Validation of Reference Genes for Studying Different Abiotic Stresses in Oat (Avena sativa L.) by RT-qPCR

Judit Tajti,Tibor Janda,Magda Pál

doi:10.3390/plants10071272

Judit Tajti, Tibor Janda + Show 1 more

Open Access

https://doi.org/10.3390/plants10071272

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Abstract

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

Highlights

Oat (Avena sativa L.) is cultivated throughout the world as a unique cereal which serves as an excellent forage due to its high protein and essential mineral level [1]
Melt curve analysis of the nine reference genes revealed that in every case a single peak was observable under different abiotic stresses in both oat genotypes, and the amplification curves showed good repeatability (Figure 2), which means that the primers amplified a single PCR product
Our work aimed to identify stable reference genes for gene expression studies under different abiotic stresses in cultivated oat

Summary

Introduction

Oat (Avena sativa L.) is cultivated throughout the world as a unique cereal which serves as an excellent forage due to its high protein and essential mineral level [1] It is a good source of dietary fiber, especially β-glucan, with the potential to improve human health [2]. This crop is less profitable than maize, soybean, or wheat crops, so it is often cultivated in areas which have a number of disadvantages, like drought or high salinity [3]. The discovery of genes playing role in abiotic stress response of oat is still ahead of us and the examination of the related gene expression changes is a great way to better understanding gene functions in oat under adverse environmental conditions

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