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. 2025 Aug 6;14(15):2434.
doi: 10.3390/plants14152434.

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

Qiuwei Lu  1 , Xueying Wang  1 , Suxuan Dong  1 , Jinghan Fu  1 , Yiqing Lin  1 , Ying Zhang  1 , Bo Zhao  1 , Fuye Guo  1
Affiliations

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

Qiuwei Lu et al. Plants (Basel). .

Abstract

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.

Keywords: Chenopodium quinoa; RT-qPCR; drought stress; expression stability; gradient stress; heat stress; low-temperature stress; reference genes.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Specificity analysis of primers for candidate reference and target genes. (a) Agarose gel electrophoresis of PCR products of 11 candidate reference genes and 4 target genes in quinoa. M: DL2000 DNA Marker; 1: DHDPS96: dihydrodipicolinate synthase 2 (201 bp); 2: SAL92: IT4 phosphatase-associated protein (167 bp); 3: ACT16: actin (176 bp); 4: NRP13: asparagine-rich protein (152 bp); 5: UBC19: ubiquitin-conjugating enzyme E2 (244 bp); 6: EF03: translation elongation factor (134 bp); 7: WLIM96: LIM domain-containing protein (187 bp); 8: SKP27: S-phase kinase-associated protein (160 bp); 9: COR72: cold-regulated protein (136 bp); 10: HSP44: heat shock protein (231 bp); 11: COR413-PM: cold-regulated 413-plasma membrane protein (124 bp); 12: DREB12: dehydration response element binding protein (225 bp); 13: TSB05: tryptophan synthase beta-subunit 2 (156 bp); 14: SSU32: Ssu72-like family protein (178 bp); 15: UBC22: ubiquitin-conjugating enzyme E2 (185 bp). (b) Melting curves of 11 candidate reference genes and 4 target genes in quinoa.
Figure 2
Figure 2
A comparison of cycle threshold (Ct) values for the 11 candidate reference genes in samples subjected to different treatments. (a) Global group: Control (CK), low-temperature (LT), heat (HT), and drought (DR). (b) LT group: Low-temperature treatment group. (c) HT group: Heat treatment group. (d) DR group: Drought treatment group.
Figure 3
Figure 3
Analysis of expression stability of candidate reference genes using multiple software. (a) Global group: Control (CK), low-temperature (LT), heat (HT), and drought (DR). (b) LT group: Low-temperature treatment group. (c) HT group: Heat treatment group. (d) DR group: Drought treatment group.
Figure 4
Figure 4
Candidate reference genes assayed using GeNorm. (a) Expression stabilities of candidate reference genes. (b) Normalized number of reference genes. Global group: Control (CK), low-temperature (LT), heat (HT), and drought (DR). LT group: Low-temperature treatment group. HT group: Heat treatment group. DR group: Drought treatment group.
Figure 5
Figure 5
Expression pattern analysis of target genes with different candidate genes as reference genes. (a) COR72. (b) COR413-PM. (c) HSP44. (d) DREB12. LT combination: ACT16, SAL92, SSU32, and TSB05. HT combination: ACT16 and NRP13. DR combination: ACT16, SKP27, and NRP13. Global combination: ACT16, NRP13, WLIM96, SSU32, SKP27, SAL92, and UBC22.
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