Quinoa (
Chenopodium 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
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Quinoa (
Chenopodium 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
ACT16 (Actin),
SAL92 (IT4 phosphatase-associated protein),
SSU32 (Ssu72-like family protein), and
TSB05 (Tryptophan synthase beta-subunit 2) for the LT group;
ACT16 and
NRP13 (Asparagine-rich protein) for the HT group; and
ACT16,
SKP27 (S-phase kinase), and
NRP13 for the DR group, with
ACT16,
NRP13,
WLIM96 (LIM domain-containing protein),
SSU32,
SKP27,
SAL92, and
UBC22 (ubiquitin-conjugating enzyme E2) demonstrating cross-stress stability (global group).
DHDPS96 (dihydrodipicolinate synthase) and
EF03 (translation elongation factor) showed minimal stability. Validation using stress-responsive markers—
COR72 (LT),
HSP44 (HT),
COR413-PM (LT), and
DREB12 (DR)—confirmed reliability;
COR72 and
COR413-PM exhibited oscillatory cold response patterns,
HSP44 peaked at 43 °C before declining, and
DREB12 showed progressive drought-induced upregulation. Crucially, normalization with unstable genes (
DHDPS96 and
EF03) distorted expression profiles. This work provides validated reference standards for quinoa transcriptomics under abiotic stresses.
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