Validation of Reliable Reference Genes for Accurate Normalization in RT-qPCR Analysis of Codonopsis pilosula

Abstract

ObjectiveTo identify reliable reference genes (RGs) for normalization of real-time PCR (RT-qPCR) data in Codonopsis pilosula. MethodsThe expression profiles of 10 candidate RGs (GAPDH, ACT, α-TUB, β-TUB, UBQ, CYP, EF-1α, NAC, F-box and PP2A) were examined in C. pilosula during the phenological period. The raw materials examined included roots, stems, leaves, and flower buds at flowering and boll-forming stages, five growth stages of untreated and treated roots with plant growth retardant. The best-suited RGs were accessed using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. ResultsThe best-ranked references genes differed across the samples. GAPDH and PP2A were the most suitable for expression analysis in untreated tissues while GAPDH, α-TUB, and PP2A were ranked as the three most stably expressed genes in untreated roots, while NAC and CYP were the most stably expressed genes in stressed (i.e., treated) roots. The expression of UGPase, a key enzyme for CPP biosynthesis, was determined to further validate the selected RGs. ConclusionA total of 10 RGs can be used as reference genes of C. pilosula, however the appropriate one should be used as it may chance.