Selection of suitable reference genes for assessing gene expression in pearl millet under different abiotic stresses and their combinations.

Radha Shivhare,Charu Lata

doi:10.1038/srep23036

Abstract

Pearl millet [Pennisetum glaucum (L.) R. Br.] a widely used grain and forage crop, is grown in areas frequented with one or more abiotic stresses, has superior drought and heat tolerance and considered a model crop for stress tolerance studies. Selection of suitable reference genes for quantification of target stress-responsive gene expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of improved stress tolerance. For precise normalization of gene expression data in pearl millet, ten candidate reference genes were examined in various developmental tissues as well as under different individual abiotic stresses and their combinations at 1 h (early) and 24 h (late) of stress using geNorm, NormFinder and RefFinder algorithms. Our results revealed EF-1α and UBC-E2 as the best reference genes across all samples, the specificity of which was confirmed by assessing the relative expression of a PgAP2 like-ERF gene that suggested use of these two reference genes is sufficient for accurate transcript normalization under different stress conditions. To our knowledge this is the first report on validation of reference genes under different individual and multiple abiotic stresses in pearl millet. The study can further facilitate fastidious discovery of stress-tolerance genes in this important stress-tolerant crop.

Highlights

IntroductionQuantification of variable transcriptomes and analysing differential expression of stress responsive genes in diverse biological samples and experimental conditions are important functional genomic approaches to investigate the molecular basis of stress tolerance involving complex regulatory gene networks[20]
It has been revealed that simultaneous occurrence of more than one stress leads to huge complexity in plant responses, as the responses and adaptation to such combination of stresses are mostly influenced by diverse and at times by antagonistic signaling pathways that may interact and impede each other[6,7]
Lately focus of several stress biology research has been directed towards understanding the molecular mechanisms of plant responses to abiotic stresses and their combinations through transcriptomics or functional genomics approaches. qRT-PCR has emerged as a useful technique for validation of transcriptomics data owing to its precision, accuracy, convenience, speed and sensitivity[20]

Summary

Introduction

Quantification of variable transcriptomes and analysing differential expression of stress responsive genes in diverse biological samples and experimental conditions are important functional genomic approaches to investigate the molecular basis of stress tolerance involving complex regulatory gene networks[20]. Considering the inevitability of crop- as well as stress-specific internal control genes for accurate normalization of transcripts in qRT-PCR, the present study was carried out to evaluate the expression stability of 10 candidate reference genes in pearl millet subjected to individual or combination of abiotic stresses and or abscisic acid (ABA), a stress hormone at early (1h) and late (24h) stress durations as well as in different developmental tissues of two contrasting genotypes

Methods

Results

Conclusion