Soil salinity is one of the most brutal environmental factors that affect crop growth and productivity. Chenopodium quinoa is known as one of the essential food crops in the future due to its agronomic and nutritive value and its strong adaptability to stress environments and soil conditions. However, the molecular aspects of salt tolerance of quinoa remain not well known. Therefore, the expression study of candidate genes related to salt tolerance has become one of the most important features for the identification of their functions. However, one of the most crucial points in Quantitative PCR (qPCR) data analysis is the selection of appropriate reference gene that should be stable and unaffected in a given condition. In this study, six candidate reference genes were analysed in order to select the most stable one under salt stress conditions. The expression stability was assessed using three different algorithms: geNorm, NormFinder and BestKeeper. The most stable and appropriate reference genes screened in this study whose expression was confirmed to be constant in different salt treated and untreated quinoa plant were Monensin sensitivity1 (MON1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Factor elongation 1-alpha (EF-1α), while Ubiquitin-conjugating enzyme (UBC) and Ubiquitin-protein ligase 7 (UPL7) had the worst stability. However, the data obtained by BestKeeper demonstrated slight differences compared to those from geNorm and NormFinder.
Overall, we designated MON1 and GAPDH as the best candidates and the most stable housekeeping gene under salt stress conditions and their geometric means would provide accurate normalization factor for expression data in Chenopodium quinoa.