Cloning and validation of reference genes for normalization of gene expression studies in pearl millet [Pennisetum glaucum (L.) R. Br.] by quantitative real-time PCR

Abstract

Cloning and validation of reference genes for normalization of gene expression studies in pearl millet [Pennisetum glaucum (L.) R. Br.] by quantitative real-time PCR Cloning and validation of reference genes for normalization of gene expression studies in pearl millet [Pennisetum glaucum (L.) R. Br.] by quantitative real-time PCR, (2015), AbstractTo facilitate gene expression studies in pearl millet (Pennisetum glaucum (L.) R. Br.), the key reference genes including ACP, ACT, TUB, CYP, EIF4A, GAPDH, MDH, PP2C, UBC and S24 were selected based on the available literature, and their expression stabilities were studied to determine their suitability for normalizing gene expression in pearl millet. Sequence information of the reference genes were obtained from the closely related species and cloned from pearl millet using homology based cloning strategy. Further, expression stabilities were validated for their accurate expression in different tissues, genotypes and abiotic stress treatments using three statistical algorithms including geNorm, NormFinder and RefFinder.Analysis showed that while the expression of EF-1α and EIF4A were most stable in different plant tissues, MDH and EIF4A were stable under different abiotic stress conditions. Amongst the different genotypes of pearl millet tested, while UBC and MDH genes exhibited most stable expression, MDH and ACP showed greater stability in all samples set. Interestingly, the widely used reference genes S24 and TUB were found to be least stable across all the tested samples.Pair-wise analysis showed that two reference genes were sufficient for proper normalization, except when analyzing the gene expression studies in all samples set. Results of this study can help in the selection of reference genes for quantitative real time PCR (qRT-PCR) normalization in pearl millet that will contribute towards more accurate and reliable quantification of transcripts in this important crop of the drylands.

Identification of Reference Genes for qRT‐PCR Gene Expression Studies during Seed Development and under Abiotic Stresses in Cyamopsis tetragonoloba

“…Hence, a normalization method is required for obtaining reliable and unbiased quantitative gene expression profiling of a target gene. (Reddy et al, 2015), and Apium graveolens L. (Li et al, 2016), have shown that the expression of the HKGs is not as stable as it was generally assumed. The reliability of the quantitative results of qRT-PCR depends on the selected reference gene(s).…”

“…(Chandna et al, 2012), Arachis hypogea L. (Reddy et al, 2013), Vitis vinifera L. (Borges et al, 2014), Pennisetum glaucum (L.) R. Br. (Reddy et al, 2015), and Apium graveolens L. (Li et al, 2016), have shown that the expression of the HKGs is not as stable as it was generally assumed. It is risky to randomly choose any HKG to serve as a reference gene for qRT-PCR.…”

Exploration of Genetic and Genomic Resources for Abiotic and Biotic Stress Tolerance in Pearl Millet

“…This transcriptome assembly also includes pearl millet ovule transcriptome data. Recently three studies have been published regarding identification and selection of suitable reference genes for assessment of gene expression through quantitative real-time (qRT)-PCR analysis under individual as well as multiple stress conditions (Saha and Blumwald, 2014; Reddy et al, 2015; Shivhare and Lata, 2016). …”

Evaluation of Endogenous Gene Validation: An Important Contrivance for Differential Gene Study in Pearl Millet

“…Analysis of genes by by qRT-PCR in pearl millet is restricted due to short of sequence information available till now, which can be provided as suitable reference genes. Therefore identification and selection of suitable reference gene remain to be a pre requisite for future gene expression studies in pearl millet and other allied species of millet (Reddy et al, 2015). With advancement and more focus on studies in pearl millet, few scientists are engrossed for screening endogenous genes in various biotic and abiotic stress condition.…”

“…EST database was used for retrieving of the sequences and degenerate primers were designed based on the homologous genes of other closely related plant species (Reddy et al, 2015). Like GenBank accession numbers of selected candidate reference genes from Arabidopsis and rice prior were used for study, also by searching locus IDs and/or GenBank accession numbers of foxtail millet (a close relative of pearlmillet) orthologous locus Ids from Phytozome v.9.0., Sequences (Saha and Blumwald, 2014;Shivhare and Lata, 2016 In conclusion, in this present paper, it is tried to show the importance of validating endogenous genes in pearl millet for further differential gene expression studies is must for precise and accurate data obtaining.…”