Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization

Reddy, P S and Reddy, D S and Sivasakthi, K and Bhatnagar-Mathur, P and Vadez, V and Sharma, K K (2016) Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization. Frontiers in Plant Science, 7 (529). pp. 1-14. ISSN 1664-462X

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Abstract

Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.

Item Type: Article
Divisions: Research Program : Genetic Gains
CRP: CGIAR Research Program on Dryland Cereals
Uncontrolled Keywords: Quantitative Real-TimeRT-PCR(qPCR), qPCR, RefFinder, Sorghum bicolor, gene expression stability, reference gene, normalization, gene studies
Subjects: Mandate crops > Sorghum
Depositing User: Mr Ramesh K
Date Deposited: 11 May 2017 05:54
Last Modified: 11 May 2017 05:54
URI: http://oar.icrisat.org/id/eprint/10001
Official URL: http://dx.doi.org/10.3389/fpls.2016.00529
Projects: UNSPECIFIED
Funders: INSPIRE Faculty Award, Department of Science and Technology, Government of India
Acknowledgement: Financial support to PS through the INSPIRE Faculty Award (IFA11-LSPA-06) and the Young Scientist Scheme SB/YS/LS-12/2013 provided by the Department of Science and Technology, Government of India, New Delhi is gratefully acknowledged. This work was performed as part of the CGIAR Research Program on Dryland Cereals.ICRISAT is a member of the CGIAR Consortium.
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