Identification and Validation of Reference Genes for Quantification of Target Gene Expression with Quantitative Real-time PCR for Tall Fescue under Four Abiotic Stresses

Abstract

Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

“…For example, EF1α was identified as the best reference gene under salinity and drought stresses in Glycine max [36]. Nevertheless, EF1α exhibited poor performance in leaves and roots during abiotic stress treatments [15]. In the present study, we also found that the most suitable reference genes varied under different environmental stimuli.…”

Selection of reference genes for quantitative real‐time PCR in postharvest tomatoes (Lycopersicon esculentum) treated by continuous low‐voltage direct current electricity to increase secondary metabolites

“…Despite the above‐mentioned results, the candidate genes should not be considered as an ultimate reference gene set for all conditions because other reports have indicated that these genes were not suitable under some stress conditions in tomatoes (Lovdal & Lillo, ), tall fescue ( Festuca arundinacea Schreb.) (Yang et al ., ) and S. europaea (Xiao et al ., ). For instance, PP2Acs / RPL2 were noted to be the most stable reference genes in tomato leaves when considering the combined data set of nitrogen, cold and light stresses, but not when considering the data set of each stress (Lovdal & Lillo, ).…”

Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani

“…This program assigns an appropriate weight to tested genes and calculates the geometric mean of their weights using ranking information from the three programs. The comprehensive ranking by RefFinder made the selection of candidate reference genes reliable [41]. …”