Validation of reference genes for RT-qPCR normalization in Iris. lactea var. chinensis leaves under different experimental conditions

Iris. lactea var. chinensis (I. lactea var. chinensis) is highly tolerant of Cu, Pb, drought, NaCl and cold. To study the molecular mechanism of I. lactea var. chinensis under these stresses, it is necessary to select reliable reference genes for RT-qPCR study. In the present study, the expression stability of nine candidate reference genes, namely, beta-tubulin (TUBLIN6), ACTIN11, translation initiation factor 4A-1 (EIF4A1), cytosolic phosphoglycerate kinase (PGK), TIP41-like family protein (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAP), phosphatase 2A (PP2Acs), cyclophilin (CYP) and ubiquitin (UBQ), was assessed in I. lactea var. chinensis plants subjected to five abiotic stresses using GeNorm and NormFinder. A combination of GAP, ACTIN11, TIP41, EIF4A1 and PGK was identified as appropriate genes for normalization under PEG stress, whereas the combination of UBQ, PGK, TIP41 and PP2Acs was the most suitable under NaCl stress. PP2Acs, ACTIN11 and UBQ exhibited the most stable expression under Pb stress. For Cu-treated leaves, CYP, GAP and TIP41 were the most stably expressed, while GAP, PP2Acs and TIP41 for cold-treated leaves. Generally, the analyses found that TIP41, CYP, PGK, GAP and PP2Acs were the most stable genes under five abiotic stress conditions. These results would contribute the more accurate and widespread use of RT-qPCR in I. lactea var. chinensis gene analysis.