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Selection of low-variance expressed Malus x domestica (apple) genes for use as quantitative PCR reference genes (housekeepers)

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Abstract

To accurately measure gene expression using PCR-based approaches, there is the need for reference genes that have low variance in expression (housekeeping genes) to normalise the data for RNA quantity and quality. For non-model species such as Malus x domestica (apples), previously, the selection of reference genes relied on using homology to reference genes in model species. In this study, a genomics approach was used to identify apple genes with low variance in expression in 217 messenger RNA (mRNA)-seq data sets covering different tissues, during fruit development, and treated with a range of different stress conditions. Ten potential reference genes were chosen for validation by quantitative PCR (qPCR) over 29 different tissue types and treatments. From the combined mRNA-seq and qPCR results, three potential reference genes are proposed that can be used as good controls for PCR based expression studies. The three genes show homology to lipid transfer proteins, phytochrome protein phosphatase and the ubiquitination pathway. With the progression of research away from non-model species, this approach provides a robust method for selecting candidate genes for use as reference genes in qPCR.

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Acknowledgments

We would like to thank Niels Nieuwenhuizen for critically reading this manuscript and acknowledge New Zealand MBIE (Pipfruit, ‘a juicy future’, contract ID C06X0705) and the US USDA:SCRI (Postharvest Toolbox) for funding this work.

Conflict of interest

The authors declare that they have no conflict of interest.

Data archiving statement

To generate the widest possible set for analysis, the data presented in this experiment are from a diverse range of independent experiments, these include data published in (Foster et al. 2013; Schaffer et al. 2013). The rest will be published and archived elsewhere.

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Authors and Affiliations

  1. The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, 1142, New Zealand

    Judith Bowen, Hilary S. Ireland, Ross Crowhurst, Zhiwei Luo & Robert J. Schaffer

  2. The New Zealand Institute for Plant & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand

    Amy E. Watson & Toshi Foster

  3. Department of Horticulture, Cornell University, Ithaca, NY, USA

    Nigel Gapper & Chris Watkins

  4. The Boyce Thompson Institute for Plant Research, Cornell University Campus, Ithaca, NY, USA

    Nigel Gapper & Jim J. Giovanonni

  5. United States Department of Agriculture – Agricultural Research Service, Tree Fruit Research Laboratory, Wenatchee, WA, USA

    James P. Mattheis & David Rudell

  6. The New Zealand Institute for Plant & Food Research Limited, Private Bag 1401, Havelock North, 4157, New Zealand

    Jason W. Johnston

  7. School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand

    Robert J. Schaffer

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  1. Judith Bowen
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  2. Hilary S. Ireland
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  6. Toshi Foster
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  7. Nigel Gapper
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  8. Jim J. Giovanonni
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  10. Chris Watkins
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  11. David Rudell
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  12. Jason W. Johnston
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  13. Robert J. Schaffer
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Corresponding author

Correspondence to Robert J. Schaffer.

Additional information

Communicated by D. Chagné

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Fig. 1

Genious alignment for primers figure (PPTX 351 kb)

Fig. 2

mRNA-seq vs qPCR CV(%) (PPTX 60 kb)

Fig. 3

Pairwise variation (Vn/n + 1) to determine the number of genes required for accurate normalisation from geNorm (PPTX 38 kb)

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Bowen, J., Ireland, H.S., Crowhurst, R. et al. Selection of low-variance expressed Malus x domestica (apple) genes for use as quantitative PCR reference genes (housekeepers). Tree Genetics & Genomes 10, 751–759 (2014). https://doi.org/10.1007/s11295-014-0720-6

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  • DOI: https://doi.org/10.1007/s11295-014-0720-6

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