Reference gene selection in Artemisia annua L., a plant species producing anti-malarial artemisinin

The selection and validation of reference genes are essential for gene expression studies by real-time quantitative PCR. The genetic map of Artemisia annua L., a Chinese medicinal plant species producing anti-malarial artemisinin, has been reported. However, few reference genes of A. annua have been estimated for real-time quantitative PCR until now. In this study, ten putative housekeeping genes, including ACT, UBQ, TUB, 18S rRNA, EF1 alpha, CYP, RPL13D, TUA, RPII and GAPDH, were chosen for identifying expression stability using geNorm and NormFinder software tools in 11 different sample pools, containing those from different plant organs and from plants treated with phytohormones and abiotic stresses. As expected, the variation in expression stability of the ten candidate reference genes tested in this study suggested there was no single reference gene that can be used for all experimental conditions in A. annua. The combination of RPII & EF1 alpha was the most stably expressed reference genes for different organs. Under phytohormone treatment, the combination of EF1 alpha & TUB was recommended as internal reference genes used for investigating target gene expression levels. In addition, the combination of ACT & EF1 alpha was suitably chosen for normalization in temperature-shocked samples. In order to further verify the reliability of the experimental results, RPII & EF1 alpha were used in combination as reference genes to examine the expression levels of ADS gene in different organs. Meanwhile, the expression levels of ADS, CYP71AV1 and DBR2 were tested by qPCR normalized with the combination of EF1 alpha & TUB in MeJA treatment samples. Our study will benefit future research on the expression of genes related to artemisinin biosynthesis under different experimental conditions.