4.7 Article

Comprehensive evaluation of candidate reference genes for quantitative real-time PCR-based analysis in Caucasian clover

Journal

SCIENTIFIC REPORTS
Volume 11, Issue 1, Pages -

Publisher

NATURE RESEARCH
DOI: 10.1038/s41598-021-82633-2

Keywords

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Funding

  1. National Natural Science Foundation of China [31802120]
  2. Research and Demonstration of Large-scale Artificial Grassland Combined Plant and Circular Mode [2017YFD0502106]
  3. Academic Backbone Fund Project of Northeast Agricultural University

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Twelve reference genes were selected based on transcriptomic data for stable expression analysis in various organs of Caucasian clover under different stress conditions. Results showed that different reference genes were most stable under different stress conditions, with RCD1 and NLI2 identified as the most stable reference genes across all organs.
The forage species Caucasian clover (Trifolium ambiguum M. Bieb.), a groundcover plant, is resistant to both cold and drought. However, reference genes for qRT-PCR-based analysis of Caucasian clover are lacking. In this study, 12 reference genes were selected on the basis of transcriptomic data. These genes were used to determine the most stably expressed genes in various organs of Caucasian clover under cold, salt and drought stress for qRT-PCR-based analysis. Reference gene stability was analyzed by geNorm, NormFinder, BestKeeper, the Ct method and RefFinder. Under salt stress, RCD1 and PPIL3 were the most stable reference genes in the leaves, and NLI1 and RCD1 were the most stable references genes in the roots. Under low-temperature stress, APA and EFTu-GTP were the most stable reference genes in the leaves, and the RCD1 and NLI2 genes were highly stable in the roots. Under 10% PEG-6000 stress, NLI1 and NLI2 were highly stable in the leaves, and RCD1 and PPIL3 were the most stable in the roots. Overall, RCD1 and NLI2 were the most stable reference genes in organs under normal conditions and across all samples. The most and least stable reference genes were validated by assessing their appropriateness for normalization via WRKY genes.

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