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Selection of suitable reference genes for qRT-PCR analysis of Begonia semperflorens under stress conditions

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Abstract

Begonia semperflorens (B. semperflorens), belonging to the family Begoniaceae, has now been widely cultivated worldwide and is famous for its ornamental plants with colourful flowers and distinctive leaves. The selection of appropriate internal reference genes is very important to accurately determine target gene expression via quantitative real-time PCR. However, internal reference gene selection has never been conducted in B. semperflorens. In this study, seven candidate reference genes of B. semperflorens, including 18S ribosomal RNA (Bs18S), pentatricopeptide repeat-containing protein (BsPPR), actin-related protein 5 isoform X2 (BsACT), DNAJ homologue subfamily C member 17 (BsDNAJ), glyceraldehyde-3-phosphate dehydrogenase (BsGAPDH), NAD-dependent malic enzyme 59 kDa isoform, mitochondria (BsNAD-ME), and peptidyl-prolyl cis–trans isomerase CYP26-2, chloroplast (BsCYP), which were obtained from our previous studies, were selected. The stabilities of these genes under stress conditions were analysed using geNorm and NormFinder. Validation of target gene expressions, including phenylalanine ammonia-lyase (BsPAL) and respiratory burst oxidase homologue D (BsRBOHD) under biotic and abiotic conditions, phenylalanine ammonia-lyase (BsPAL), anthocyanidin synthase (BsANS), chalcone synthase (BsCHS), and flavanone-3-hydroxylase (BsF3H) under low temperature, using these seven internal reference genes for normalisation further confirmed the stabilities of the selected genes and indicated the need for reference gene selection for normalising gene expressions in B. semperflorens. Of the seven candidate reference genes, the combination of BsACT, BsDNAJ, and BsNAD-ME was the ideal reference gene set for normalising gene expression in samples under biotic conditions. BsCYP combined with BsACT or BsGAPDH was the best reference gene pair under abiotic conditions. BsACT and BsPPR could be combined to normalise gene expression under low temperature. Our results will benefit future studies on gene expression in plants of Begoniaceae.

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Funding

This research was supported by the Natural Science Foundation of Henan Province (Grant No. 182300410091) and the Key Scientific Research Project of High Education in Henan Province (Grant No. 18B220004).

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Author notes

  1. Yanman Li and Ying Qu have contributed equally to this work.

Authors and Affiliations

  1. College of Forestry, Henan Agricultural University, 63 Nongye Road, Zhengzhou, 450002, China

    Ying Qu, Yang Wang, Xue Bai, Geng Tian, Zhirou Liu, Yonghua Li & Kaiming Zhang

  2. College of Horticulture, Henan Agricultural University, 63 Nongye Road, Zhengzhou, 450002, China

    Yanman Li

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  1. Yanman Li
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Contributions

KMZ planned the research. YQ and GT performed the experiments. YW, RQ, XB, and YHL analysed and discussed the data. YML and KMZ wrote the article.

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Correspondence to Kaiming Zhang.

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The authors declare that they all contribute significantly to the work and have no conflicts of interest. The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Additionally, neither the manuscript nor any part of its content has been published or submitted for publication elsewhere.

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Li, Y., Qu, Y., Wang, Y. et al. Selection of suitable reference genes for qRT-PCR analysis of Begonia semperflorens under stress conditions. Mol Biol Rep 46, 6027–6037 (2019). https://doi.org/10.1007/s11033-019-05038-5

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