Selection of reference genes for quantitative real-time PCR normalization in creeping bentgrass involved in four abiotic stresses.

KEY MESSAGE: This study identified stable reference genes for normalization of gene expression data in qRT-PCR analysis of leaf and root tissues in creeping bentgrass under four abiotic stresses. Examination of gene expression using quantitative real-time PCR (qRT-PCR) in plant responses to abiotic stresses can provide valuable information for stress-tolerance improvement. Selecting stable reference genes for qRT-PCR analysis is critically important. The objective of this study was to determine the stability of expression for eight candidate reference genes (ACT, EF1a, TUB, UPL7, GAPDH, PP2A, PEPKR1, and CACS) in two tissues (roots and leaves) of a perennial grass species under four abiotic stresses (salt, drought, cold, and heat) using four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). The results showed that (1) the combinations of CACS and UPL7 or PP2A and ACT were stably expressed in salt-treated roots or leaves; (2) the combinations of GAPDH and CACS or PP2A and PEPKR1 were stable in roots and leaves under drought stress; (3) CACS and PP2A exhibited stable expression in cold-treated roots and the combination of EF1a and UPL7 was also stable in cold-treated leaves; and (4) CACS and PP2A were the two most stable reference genes in heat-stressed roots and UPL7 combined with GAPDH and PP2A was stably expressed in heat-stressed leaves. The qRT-PCR analysis of a target gene, AsSAP expression patterns in response to salinity and drought stress, confirmed the reliability of those selected and stable reference genes. Identification of stable reference genes in creeping bentgrass will improve assay accuracy for selecting stress-tolerance genes and identifying molecular mechanisms conferring stress tolerance in this species.