Reference gene selection for real-time quantitative PCR normalization in Hemarthria compressa and Hemarthria altissima leaf tissue

Hemarthria compressa and Hemarthria altissima are widely used as livestock forage and play important roles in tropical and subtropical grassland agricultural systems promoting healthy ecological environment and the development of animal husbandry. Leaf tissue of "Yaan" limpograss (H. compressa) and "H255" whip grass (H. altissima) were used to test the mRNA expression levels of 12 reference genes using RT-qPCR. The Delta-Ct method, BestKeeper ( ver. 1.0), Genorm (ver. 3.5), Normfinder (ver. 0.953) and RefFinder were used to analyze the expression stability of the 12 reference genes under drought, salt, acid-aluminum and cold stresses to provide significant technical support for the study of gene expression under various abiotic stresses in Hemarthria. The results showed that the candidate reference genes showed divergent expression levels under various abiotic stresses. Among the genes that were selected, CL18892 showed the highest expression stability under salt stress in the leaf tissue. eEF-1a was the most stable gene under cold and acid-aluminum stresses and CL16384 was comparatively the most suitable genes under drought stress. As a whole, according to RefFinder analysis, CYP5, BMK.74327 and CL21527 were the most suitable reference genes for studying the effects of abiotic stress in Hemarthria. In general, CL16812 and CL18038 were not suitable reference genes under abiotic stress conditions that were examined in this study.