Selection and validation of reference genes in all-red Amaranth (Amaranthus tricolor L.) seedlings under different culture conditions

Amaranth is rich in betalain. Both betalain metabolites and plant growth are affected by external factors. However, studies of amaranth seedling growth and betalain metabolism are limited by a lack of appropriate reference genes for qRT-PCR analyses. We selected 13 candidate reference genes from the amaranth transcriptome database and tested their expression stability under various environmental conditions and plant growth regulators. Five methods were used for the analysis: geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder. The most stable reference genes were SAND and RAN1 for light quality, SAND and EIF5a for photoperiod, Actin-7 and TuA for temperature, SAND and Actin-7 for different tissue, SAND and EIF5a for NaCl, SAND and 60S RPL for GA3, 28S rRNA and 18S rRNA for 6-BA, 18S rRNA and 60S RPL for 2,4-D, EIF5a and 28S rRNA for ABA, Actin-11 and SKIP35 for SA, 28S rRNA and Actin-7 for MeJA, SAND and EIF5a for all samples. To further verify the reliability of the reference genes identified in this study, the expression levels of CYP76AD1 were studied in amaranth. This is the first systematic analysis aimed at the selection of reference genes for qRT-PCR in A. tricolor under different culture conditions.