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Elymus sibiricus , which is a perennial and self-pollinated grass, is the typical species of the genus  Elymus , which plays an important role in forage production and ecological restoration. No reports have, so far, systematically described the selection of optimal reference genes for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis in  E. sibiricus . The goals of this study were to evaluate the expression stability of 13 candidate reference genes in different experimental conditions, and to determine the appropriate reference genes for gene expression analysis in  E. sibiricus . Five methods including Delta Ct (ΔCt), BestKeeper, NormFinder, geNorm, and RefFinder were used to assess the expression stability of 13 potential reference genes. The results of the RefFinder analysis showed that  TBP2  and  HIS3  were the most stable reference genes in different genotypes.  TUA2  and  PP2A  had the most stable expression in different developmental stages.  TBP2  and  PP2A  were suitable reference genes in different tissues. Under salt stress,  ACT2  and  TBP2  were identified as the most stable reference genes.  ACT2  and  TUA2  showed the most stability under heat stress. For cold stress,  PP2A  and  ACT2  presented the highest degree of expression stability.  DNAJ  and  U2AF  were considered as the most stable reference genes under osmotic stress. The optimal reference genes were selected to investigate the expression pattern of target gene  CSLE6  in different conditions. This study provides suitable reference genes for further gene expression analysis using RT-qPCR in  E. sibiricus .

genes

Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions