Validation of reference genes for quantitative real-time PCR during Chinese wolfberry fruit development

Lycium barbarum L., a woody bush that grows in Eurasia and North Africa, is an ornamental and medicinal plant. Its fruits have been used for centuries in China as a traditional herbal medicine and as a valuable nourishing tonic. There has been no report describing the selection of reference genes for stringent normalization for quantitative PCR (qPCR) in L. barbarum. The present study identified reliable reference genes for normalization of qPCR data in L. barbarum during fruit development from among eight candidate genes (GAPDH, TEF G, EF 1a, UBQ, TUB a, SAMS, EF2 and Hsp80) using the geNorm and NormFinder statistical algorithms. The results showed that the best-ranked references genes differed across the samples. A combination of GAPDH and EF1a would be appropriate as a reference panel for normalizing gene expression data across fruit developmental stages. A combination of EF 1a and SAMS would be appropriate as a reference panel for normalizing gene expression data at the stage A tested, whereas the combination of TUB a, and TEF G was the most suitable for stage B. EF2 and Hsp80 exhibited the most stable expression under stage C and stage D. NormFinder ranking of reference gene candidates was slightly different from that determined by geNorm. These results provide guidelines for the selection of reference genes under different development stages and also represent a foundation for more accurate and widespread use of qRT-PCR in L. barbarum gene analysis.