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.
Highlights
Sorghum is the fifth most important cereal crop that is widespread in the semi-arid regions of the world with an annual production of 65.5 Mt (FAO, 2011), possesses strong drought-tolerance traits and a high forage value
Primer pairs for 15 candidate reference genes were used for quantitative real-time PCR (qPCR) amplification of sorghum cDNA and yielded single PCR products of the expected sizes (Figure 1A), as well as single melting curve peaks (Figure 1B)
A total of 15 candidate reference genes were selected for qPCR normalization
Summary
Sorghum is the fifth most important cereal crop that is widespread in the semi-arid regions of the world with an annual production of 65.5 Mt (FAO, 2011), possesses strong drought-tolerance traits and a high forage value. Sorghum seeds are utilized for both human and animal feed, and cultivars are processed for syrup, sugar, and alcohol. Reference Genes for qRT-PCR in Sorghum bicolor potential as they can be valorized for second-generation biofuels to produce environment-friendly energy (Vermerris, 2011; Calviño and Messing, 2012). Sorghum is a diploid with a small genome (750 Mbp) and possesses an extraordinary germplasm diversity that greatly aids gene discovery and analysis through comparative and functional genomics, making it highly useful as model cereal for structural and functional genomic studies aimed at improving agronomically important traits (Paterson et al, 2009)
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