|GINES, MICHAEL - Brigham Young University|
|JELLEN, ERIC - Brigham Young University|
|MAUGHAN, JEFF - Brigham Young University|
|Esvelt Klos, Kathy|
Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/2017
Publication Date: 1/1/2018
Citation: Gines, M.C., Baldwin, T.T., Rashid, A., Bregitzer, P.P., Jellen, E., Maughan, J., Esvelt Klos, K.L. 2018. In silico selection of expression reference genes with demonstrated stability in barley among a diverse set of tissues and cultivars. Crop Science. 58(1):332-341. https://doi.org/10.2135/cropsci2017.07.0443.
Interpretive Summary: An objective of crop research is to find those lines that best meet specific end-use needs. This is often associated with gene expression activity. Measuring gene expression helps to explain what makes one barley variety better than another. In this study, we identified tools to better measure gene expression activity in barley. Reference genes are genes whose proteins occur at relatively similar levels throughout all parts of an organism. Because their expression is stable, they can be used to measure the expression of other genes that experience greater changes. We used public gene expression data to make a list of genes that are expressed in every barley tissue. We then measured gene expression directly in different barley tissues. By comparing the transcript measurements across all of the barley tissues, we ranked the reference gene candidates by their stability. Our study returned three reference genes which performed optimally: Actin, malate dehydrogenase, and elongation factor 2. These genes were most stable overall and are recommended for future gene expression studies.
Technical Abstract: Premise of the study: Reference genes are selected based on the assumption of temporal and spatial expression stability and on their widespread use in model species. They are often used in new target species without validation, presumed as stable. For barley, reference gene validation is lacking, but publicly-available bioinformatic resources are available to predict the expression stability of experimental candidates. Methods: EST profile viewer data from the UniGene library were used to estimate the expression stability of 655 barley genes. Twenty gene candidates predicted as most-stable were evaluated in the barley cultivar ‘Conlon’ by qRT-PCR across eight tissues. The five most-stable genes were then tested in the barley cultivars ‘Golden Promise’ and ‘Harrington,’ and (to test potential applicability to other Poaceae species) in the oat cultivar ‘HiFi.’ Results: The traditional candidate actin (Hv.23088) and novel candidates elongation factor 2 (Hv.9509) and malate dehydrogenase (Hv.22901) demonstrated the most stable expression across barley and oat. Discussion: The predictive capacity of bioinformatics to identify suitable reference genes was demonstrated. Several novel reference genes were found to have similar stability to the top candidate, actin. These reference genes are recommended for barley under normal conditions and should be validated under experimental conditions if used.