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

Abstract

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.

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

“…Moreover, high stability levels were observed using geNorm analysis; all the M values were below the accepted threshold of 1.5 (Fig 2), which indicated that all the genes could be used as internal control genes for normalization. However, according to different algorithms and analytical procedures, several differences were observed in the ranking of the most stable genes, which was similar to the results for other species [19–21]. For wounding treatment in S .…”

“…Traditional reference genes, such as ACT , GAPDH , and EF1α , which are involved in primary metabolism or other cellular processes [19], as well as new candidate reference genes derived from public databases [12, 17, 22, 25, 26], have been validated to allow precise interpretation of the qRT-PCR results for specific experimental conditions. However, fluctuating expression of reference genes has been observed under certain experimental conditions.…”

Reference Gene Selection for qRT-PCR Normalization Analysis in kenaf (Hibiscus cannabinus L.) under Abiotic Stress and Hormonal Stimuli

“…For example, PP2A expressed stably in Pennisetum glaucum under different abiotic stresses and hormonal stimuli (Saha and Blumwald, 2014; Shivhare and Lata, 2016), Caragana intermedia under osmotic and heat stress (Zhu et al, 2013), Agrostis stolonifera under different abiotic stresses (Chen et al, 2015), Brassica napus under drought, salinity and JA stresses (Wang et al, 2014b), Cynodon dactylon under different abiotic stresses (Chen et al, 2014). ACT7 , one of the best reference genes in this study, was also stably expressed under different abiotic stresses and hormonal stimuli of Daucus carota (Tian et al, 2015), JA and ABA stresses of Brassica napus (Wang et al, 2014b), drought stress of Populus euphratica (Wang et al, 2014a), and Agrostis stolonifera (Chen et al, 2015). Another stable gene is PTB , a novel reference gene identified in A. thaliana , has been identified as one of the best reference for Gossypium hirsutum (Artico et al, 2010) under abiotic stress.…”

Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis

“…Thus, it is necessary to validate the stability of the reference gene under determined experimental condition to ensure proper normalization and a robust RT-qPCR analysis14. Several studies have been published with the aim of identifying suitable reference genes for expression analysis under different stages of the plant life cycle15161718 or under biotic and abiotic stresses19202122.…”