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Identification of Suitable Reference Genes for Barley Gene Expression Under Abiotic Stresses and Hormonal Treatments

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Abstract

Barley (Hordeum vulgare L.) is an important cereal crop and also can be considered as a model plant for the Triticeae family. Quantitative real-time PCR (qRT-PCR) has emerged as a more practical technique to study barley gene expression, and the data normalization with reference genes is essential for accurate and reliable results. In this study, 17 candidate reference genes were evaluated from two distinct plant tissues (roots and leaves) at the barley seedling stage under different abiotic stresses (osmotic, salt, and heat) and hormonal treatments (gibberellin A3 (GA3) and methyl jasmonate (JA)). Our qRT-PCR data were analyzed with three commonly used software packages: geNorm, NormFider, and BestKeeper. In combination with the analysis of these three packages, four to five candidate genes were selected as suitable reference genes in each experimental set. These results confirmed that expression stability of reference genes depends on the experimental conditions. This study is the systematic analysis for the selection of superior reference genes for qRT-PCR in barley under different abiotic and hormonal treatments and will benefit further studies on gene expression in barley and other species of Triticeae family.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 31101149), Natural Science Foundation of Zhejiang Province (Grant No. Y3110574), Public Benefit Technology Applied Research Project of Zhejiang Province (Project No. 2012C22029), China Agriculture Research System (CARS-05), the Key Research Foundation of Science and Technology Department of Zhejiang Province of China (2012C12902-2), and Agro-scientific Research in the Public Interest (201303016).

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  1. Zhejiang Academy of Agricultural Science, Hangzhou, 310021, China

    Wei Hua, Jinghuan Zhu, Yi Shang, Junmei Wang, Qiaojun Jia & Jianming Yang

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  1. Wei Hua
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  2. Jinghuan Zhu
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  3. Yi Shang
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  4. Junmei Wang
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Correspondence to Jianming Yang.

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Fig. S1

Specificity of primer pairs for qRT-PCR amplification. (A) Melting curve of the 17 candidate reference genes showing a single peak. (B) Agarose gel (2%) showing amplification of a specific PCR product of the expected size for each gene tested in this study. M: Marker 1000. (JPEG 1,532 kb)

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Hua, W., Zhu, J., Shang, Y. et al. Identification of Suitable Reference Genes for Barley Gene Expression Under Abiotic Stresses and Hormonal Treatments. Plant Mol Biol Rep 33, 1002–1012 (2015). https://doi.org/10.1007/s11105-014-0807-0

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