Abstract
BackgroundWild peanut species (Arachis spp.) are a rich source of new alleles for peanut improvement. Plant transcriptome analysis under specific experimental conditions helps the understanding of cellular processes related, for instance, to development, stress response, and crop yield. The validation of these studies has been generally accomplished by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) which requires normalization of mRNA levels among samples. This can be achieved by comparing the expression ratio between a gene of interest and a reference gene which is constitutively expressed. Nowadays there is a lack of appropriate reference genes for both wild and cultivated Arachis. The identification of such genes would allow a consistent analysis of qRT-PCR data and speed up candidate gene validation in peanut.ResultsA set of ten reference genes were analyzed in four Arachis species (A. magna; A. duranensis; A. stenosperma and A. hypogaea) subjected to biotic (root-knot nematode and leaf spot fungus) and abiotic (drought) stresses, in two distinct plant organs (roots and leaves). By the use of three programs (GeNorm, NormFinder and BestKeeper) and taking into account the entire dataset, five of these ten genes, ACT1 (actin depolymerizing factor-like protein), UBI1 (polyubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L10) and UBI2 (ubiquitin/ribosomal protein S27a) emerged as top reference genes, with their stability varying in eight subsets. The former three genes were the most stable across all species, organs and treatments studied.ConclusionsThis first in-depth study of reference genes validation in wild Arachis species will allow the use of specific combinations of secure and stable reference genes in qRT-PCR assays. The use of these appropriate references characterized here should improve the accuracy and reliability of gene expression analysis in both wild and cultivated Arachis and contribute for the better understanding of gene expression in, for instance, stress tolerance/resistance mechanisms in plants.
Highlights
Wild peanut species (Arachis spp.) are a rich source of new alleles for peanut improvement
RNA quality and cDNA synthesis A set of 24 pooled samples including two different tissues of four Arachis species submitted to three different stresses was used to analyze the expression stability of ten candidate genes for normalization of quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
The values of primer pair efficiencies were used in subsequent qRT-PCR analysis
Summary
Wild peanut species (Arachis spp.) are a rich source of new alleles for peanut improvement. Plant transcriptome analysis under specific experimental conditions helps the understanding of cellular processes related, for instance, to development, stress response, and crop yield The validation of these studies has been generally accomplished by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) which requires normalization of mRNA levels among samples. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is currently the most sensitive technique for quantification of low abundance transcripts, and at the same time is suitable for abundant transcripts For these reasons, and because of relative ease of use, qRTPCR has become widely preferred to classic transcriptome analysis tools, such as Northern blotting, semi-quantitative RT-PCR, micro and macroarrays, RNase protection analysis, and in situ hybridization [13,14]. The method continues to be improved, with recent developments enabling qRT-PCR reactions to be performed at lower reagents cost, less hands-on time and with higher throughput than previously possible [15,16]
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