Abstract
Pearl millet [Pennisetum glaucum (L.) R.Br.], a close relative of Panicoideae food crops and bioenergy grasses, offers an ideal system to perform functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) provides a sensitive platform to conduct such gene expression analyses. However, the lack of suitable internal control reference genes for accurate transcript normalization during qRT-PCR analysis in pearl millet is the major limitation. Here, we conducted a comprehensive assessment of 18 reference genes on 234 samples which included an array of different developmental tissues, hormone treatments and abiotic stress conditions from three genotypes to determine appropriate reference genes for accurate normalization of qRT-PCR data. Analyses of Ct values using Stability Index, BestKeeper, ΔCt, Normfinder, geNorm and RefFinder programs ranked PP2A, TIP41, UBC2, UBQ5 and ACT as the most reliable reference genes for accurate transcript normalization under different experimental conditions. Furthermore, we validated the specificity of these genes for precise quantification of relative gene expression and provided evidence that a combination of the best reference genes are required to obtain optimal expression patterns for both endogeneous genes as well as transgenes in pearl millet.
Highlights
Increasing global population has raised the need of both food and fuel production
Identification of candidate reference genes We found locus identifiers and/or GenBank accession numbers of selected Arabidopsis and rice candidate reference genes from previous published work (Table 1)
We identified orthologous locus IDs and/or GenBank accession numbers of these potential candidate reference genes from foxtail millet, a close relative of pearl millet, using orthologous group search in Phytozome and/or BLASTN search in National Center for Biotechnology Information (NCBI) GenBank
Summary
Increasing global population has raised the need of both food and fuel production. The growing use of fossil fuel is contributing to global climate changes due to elevated greenhouse gas emission. Br., formerly P. americanum] is an excellent food and forage crop of arid to semiarid regions of the world [1,2] and a close relative of Panicoideae bioenergy grasses like switchgrass and foxtail millet [3]. Thereby, pearl millet provides an ideal crop for functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Several genetic engineering studies have been conducted in pearl millet [5,6], functional genomic studies under abiotic stress conditions are scanty [7]
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