Selection of Expression Reference Genes with Demonstrated Stability in Barley among a Diverse Set of Tissues and Cultivars

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 without validation in the studied species. For barley ( Hordeum vulgare L.), previous reference gene validation studies have been limited by the number of tissues, cultivars, and/or reference gene candidates assayed. Publically available bioinformatic resources are now available that enable efficient assessment of much larger numbers of candidate genes than is possible with empirical approaches. Expression sequence tag profile viewer data from the UniGene library were used to predict the expression stability of 655 barley genes. The expression profiles of 20 gene candidates predicted to be stable across tissue types were evaluated in eight tissues of the cultivar ‘Conlon’ by real‐time quantitative polymerase chain reaction. 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 ( Avena sativa L.) cultivar ‘Hifi’. The traditional reference gene actin and the candidate genes elongation factor EF‐2 and malate dehydrogenase demonstrated the most stable expression. The predictive capacity of bioinformatics to identify suitable reference genes was demonstrated. Four genes with better stability than all except one traditionally used reference gene were discovered.