Selection of Appropriate Reference Genes for Gene Expression Analysis under Abiotic Stresses in Salix viminalis.

Valentin Ambroise,Jean-Francois Hausman,Kjell Sergeant,Ann Cuypers,Sylvain Legay,Gea Guerriero

doi:10.3390/ijms20174210

Abstract

Salix viminalis is a fast growing willow species with potential as a plant used for biomass feedstock or for phytoremediation. However, few reference genes (RGs) for quantitative real-time polymerase chain reaction (qPCR) are available in S. viminalis, thereby limiting gene expression studies. Here, we investigated the expression stability of 14 candidate reference genes (RGs) across various organs exposed to five abiotic stresses (cold, heat, drought, salt, and poly-metals). Four RGs ranking algorithms, namely geNormPLUS, BestKeeper, NormFinder, and GrayNorm were applied to analyze the qPCR data and the outputs were merged into consensus lists with RankAggreg, a rank aggregation algorithm. In addition, the optimal RG combinations were determined with geNormPLUS and GrayNorm. The genes that were the most stable in the roots were TIP41 and CDC2. In the leaves, TIP41 was the most stable, followed by EF1b and ARI8, depending on the condition tested. Conversely, GAPDH and β-TUB, two genes commonly used for qPCR data normalization were the least stable across all organs. Nevertheless, both geNormPLUS and GrayNorm recommended the use of a combination of genes rather than a single one. These results are valuable for research of transcriptomic responses in different S. viminalis organs.

Highlights

Perennial woody plants are present across the world and cover about 30% of the land surface [1]
In addition to fine roots and leaves, the stele and cortex of coarse roots, stem cortex and bark were collected from control plants on the 12th day to study the stability of the candidate reference genes in various organs under control condition
The expression stability of 14 candidate reference genes (RGs) was assessed in Salix viminalis leaves and roots exposed for 12 days to five abiotic stress conditions

Summary

Introduction

Perennial woody plants are present across the world and cover about 30% of the land surface [1]. Trees are a source of numerous economical products, such as timber, fuel, food, resins, and oil, but they provide a range of ecosystem services including carbon storage, maintenance of wildlife habitats, and soil stabilization [2]. Unlike herbaceous plants, they usually have a longer life cycle, spanning from decades to centuries. Abiotic stresses are the main factors affecting plant distribution and primary production [3]. From this perspective, unravelling stress acclimation and resistance mechanisms could help us to optimize plant primary production [4]

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Conclusion