Sword-leaf dogbane (Apocynum venetum) is a traditional Chinese herb with increasingly recognized potential to enhance health, but no study of stable reference genes in this herb has been reported. Based on a homologous cloning strategy, we have successfully cloned five candidate reference genes from sword-leaf dogbane: glyceraldehyde-3-phosphate dehydrogenase (AvGAPDH), beta tubulin (AvbTUB), polyubiquitin (AvUBQ), elongation factor 1-alpha (AvEF1α), and actin (AvACTIN). Three distinct algorithms, geNorm, NormFinder, and BestKeeper, were used to estimate the expression stability of candidate reference primer pairs. We found that AvACTIN-2 and AvACTIN-3 presented the highest stability of expression in different tissue samples, and AvGAPDH-2 was most stable under salinity stress. In addition, we illustrated the application of these new reference genes by assaying the expression levels of two hyperoside biosynthesis terminal enzyme genes, flavonoid 3′-hydroxylase (F3′H) and flavonol synthase (FLS), under salinity stress. Our study is the first to report stable expression of internal reference genes in sword-leaf dogbane in multiple experimental sample sets.

Background Ginsenosides accumulation responses to temperature are critical to quality formation in cold-dependent American ginseng. However, the studies on cold requirement mechanism relevant to ginsenosides have been limited in this species. Methods Two experiments were carried out: one was a multivariate linear regression analysis between the ginsenosides accumulation and the environmental conditions of American ginseng from different sites of China and the other was a synchronous determination of ginsenosides accumulation, overall DNA methylation, and relative gene expression in different tissues during different developmental stages of American ginseng after experiencing different cold exposure duration treatments. Results Results showed that the variation of the contents as well as the yields of total and individual ginsenosides Rg1, Re, and Rb1 in the roots were closely associated with environmental temperature conditions which implied that the cold environment plays a decisive role in the ginsenoside accumulation of American ginseng. Further results showed that there is a cyclically reversible dynamism between methylation and demethylation of DNA in the perennial American ginseng in response to temperature seasonality. And sufficient cold exposure duration in winter caused sufficient DNA demethylation in tender leaves in early spring and then accompanied the high expression of flowering gene PqFT in flowering stages and ginsenosides biosynthesis gene PqDDS in green berry stages successively, and finally, maximum ginsenosides accumulation occurred in the roots of American ginseng. Conclusion We, therefore, hypothesized that cold-induced DNA methylation changes might regulate relative gene expression involving both plant development and plant secondary metabolites in such cold-dependent perennial plant species.