosomal RNA

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Acknowledgments

The research was supported by the National Natural Science Foundation of China (No. 31270660), the Doctor Western-funded Projects of Chinese Academy of Sciences (No. XBBS 201201), the Outstanding Youth Foundation of Science and Technology in Xinjiang of China (No. 2013711018), and the 100 Talents Program of Chinese Academy of Science.

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Authors and Affiliations

  1. Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Science, Ürümqi, China

    Xinlong Xiao, Xiaomeng Wu, Jinbiao Ma, Pengbo Li, Taotao Li & Yinan Yao

  2. University of Chinese Academy of Sciences, Beijing, China

    Xinlong Xiao, Xiaomeng Wu, Pengbo Li & Taotao Li

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  1. Xinlong Xiao
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  2. Xiaomeng Wu
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  3. Jinbiao Ma
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  4. Pengbo Li
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Correspondence to Yinan Yao.

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Communicated by M. Stobiecki.

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11738_2015_1922_MOESM1_ESM.tif

Fig. S1. Distribution of CV values of unigenes expression for various reference genes. The Box plot contains the mean, interquartile range, non-outlier range, and outlier. The number following the gene name represents the unigene number, (TIFF 64 kb)

Table S1. geNorm rankings extracted from researches which evaluated reference genes under salt stress. (XLS 27 kb)

Table S2. geNorm rankings extracted from researches which evaluated reference genes under drought stress. (XLS 28 kb)

11738_2015_1922_MOESM4_ESM.xls

Table S3. geNorm rankings extracted from the researches which evaluated reference genes simultaneously under salt stress and drought stress. (XLS 25 kb)

11738_2015_1922_MOESM5_ESM.xls

Table S4. Unigenes with RPKM and annotation were collected for each kind of housekeeping genes from Salicornia europaea transcriptome(XLS 519 kb)

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Xiao, X., Wu, X., Ma, J. et al. Systematic assessment of reference genes for RT-qPCR across plant species under salt stress and drought stress. Acta Physiol Plant 37, 186 (2015). https://doi.org/10.1007/s11738-015-1922-8

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  • DOI: https://doi.org/10.1007/s11738-015-1922-8

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