Abstract
Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study proposed to test the expression stability of 13 candidate reference genes in Setaria viridis, a monocot species recently proposed as a new C4 model plant. Gene expression stability of these genes was assayed across different tissues and developmental stages of Setaria and under drought or aluminum stress. In general, our results showed Protein Kinase, RNA Binding Protein and SDH as the most stable genes. Moreover, pairwise analysis showed that two reference genes were sufficient to normalize the gene expression data under each condition. By contrast, GAPDH and ACT were the least stably expressed genes tested. Validation of suitable reference genes was carried out to profile the expression of P5CS and GolS during abiotic stress. In addition, normalization of gene expression of SuSy, involved in sugar metabolism, was assayed in the developmental dataset. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant.
Highlights
Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization
Gene expression analysis is an important tool towards the understanding of the complex signaling networks that regulate the different responses observed during the plant life cycle or when they are submitted to different stimulli, and it has been used in many studies for this purpose[8,9,10,11]
RT-qPCR has emerged as the standard method for gene expression profiling due to its high sensibility, reproducibility and large dynamic range with a potential increasing sample throughput[13,40]
Summary
Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant. Engineered S. viridis plants can be utilized in a proof-of-concept approach to evaluate phenotypes related to important agricultural traits such as abiotic stress tolerance, resistance to pathogens and improved yield and biomass[5,6,7], and the promising genes could be further transferred to a target crop To achieve this goal, it is important to establish suitable genetic tools, including a reliable gene expression analysis in this species. The knowledge about the physiology, biochemistry and molecular responses involved in abiotic stresses contributes to the discovery of new genes and signaling networks that plants use to cope with these challenges, and it is pivotal for the development of new crop varieties with enhanced tolerance to stress[23,24,25,26]
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