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Reference gene selection for quantitative RT-PCR in Miscanthus sacchariflorus under abiotic stress conditions

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Abstract

Background

Reference genes are necessary for quantitative real-time PCR (qRT-PCR) analysis and their stability can directly influence the accuracy of gene expression result. Miscanthus sacchariflorus, a perennial C4 grass that serves as promising biofuel plant for temperate climates, has not been explored for the identification of stable reference genes yet.

Materials and methods

Nine potential reference genes (ACT, EF1a, FBOX, GAPDH, PP2A, SAND, TIP41, TUB and UBC) of M. sacchariflorus under different abiotic (salinity, drought and cadmium) stresses, as well as in two tissues (roots and leaves) were evaluated. The expression stability of these genes were analyzed by four commonly used software programs (geNorm, NormFinder, BestKeeper, ΔCt method and RefFinder).

Results

Our results found that FBOX and SAND are the most stable genes among all tested samples. FBOX and EF1a are suitable for gene expression normalization of cadmium-treated samples and salinity-treated leaves. FBOX and PP2A are appropriate reference genes for salt-stressed roots and PEG-treated leaves. The traditional reference gene ACT and GAPDH exhibited the most variable pattern, which would not be recommended for qRT-PCR analysis under different abiotic stresses. Furthermore, the expression levels of PIP2, NHX1 and MT2a under drought, salt and cadmium treatment were detected with above reference genes.

Conclusions

This work identified the appropriate reference genes for qRT-PCR in M. sacchariflorus and FBOX was recommended to be effective internal control for gene expression normalization in M. sacchariflorus in response to different abiotic stresses.

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Funding

This work was supported by the the Program of National Natural Science Foundation of China (31771870, 31201262), Natural Science Foundation of Jiangsu Province (BK2012790) and Public Science and Technology Research Funds Projects of Ocean (201505023).

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Authors and Affiliations

  1. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China

    Junqin Zong, Jingbo Chen, Ling Li, Jianjian Li, Dandan Li, Jingjing Wang & Jianxiu Liu

  2. College of Agro-grassland Science, Nanjing Agricultural University, Nanjing, 210095, China

    Jun Liu

Authors
  1. Junqin Zong
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  2. Jingbo Chen
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  3. Ling Li
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  4. Jianjian Li
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  5. Dandan Li
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  6. Jingjing Wang
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  7. Jun Liu
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Contributions

JZ, JC, JL and JxL conceived the study and designed the experiments. JZ, JC and LL performed the experiments. JjL and DL analyzed the data with suggestions by JL and JxL. JW provided in material. JZ and JL wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jun Liu or Jianxiu Liu.

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The present research work did not involve human participants and/or animals.

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Zong, J., Chen, J., Li, L. et al. Reference gene selection for quantitative RT-PCR in Miscanthus sacchariflorus under abiotic stress conditions. Mol Biol Rep 49, 907–915 (2022). https://doi.org/10.1007/s11033-021-06902-z

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