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Genome-wide identification and validation of optimal reference genes for gene expression normalization in pear peel

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Abstract

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the preferred method for gene expression research, but normalization based on suitable reference genes (RGs) is the key to obtaining reliable gene expression results. In this study, we selected six “commonly used” RGs, two “traditional” housekeeping genes (HKGs), and four novel genes as candidate RGs based on 54 publicly available peel transcriptome libraries that include data from development, bagging, and post-harvest cryopreservation studies of four pear cultivars. The results of this multifaceted assessment from transcriptome and qRT-PCR analyses consistently revealed that ACT6/7/8/9 had the best expression stability among all candidate RGs, and expression of the novel RGs showed greater stability compared with the other “commonly used” RGs and “traditional” HKGs. Among the candidate RGs, ACT6/7/8/9 and Nucleosome Assembly Protein 1 (NAP1) showed superior expression stability and abundance, and they were recommended as the optimal RG combination for gene expression normalization in pear peel. These genome-wide findings provide more reasonable RG usage specifications, and additional useful and reliable RGs as resources for accurate study of gene expression in pear peel studies of different cultivars.

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Data availability

Genomic and publicly available annotated protein databases for pear (P. bretschneideri) were downloaded from the pear genome project (https://peargenome.njau.edu.cn/). The Arabidopsis data were downloaded from the Arabidopsis Information Resource (https://www.arabidopsis.org/). The gene identity information is listed in the supplemental material (Table S2).

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Acknowledgements

This work was supported by Natural Science Foundation of Fujian Province, China (2018J01702); the National Natural Science Foundation of China (31801814); the Fundamental Research Funds for the Fujian Universities (JK2017017); and the Central Government Guides Local Science and Technology Development Projects (2017L3001). We thank Mallory Eckstut, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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  1. Changmei Chen, Tengyun Wang and Zhenmei Cai are Equal contributors.

Authors and Affiliations

  1. College of Horticulture, Fujian Agriculture and Forestry University, No. 15 Shangxiadian Road, Fuzhou, 350002, China

    Changmei Chen, Tengyun Wang, Zhenmei Cai, Guifeng Xie, Zhiyi Chen, Wei Wang, Qian Xie, Xiayu Guan, Qingxi Chen & Jianqing Chen

  2. Department of Horticulture and Landscape Architecture, Fujian Vocational College of Agriculture, Fuzhou, 350119, China

    Yafang Yuan

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  1. Changmei Chen
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Contributions

CJQ, CQX, and CCM conceived and designed the experiments, analyzed the data, and wrote the manuscript; CCM, WTY, and CZM performed the experiments; XGF and CZY contributed plant materials; YYF and WW contributed analytical tools; XQ and GXY helped analyze data; CJQ revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Qingxi Chen or Jianqing Chen.

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Chen, C., Wang, T., Cai, Z. et al. Genome-wide identification and validation of optimal reference genes for gene expression normalization in pear peel. Plant Growth Regul 92, 249–261 (2020). https://doi.org/10.1007/s10725-020-00634-z

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