Evaluation of Reference Genes for Quantitative PCR in Eustoma grandiflorum under Different Experimental Conditions

Abstract

Eustoma grandiflorum, commonly known as prairie gentian or Texas bluebells, is among the most popular agriculturally propagated species of cut flowers. Due to its widespread appeal, there is increasing interest in understanding the molecular genetic factors underlying floral development and resistance to abiotic stresses. We analyzed 18 potential reference genes in different organs, at different floral developmental stages and under drought- and salt-stress treatments, for use in RT-qPCR analysis. A total of four analytical tool packages, including geNorm, NormFinder, BestKeeper, and RefFinder were employed to determine the most appropriate reference genes under each treatment condition. The results demonstrate that different reference genes should be used for normalization under different experimental treatments. EgPP and EgPP2A2 were the most stable internal control genes across different organ types, EgPP and Eg18S were the most stable under salt-stress, EgPP and EgACT1 were the most stable across different floral development stages, and EgEF1A and EgTUA were the most stable reference genes under drought-stress. Additional gene expression analyses of EgMIXTA1, EgTOE1, and EgP5CS1 further confirmed the applicability of these reference genes. The results represent a significant contribution to future studies of reference gene selection for the normalization of gene expression in Eustoma grandiflorum.