Dataset Information

Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L.) R. Br].

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, ?Ct, 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.

SUBMITTER: Saha P

PROVIDER: S-EPMC4149553 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L.) R. Br].

Saha Prasenjit P Blumwald Eduardo E

PloS one 20140829 8

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-PC ...[more]

PMID: 25170776

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Project description:Pearl millet is a staple food for people in arid and semi-arid regions of Africa and South Asia due to its high drought tolerance and nutritional qualities. A better understanding of the genomic diversity and population structure of pearl millet germplasm is needed to support germplasm conservation and genetic improvement of this crop. Here we characterized two pearl millet diversity panels, (i) a set of global accessions from Africa, Asia, and the America, and (ii) a collection of landraces from multiple agro-ecological zones in Senegal.We identified 83,875 single nucleotide polymorphisms (SNPs) in 500 pearl millet accessions, comprised of 252 global accessions and 248 Senegalese landraces, using genotyping by sequencing (GBS) of PstI-MspI reduced representation libraries. We used these SNPs to characterize genomic diversity and population structure among the accessions. The Senegalese landraces had the highest levels of genetic diversity (?), while accessions from southern Africa and Asia showed lower diversity levels. Principal component analyses and ancestry estimation indicated clear population structure between the Senegalese landraces and the global accessions, and among countries in the global accessions. In contrast, little population structure was observed across in the Senegalese landraces collections. We ordered SNPs on the pearl millet genetic map and observed much faster linkage disequilibrium (LD) decay in Senegalese landraces compared to global accessions. A comparison of pearl millet GBS linkage map with the foxtail millet (Setaria italica) and sorghum (Sorghum bicolor) genomes indicated extensive regions of synteny, as well as some large-scale rearrangements in the pearl millet lineage.We identified 83,875 SNPs as a genomic resource for pearl millet improvement. The high genetic diversity in Senegal relative to other regions of Africa and Asia supports a West African origin of this crop, followed by wide diffusion. The rapid LD decay and lack of confounding population structure along agro-ecological zones in Senegalese pearl millet will facilitate future association mapping studies. Comparative population genomics will provide insights into panicoid crop evolution and support improvement of these climate-resilient crops.

Project description:BACKGROUND: Pearl millet landraces display an important variation in their cycle duration. This diversity contributes to the stability of crop production in the Sahel despite inter-annual rainfall fluctuation. Conservation of phenological diversity is important for the future of pearl millet improvement and sustainable use. Identification of genes contributing to flowering time variation is therefore relevant. In this study we focused on three flowering candidate genes, PgHd3a, PgDwarf8 and PgPHYC. We tested for signatures of past selective events within polymorphism patterns of these three genes that could have been associated with pearl millet domestication and/or landraces differentiation. In order to implement ad hoc neutrality tests, a plausible demographic history of pearl millet domestication was inferred through Approximate Bayesian Computation by using eight neutral STS loci. RESULTS: Domesticated pearl millet exhibited 84% of the nucleotide diversity level found in the wild population. No specific polymorphisms were found either in the wild or in the domestic populations. The bayesian approach and previous studies suggest that gene flow between wild relatives and domesticated pearl millets is a main factor explaining these results. Early and late landraces did not show significant genetic differentiation at both the neutral and the candidate loci. A positive selection was evidenced in PgHd3a and PgDwarf8 genes of domestic forms but not in the wild population. CONCLUSION: Our results strongly suggest that PgHd3a and PgDwarf8 were likely targeted by selection during domestication. However, a potential role of any of the three candidate genes in the phenological differentiation between early and late landraces was not supported by our data. Reasons why these results contrast with previous results that have shown a slight but significant association between PgPHYC polymorphisms and variation in flowering time in pearl millet are discussed.

Dataset Information

Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L.) R. Br].

Publications

Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L.) R. Br].

Similar Datasets

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