Key message

This study proposed the combination of PR26S and PP1 as a good choice for RT-qPCR normalization in pecan under abiotic stress, developing kernels, grafting, and various tissues.

Abstract

Reference gene selection is an essential pre-requisite to generate reliable results in real-time quantitative polymerase chain reaction (RT-qPCR) analysis. However, studies regarding systematic validation of suitable reference genes in pecan are still lacking. In this study, 17 candidate reference genes were selected and evaluated for their expression stabilities in pecan under various experimental conditions, including various tissues, developing kernels, grafting, and two plant tissues (leaves and roots) subjected to three abiotic stresses (salt, drought, and Zn deficiency). The stability of the candidate genes was assessed by geNorm, NormFinder, and BestKeeper, and their outputs were integrated to obtain a final comprehensive rank of stability based on the geometric mean. The results indicated that samples under different experimental conditions possessed their own best reference genes, and using two reference genes for RT-qPCR normalization was recommended for the tested experiments. Overall, the combination of 26S protease regulatory subunit 7A (PR26S) and serine/threonine-protein phosphatase-1 (PP1) was recognized as a good choice for RT-qPCR normalization in pecan across all the treatments. More importantly, the widely used alpha-tubulin (α-TUB), ubiquitin (UBQ), and actin (ACT) genes were not the best suitable reference genes in most of our experiments. Our results will be helpful for future gene-expression studies in pecan.

中文翻译：

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鉴定合适的参考基因以标准化山核桃（伊利诺伊州山核桃）的实时定量PCR数据

关键信息

这项研究提出PR26S和PP1的组合是在非生物胁迫，发育粒，嫁接和各种组织下山核桃RT-qPCR标准化的良好选择。

抽象

参考基因选择是在实时定量聚合酶链反应（RT-qPCR）分析中产生可靠结果的必要先决条件。但是，仍然缺乏有关对山核桃中合适的参考基因进行系统验证的研究。在这项研究中，选择了17个候选参考基因，并评估了它们在山核桃在各种实验条件下的表达稳定性，包括各种组织，发育中的籽粒，嫁接以及遭受三种非生物胁迫（盐，干旱）的两种植物组织（叶和根）。和锌缺乏症）。候选基因的稳定性由geNorm，NormFinder和BestKeeper进行了评估，并将它们的输出进行整合以获得基于几何平均值的最终综合稳定性等级。结果表明，在不同实验条件下的样品均具有自己的最佳参考基因，推荐将两个参考基因用于RT-qPCR的标准化。总体而言，26S蛋白酶调节亚基7A（PR26S）和丝氨酸/苏氨酸蛋白磷酸酶-1（PP1）被认为是在所有治疗中山核桃RT-qPCR标准化的良好选择。更重要的是，在我们的大多数实验中，广泛使用的α-微管蛋白（α-TUB），泛素（UBQ）和肌动蛋白（ACT）基因并不是最合适的参考基因。我们的研究结果将有助于山核桃今后的基因表达研究。