Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress

Abstract

Chenopodium quinoa Willd. (quinoa), a member of the Amaranthaceae family, is an allotetraploid annual plant, endemic to South America. The plant of C. quinoa presents significant ecological plasticity with exceptional adaptability to several environmental stresses, including salinity. The resilience of quinoa to several abiotic stresses, as well as its nutritional attributes, have led to significant shifts in quinoa cultivation worldwide over the past century. This work first defines germination sensu stricto in quinoa where the breakage of the pericarp and the testa is followed by endosperm rupture (ER). Transcriptomic changes in early seed germination stages lead to unstable expression levels in commonly used reference genes that are typically stable in vegetative tissues. Noteworthy, no suitable reference genes have been previously identified specifically for quinoa seed germination under salt stress conditions. This work aims to identify these genes as a prerequisite step for normalizing qPCR data. To this end, germinating seeds from UDEC2 and UDEC4 accessions, with different tolerance to salt, have been analyzed under conditions of absence (0 mM NaCl) and in the presence (250 mM NaCl) of sodium chloride. Based on the relevant literature, six candidate reference genes, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Monensin sensitivity1 (MON1), Polypyrimidine tract-binding protein (PTB), Actin-7 (ACT7), Ubiquitin-conjugating enzyme (UBC), and 18S ribosomal RNA (18S), were selected and assessed for stability using the RefFinder Tool encompassing the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt in the evaluation. The data presented support the suitability of CqACT7 and CqUBC as reference genes for normalizing gene expression during seed germination under salinity stress. These recommended reference genes can be valuable tools for consistent qPCR studies on quinoa seeds.