Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions

Linkai Huang,Xinquan Zhang,Yu Zhang,Haidong Yan,Xiaomei Jiang,Yan Peng,Yanhong Yan,Xiao Qi,Guohua Yin,Xiao Ma,Robert Dettman

doi:10.1371/journal.pone.0093724

Abstract

BackgroundQuantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied.Methodology/Principal FindingsIn this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches – Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments.Conclusions/SignificanceThese results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies.

Highlights

Perennial ryegrass (Lolium perenne L.) is dominant forage and turf grass specie in temperate regions due to its good grazing tolerance, extraordinarily high digestibility and adequate seed production [1,2]
This and the single peak melting curves of the Quantitative real-time reverse-transcriptase PCR (qRT-PCR) products (Figure 1B) demonstrated that a single PCR amplification product of the expected size was obtained for each reference gene
The results showed that Transcription elongation factor 1 (TEF1) and UBQ were most stable in expression, and they were the most appropriate reference genes, under drought and waterlogging stresses, with M2 values of 0.568 and 0.700, respectively

Summary

Introduction

Perennial ryegrass (Lolium perenne L.) is dominant forage and turf grass specie in temperate regions due to its good grazing tolerance, extraordinarily high digestibility and adequate seed production [1,2]. Marginal lands are usually afflicted by abiotic stresses such as heat, cold, drought, waterlogging and high salinity, all of which may negatively impact yield, quality, and growth of perennial ryegrass. Perennial ryegrass is a drought-susceptible grass species [5] and its leaf extension and appearance rates are both reduced under drought stress [6]. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied

Methods

Results

Conclusion