Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments.

Huihui Zhuang,Yahui Wei,Wei He,Lin Wang,Yanping Fu

doi:10.3389/fpls.2015.00475

Abstract

Background: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala.Results: We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA), actin2/7 (ACT7), β-actin (ACTB), actin101 (ACT101), actin11 (ACT11), β-tubulin (TUB), α-tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1), GAPDH2, metallothionein-like protein (MET), fructose-bisphosphate aldolase (FBA) and histone H3 (HIS), from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7, and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable.Conclusions: The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation.

Highlights

Oxytropis ochrocephala Bunge (Fabaceae), a perennial grass species, grows aggressively on the grassland of northwestern China
Identification of Putative Reference Genes Based on Transcriptome Datasets Based on the FPKM value, we selected 12 genes with low coefficient of variation (CV) values (18S, ACT101, ACTB, ACT7, ACT11, GAPDH1, GAPDH2, TUB, TUA, MET, FBA, and HIS) and these genes have been reported as stable genes in other species
The sequences of the 12 reference genes were obtained from the transcriptome data

Summary

Introduction

Oxytropis ochrocephala Bunge (Fabaceae), a perennial grass species, grows aggressively on the grassland of northwestern China. It is the most widespread locoweed (the collective name for toxic Oxytropis and Astragalus species) in China. In order to better understand the role of the plant in production of swainsonine, the research on its stress response mechanism to harsh environment conditions is needed. Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala

Objectives

Methods

Results

Conclusion