Identification and evaluation of reference genes for quantitative real-time PCR analysis in Passiflora edulis under stem rot condition

Passion fruit (Passiflora edulis), an important tropical and subtropical fruit, has a high edible and medicinal value. Stem rot disease is one of the most important diseases of passion fruit. An effective way for control and prevention of this disease is to identify the genes associated with resistance to this disease. Quantitative real-time PCR (RT-qPCR) has mainly been widely applied to detect gene expression because of its simplicity, fastness, low cost and high sensitivity. One of the requirements for RT-qPCR is the availability of suitable reference genes for normalization of gene expression. However, currently, no Passiflora edulis reference genes have been identified andthus it has hindered the gene expression studies in this plant. The present study aimed to address this issue. We analyzed sixteen candidate reference genes, including nine common (GAPDH, UBQ, ACT1, ACT2, EF-1 alpha-1, EF-1 alpha-2, TUA, NADP, and GBP) and seven novel genes (C13615, C24590, C27182, C10445, C21209, C22199, and C22526), in different tissues (stem, leaf, flower and fruit) of two accessions under stem rot condition. We calculated the expression stability in twenty-four samples using the Delta Ct, GeNorm, NormFinder, BestKeeper and RefFinder. The results showed that both C21209 and EF-1 alpha-2 were sufficient to normalize gene expression under stem rot, whereas the commonly used reference genes, GAPDH and UBQ, were the least stable ones. The expression patterns of PeUFC under stem rot condition normalized by stable and unstable reference genes indicated the suitability of using the optimal reference genes. To our knowledge, this is the first systematic study of reference genes in Passiflora edulis, which identified a number of reliable reference genes suitable for gene expression studies in Passiflora edulis by RT-qPCR.