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Selection of suitable reference genes for abiotic stress-responsive gene expression studies in peanut by real-time quantitative PCR
He, Meijing; Cui, Shunli; Yang, Xinlei; Mu, Guojun; Chen, Huanying & Liu, Lifeng
Abstract
Background: Because of its strong specificity and high accuracy, real-time quantitative PCR (RT-qPCR) has been a
widely used method to study the expression of genes responsive to stress. It is crucial to have a suitable set of
reference genes to normalize target gene expression in peanut under different conditions using RT-qPCR. In
this study, 11 candidate reference genes were selected and examined under abiotic stresses (drought, salt,
heavy metal, and low temperature) and hormone (SA and ABA) conditions as well as across different organ
types. Three statistical algorithms (geNorm, NormFinder and BestKeeper) were used to evaluate the
expression stabilities of reference genes, and the comprehensive rankings of gene stability were generated.
Results: The results indicated that ELF1B and YLS8 were the most stable reference genes under PEG-simulated
drought treatment. For high-salt treatment using NaCl, YLS8 and GAPDH were the most stable genes. Under
CdCl2 treatment, UBI1 and YLS8 were suitable as stable reference genes. UBI1, ADH3, and ACTIN11 were
sufficient for gene expression normalization in low-temperature experiment. All the 11 candidate reference
genes showed relatively high stability under hormone treatments. For organs subset, UBI1, GAPDH, and ELF1B
showed the maximum stability. UBI1 and ADH3 were the top two genes that could be used reliably in all the
stress conditions assessed. Furthermore, the necessity of the reference genes screened was further confirmed
by the expression pattern of AnnAhs.
Conclusions: The results perfect the selection of stable reference genes for future gene expression studies in
peanut and provide a list of reference genes that may be used in the future.
Keywords
Abiotic stress; Drought stress; Evaluation; Expression analysis; Expression stability; Genes responsive to stress; Heavy metal; High salt; Higher-plants; Low temperature; Plant hormones
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