Sowa Sylwia, Toporowska Joanna, Paczos-Grzęda Edyta
Institute of Plant Genetics, Breeding and Biotechnology, University of Life Sciences in Lublin, Akademicka 13, Lublin, 20-950, Poland.
Sci Rep. 2024 Dec 3;14(1):30006. doi: 10.1038/s41598-024-81348-4.
A repeatable and reliable reverse transcription quantitative PCR (RT-qPCR) experiment depends upon proper reference genes (RGs) selection. This study aims to examine the expression stability of nine candidate RGs for the Avena sativa - Blumeria graminis experimental setup. B. graminis causes powdery mildew - the most devastating and economically important fungal disease of crops worldwide. RGs were evaluated in Pm3 and Pm4 oat differential lines and the susceptible cultivar Fuchs during compatible and incompatible interactions with different pathotypes of Blumeria graminis f. sp. avenae in six-time points post inoculation. The identification of genes exhibiting high expression stability was done by four algorithms (geNorm, NormFinder, BestKeeper and deltaCt). The results indicated that regardless of the analysed group, two most stable RGs are required for data normalization. The most sufficient RGs combination was HNR (heterogeneous nuclear ribonucleoprotein 27 C) + EIF4A (eukaryotic initiation factor 4 A‑3). ARF (ADP‑ribosylation factor) could also be pondered as demonstrating high expression stability. These genes can be considered universal candidates for RT-qPCR normalization to study interaction with B. graminis as well as Puccinia coronata and Puccinia graminis, as confirmed by our previous research. The worst candidate for data standardisation was TUA (α- tubulin). To our best knowledge, this is the first report regarding RGs' selection in this pathosystem. Identified RGs are proper normalisation candidates for gene expression studies in the A. sativa infected by B. graminis as well as other related pathogens.
一个可重复且可靠的逆转录定量PCR（RT-qPCR）实验取决于合适的内参基因（RGs）选择。本研究旨在检测9个候选内参基因在燕麦-禾本科布氏白粉菌实验体系中的表达稳定性。禾本科布氏白粉菌引发白粉病，这是全球范围内对作物最具毁灭性且在经济上最为重要的真菌病害。在接种后六个时间点，对Pm3和Pm4燕麦鉴别系以及感病品种富克斯在与禾本科布氏白粉菌燕麦专化型不同致病型的亲和与非亲和互作过程中评估了内参基因。通过四种算法（geNorm、NormFinder、BestKeeper和deltaCt）鉴定出表达稳定性高的基因。结果表明，无论分析的组别如何，数据标准化都需要两个最稳定的内参基因。最适用的内参基因组合是HNR（不均一核糖核蛋白27 C）+ EIF4A（真核生物翻译起始因子4 A-3）。ARF（ADP-核糖基化因子）也可被认为具有高表达稳定性。正如我们之前的研究所证实的，这些基因可被视为用于RT-qPCR标准化的通用候选基因，以研究与禾本科布氏白粉菌以及冠柄锈菌和禾柄锈菌的相互作用。数据标准化的最差候选基因是TUA（α-微管蛋白）。据我们所知，这是关于该病理系统中内参基因选择的首篇报道。鉴定出的内参基因是用于研究被禾本科布氏白粉菌以及其他相关病原体感染的燕麦中基因表达的合适标准化候选基因。
