Plant Breeding Institute, Christian-Albrechts-University of Kiel, Kiel, Germany.
King Abdullah University of Science and Technology (KAUST), Biological and Environmental Sciences & Engineering Division (BESE), Thuwal, Saudi Arabia.
PLoS One. 2021 Mar 11;16(3):e0233821. doi: 10.1371/journal.pone.0233821. eCollection 2021.
Quinoa depicts high nutritional quality and abiotic stress resistance, attracting strong interest in the last years. To unravel the function of candidate genes for agronomically relevant traits, studying their transcriptional activities by RT-qPCR is an important experimental approach. The accuracy of such experiments strongly depends on precise data normalization. To date, validation of potential candidate genes for normalization of diurnal expression studies has not been performed in C. quinoa. We selected eight candidate genes based on transcriptome data and literature survey, including conventionally used reference genes. We used three statistical algorithms (BestKeeper, geNorm and NormFinder) to test their stability and added further validation by a simulation-based strategy. We demonstrated that using different reference genes, including those top ranked by stability, causes significant differences among the resulting diurnal expression patterns. Our results show that isocitrate dehydrogenase enzyme (IDH-A) and polypyrimidine tract-binding protein (PTB) are suitable genes to normalize diurnal expression data of two different quinoa accessions. Moreover, we validated our reference genes by normalizing two known diurnally regulated genes, BTC1 and BBX19. The validated reference genes obtained in this study will improve the accuracy of RT-qPCR data normalization and facilitate gene expression studies in quinoa.
藜麦具有较高的营养价值和抗非生物胁迫特性，近年来引起了广泛关注。为了揭示与农艺性状相关的候选基因的功能，通过 RT-qPCR 研究其转录活性是一种重要的实验方法。此类实验的准确性在很大程度上取决于精确的数据归一化。迄今为止，尚未在 C. quinoa 中对昼夜表达研究的潜在候选基因进行归一化验证。我们基于转录组数据和文献综述选择了 8 个候选基因，包括常规使用的参考基因。我们使用了三种统计算法（BestKeeper、geNorm 和 NormFinder）来测试它们的稳定性，并通过基于模拟的策略进行了进一步验证。结果表明，使用不同的参考基因，包括那些稳定性排名较高的基因，会导致昼夜表达模式的显著差异。我们的结果表明，异柠檬酸脱氢酶酶（IDH-A）和多嘧啶 tract 结合蛋白（PTB）是两种不同藜麦品系昼夜表达数据归一化的合适基因。此外，我们还通过归一化两个已知的昼夜调控基因 BTC1 和 BBX19 验证了我们的参考基因。本研究中获得的经过验证的参考基因将提高 RT-qPCR 数据归一化的准确性，并促进藜麦基因表达研究。
