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Background  Real-time quantitative PCR has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. Wild barley ( Hordeum brevisubulatum  (Trin.) Link) is a wild relative species in Triticeae that has strong tolerance to abiotic stresses and extremely wide adaptation. However, suitable references gene have not been documented for standardization of gene expression in wild barley under abiotic stress.    Results  Here we report the first systematic and comprehensive analysis of reference genes for quantitative real-time PCR standardization in wild barley. We selected 11 genes, including  ACT  ( Actin ),  ADP  ( ADP - ribosylation factor 1 ),  CYP2  ( Cyclophilin 2 ),  EF - 1α  ( Elongation factor 1 - alpha ),  GAPDH  ( Glyceraldehyde 3 - phosphate dehydrogenase ),  HSP90  ( Heat shock protein 90 ),  TUBα  ( Alpha - tubulin ),  TUBβ6  ( Beta - tubulin 6 ),  UBI  ( Ubiquitin ),  18SrRNA - 1  ( guanine1575 - N7 - methyltransferase ) and  18SrRNA - 3  ( adenine1779 - N6 - dimethyltransferase ) from a wild barley transcriptome database and analyzed their expression stabilities in shoots and roots of wild barley seedling under various stress conditions using comparative ΔCt, BestKeeper, Normfinder and geNorm software. The results demonstrated that  ADP  was the most suitable reference gene in salt stress while  UBI  showed peak stability under mannitol and ABA stress;  EF - 1α  was the most appropriate reference gene for PEG, GA 3 , ethylene and heat stress;  18SrRNA - 3  was the best choice for cold stress; and  TUBα  was the first stable gene across different tissues.    Conclusions  Our main contribution was to identify reference genes with suitable and stable expression in wild barley under various stress conditions and in different tissues to provide a useful resource for future studies. The results demonstrate the importance of transcriptome data as a useful resource for the screening of candidate reference genes and highlight the need for specific reference genes for specific conditions. Furthermore, these findings will provide valuable information for wild barley and relative species for future research.    Electronic supplementary material  The online version of this article (10.1186/s13007-018-0379-3) contains supplementary material, which is available to authorized users.

Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum