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

www.nature.com/scientificreports OPEN received: 01 October 2015 accepted: 23 February 2016 Published: 14 March 2016 Selection of suitable reference genes for assessing gene expression in pearl millet under different abiotic stresses and their combinations Radha Shivhare1,* & Charu Lata1,2,* 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. Plants being sessile in nature are forced to thrive in adverse environmental conditions. Drought, salinity and temperature extremes (heat and cold) are major abiotic stresses that challenge production and productivity of crop plants1,2. The adverse effects of these stresses on crop plants are further compounded due to changing climate worldwide3. Further it has also been reported that levels of abscisic acid (ABA), a plant growth regulator, also increases under abiotic stresses4,5. Though individual stress conditions such as drought, salinity or heat have been the focus of intense research, not much study has been carried out on the combination of different abiotic stresses such as drought and heat, drought and salinity, salinity and heat etc. which frequently affect growth and yield potential of crops and other plants in field conditions across the globe6,7. There have been quite a few reports suggesting that response of plants to two or more different abiotic stresses is unique and quite distinct from the response of those exposed to individual stress8. Generally, more than one stress factors occurring simultaneously, interact with each other in additive manner and trigger more damage than when present individually9,10. Therefore it is necessary to focus on development of crops varieties that can endure multiple environmental stress factors. Since crop plants are subjected to one or more abiotic stresses concurrently under field conditions, it is thus necessary to make conscious efforts towards mimicking these conditions in laboratory as well7,9,11. Further an overlap in the expression patterns of stress responsive genes after drought, salt, heat, cold, or ABA treatments or after combinations of stresses has also been reported8,12. Subsequently several studies have been taken up recently to monitor the effects of abiotic stress combinations on crop plants as well as to elucidate the molecular mechanism(s) of stress tolerance8,12–15. 1 CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, India. 2National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi-110012, India. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to C.L. (email: or ) Scientific Reports | 6:23036 | DOI: 10.1038/srep23036 1 www.nature.com/scientificreports/ Gene name Description Accession no. Ct ± SD CV ± SD ACT Actin HM243500 26.4 ±  2.3 5.8 ±  0.3 EF-1α Elongation factor-1 alpha EF694165 23.4 ±  2.1 4.5 ±  0.2 eIF4A Eukaryotic initiation factor 4A EU856535 23.8 ±  3.1 6.6 ±  0.4 GlutR Glutaredoxin GD180652 21.9 ±  2.5 5.3 ±  0.2 HSP 90 Heat shock protein 90 ADP89125 22.8 ±  2.9 9.6 ±  0.9 MDH Malate dehydrogenase CD724779 22.2 ±  2.3 15.7 ±  2.6 PP2A Protein phosphatase 2A Si017892m 25.6 ±  2.3 5.1 ±  0.2 RPL20 Ribosomal protein L20 KJ490012 21.7 ±  3.4 6.5 ±  0.4 TIP41 Tonoplast intrinsic protein Si036884m 28.9 ±  2.5 6.7 ±  0.6 UBC-E2 Ubiquitin-conjugating enzyme E2 CD724586 23.2 ±  2.5 5.7 ±  0.3 Table 1. Details of Ct and CV values of each of the selected candidate reference genes tested in pearl millet. Pearl millet [Pennisetum glaucum (L.) R. Br.] is an important small-grained C4 panicoid crop grown for forage, grain and stover in the arid and semi-arid regions of Asia and Africa16. It is the sixth most important cereal crops after rice, wheat, maize, barley and sorghum with excellent nutrient composition17 and is also considered a potential biofuel grain feedstock2,18; http://ag.fvsu.edu/index.php/research/bioenergy/). It usually thrive in areas with scanty rainfall and is also well adapted to various abiotic stresses such as drought, high temperature, salinity etc. whether occurring individually or in combination making it an ideal crop for functional genomic studies to understand the molecular basis of abiotic stress tolerance and adaptation2,16,19. Only a limited amount of genome sequence information is available in pearl millet that greatly hinders gene discovery, functional validation, expression profiling, and ultimately crop improvement programs. 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 networks20. In this regard, qRT-PCR is a widely used technique to quantify transcriptional abundance of numerous individual genes owing to its high sensitivity, specificity and synchronized quantification of gene expression in diverse samples with a broad quantification range of up to several orders of magnitude in comparison to conventional techniques such as reverse transcription (RT)-PCR or northern hybridization20. However relative quantification of gene expression using qRT-PCR is highly influenced by the expression stability of internal control or reference genes used for transcript normalization of target genes. The use of inappropriate or unstable reference gene(s) can seriously impact the transcript quantification results leading to false inferences or misinterpretations21,22. Accurate normalization is thus necessary for obtaining biologically m (...truncated)