Abstract
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.
Highlights
IntroductionThe dense rhizome system of Caucasian clover can store large amounts of metabolic energy that enables the perennial persistence and rapid reestablishment of this s pecies[2], and a unique characteristic of this species is the formation of daughter plants from the rhizomes that develop from the taproot
The cycle threshold (Ct) values of all the candidate reference genes are shown in Supplementary Table S1 and Fig. 2, and the expression levels of the candidate reference genes were measured
The variable Ct values of all the candidate reference genes among the different treatment conditions demonstrated that the expression levels varied on the basis of the conditions and experimental treatments
Summary
The dense rhizome system of Caucasian clover can store large amounts of metabolic energy that enables the perennial persistence and rapid reestablishment of this s pecies[2], and a unique characteristic of this species is the formation of daughter plants from the rhizomes that develop from the taproot. This characteristic confers an advantage to Caucasian clover under drought conditions[3]. There are no reports in the literature on the evaluation of candidate reference genes for qRT-PCR in Caucasian clover. The results of this study will advance research on Caucasian clover
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