Superior cross-species reference genes: a blueberry case study

Authors: DIE, JOSE - Consejo Superior De Investigaciones Cientificas (CSIC); Rowland, Lisa

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/18/2013
Publication Date: 9/18/2013
Publication URL: http://handle.nal.usda.gov/10113/58535
Citation: Die, J.V., Rowland, L.J. 2013. Superior cross-species reference genes: a blueberry case study. PLoS One. 8:9 e73354.

Interpretive Summary: Blueberry is an important fruit crop because of its high nutritional value and it is one of the major berry crops grown in the United States. There is interest in characterizing genes related to fruit quality and nutritive value that are turned on during fruit development. There is also interest in studying genes that are activated during flower bud development and the ability of developing buds to survive winter temperatures. In order to reliably assess changes in activity of genes that are turned on and off, genes whose expression levels are stable and do not change during fruit and flower bud development and thus can be used as reference points, must be identified. Here we report on the identification of several genes whose expression levels are stable and are useful to assess changes in expression of genes that are turned on and off in fruits and flowers of blueberry. This collection of reference genes will be a valuable resource for scientists attempting to identify genes that play important roles in flower bud development, winter hardiness, fruit ripening, and fruit quality in blueberry and related species.

Technical Abstract: The advent of affordable Next Generation Sequencing technologies has had major impact on studies of many crop species, where access to genomic technologies and genome-scale data sets has been extremely limited until now. The recent development of genomic resources in blueberry will enable the application of high throughput gene expression approaches that should relatively quickly increase our understanding of blueberry physiology. These studies, however, require a highly accurate and robust workflow and make necessary the identification of reference genes with high expression stability for correct target gene normalization. To create a set of superior reference genes for blueberry expression analyses, we mined a publicly available transcriptome data set from blueberry for orthologs to a set of Arabidopsis genes that showed the most stable expression in a developmental series. In total, the expression stability of 13 putative reference genes was evaluated by qPCR and a set of new references with high stability values across a developmental series in fruits and floral buds of blueberry were identified. We also demonstrated the need to use at least two, preferably three, reference genes to avoid inconsistencies in results, even when superior reference genes are used. The new references identified here provide a valuable resource for accurate normalization of gene expression in Vaccinium spp. and may be useful for other members of the Ericaceae family as well.