Screening of qPCR Reference Genes in Quinoa Under Cold, Heat, and Drought Gradient Stress.

Quinoa (), a stress-tolerant pseudocereal ideal for studying abiotic stress responses, was used to systematically identify optimal reference genes for qPCR normalization under gradient stresses: low temperatures (LT group: -2 °C to -10 °C), heat (HT group: 39° C to 45 °C), and drought (DR group: 7 to 13 days). Through multi-algorithm evaluation (GeNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder) of eleven candidates, condition-specific optimal genes were established as (Actin), (IT4 phosphatase-associated protein), (Ssu72-like family protein), and (Tryptophan synthase beta-subunit 2) for the LT group; and (Asparagine-rich protein) for the HT group; and , (S-phase kinase), and for the DR group, with , , (LIM domain-containing protein), , , , and (ubiquitin-conjugating enzyme E2) demonstrating cross-stress stability (global group). (dihydrodipicolinate synthase) and (translation elongation factor) showed minimal stability. Validation using stress-responsive markers- (LT), (HT), (LT), and (DR)-confirmed reliability; and exhibited oscillatory cold response patterns, peaked at 43 °C before declining, and showed progressive drought-induced upregulation. Crucially, normalization with unstable genes ( and ) distorted expression profiles. This work provides validated reference standards for quinoa transcriptomics under abiotic stresses.