Submitted to: Tree Genetics and Genomes
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/7/2014
Publication Date: 3/26/2014
Citation: Bowen, J., Ireland, H.S., Crowhurst, R., Luo, Z., Schaffer, R.J., Watson, A.E., Foster, T., Gapper, N., Watkins, C., Giovannoni, J.J., Mattheis, J.P., Rudell Jr, D.R., Johnston, J.W. 2014. Selection of low-variance expressed Malus x domestica (apple)genes for use as quantitative PCR reference genes (housekeepers). Tree Genetics and Genomes. 10:751-759. Interpretive Summary: Quantitative polymerase chain reaction (qPCR) is a common means of quantifying gene expression which are some of the governing components of metabolism. Standardization of gene expression values across a single or multiple experiments can be difficult leading to inaccurate and imprecise data and erroneous experimental interpretation using this method. To standardize gene expression readings, genes that are expected to be expressed the same across the conditions of the experiment are used as references. Genes with similar expression across multiple conditions and tissues in apples were previously unknown. This experiment looked at multiple large experimental data sets from apple tissues generated using RNAseq, an untargeted gene expression profiling protocol to discover genes whose expression levels are most equal in every instance. This provided a novel list of genes that provide the best means to standardize qPCR protocols in apple.
Technical 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.