Selection and validation of reference genes in all-red Amaranth (<i>Amaranthus tricolor</i> 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 <i>SAND</i> and <i>RAN1</i> for light quality, <i>SAND</i> and <i>EIF5a</i> for photoperiod, <i>Actin-7</i> and <i>TuA</i> for temperature, <i>SAND</i> and <i>Actin-7</i> for different tissue, <i>SAND</i> and <i>EIF5a</i> for NaCl, <i>SAND</i> and <i>60S RPL</i> for GA₃, <i>28S rRNA</i> and <i>18S rRNA</i> for 6-BA, <i>18S rRNA</i> and <i>60S RPL</i> for 2,4-D, <i>EIF5a</i> and <i>28S rRNA</i> for ABA, <i>Actin-11</i> and <i>SKIP35</i> for SA, <i>28S rRNA</i> and <i>Actin-7</i> for MeJA, <i>SAND</i> and <i>EIF5a</i> for all samples. To further verify the reliability of the reference genes identified in this study, the expression levels of <i>CYP76AD1</i> were studied in amaranth. This is the first systematic analysis aimed at the selection of reference genes for qRT-PCR in <i>A. tricolor</i> under different culture conditions.