College of Animal Science and Technology, Northeast Agricultural University, No. 600 Changjiang Street, Xiangfang District, Harbin City, Heilongjiang, China.
Sci Rep. 2021 Feb 8;11(1):3269. doi: 10.1038/s41598-021-82633-2.
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.
饲料物种高加索三叶草（Trifolium ambiguum M. Bieb.），一种地被植物，对寒冷和干旱都有抗性。然而，用于高加索三叶草 qRT-PCR 分析的参考基因尚缺乏。在这项研究中，根据转录组数据选择了 12 个参考基因。这些基因用于确定高加索三叶草在冷、盐和干旱胁迫下各种器官中最稳定表达的基因，以便进行 qRT-PCR 分析。通过 geNorm、NormFinder、BestKeeper、∆Ct 法和 RefFinder 分析参考基因的稳定性。在盐胁迫下，RCD1 和 PPIL3 是叶片中最稳定的参考基因，而 NLI1 和 RCD1 是根中最稳定的参考基因。在低温胁迫下，APA 和 EFTu-GTP 是叶片中最稳定的参考基因，而 RCD1 和 NLI2 基因在根中高度稳定。在 10%PEG-6000 胁迫下，NLI1 和 NLI2 在叶片中高度稳定，而 RCD1 和 PPIL3 在根中最稳定。总体而言，RCD1 和 NLI2 是正常条件下器官和所有样本中最稳定的参考基因。通过评估 WRKY 基因对其归一化的适宜性，验证了最稳定和最不稳定的参考基因。
