Genetic Diversity And DNA Methylation Polymorphisms In Wild Plants And Major Domesticated Landraces Of Orient Ginseng (Panax Ginseng C.A.Meyer)

Orient Ginseng (Panax ginseng C. A. Meyer) is one of the most important global plants used in traditional medicine. The demand of this slow growing plant has exceeded its resources in the wild; this has necessitated a commercial cultivation although the wild type is still ten thousands costlier than the cultivated type in the markets. Hence, this valuable wild plant remains endangered due to the commercial differences between the wild and the domesticated types. Although studies in other plants have shown that the domestication of a plant has a tendency of narrowing genetic diversity, the genetic diversity and epigenetic variation between the wild plants and the cultivated landraces of the Orient ginseng remains unexplored.To address these issues, amplified fragment length polymorphism (AFLP) analysis was performed with 24 selected primers on seventeen Orient ginseng plants to represent: three natural wild plants (WLG), three cultivated wild plants (WDM), and eleven domesticated landraces; (Damaya (DMY), Yuanbangyuanlu (YUAN), Changbo (CHB), and Shizhu (SHIZHU), together with four cultivated American ginseng plants (AMG) and one Acanthopanax sessiliflorus as an outgroup. The selected AFLP primers generated a total of 1490 loci (average 62 loci per primer) of which 1342 loci were polymorphic among 22 plants with a high level of polymorphism (90.06%). UPGMA Cluster analysis showed that, apart from the American ginseng that formed a distinct cluster, no clear grouping for all studied plants of ginseng was evident either for the wild or the domesticated Orient ginseng landraces. This was corroborated by principal coordinate analysis (PCOORDA) that showed the same grouping. High levels of genetic distance were observed between American ginseng and each sub-group of the Orient ginseng, ranging from 0.3699 (AMG vs WLG) to 0.4715 (AMG vs CHB). Lower levels of genetic distance were observed between WLG and WDM and the cultivated plant groups (DMY, YUAN, CHB, and SHIZHU) and were almost in the same trend though Changbo and Shizhu tended to be distant from the others. In addition, the coefficient of genetic differentiation between the two groups (Wild and Cultivated ginsengs) was low (Gst = 0.2057), showing that only 20.57% of genetic differentiation resided between the two groups, while 79.43% resided in different plants of the two groups. Analysis of molecular variance (AMOVA) showed that a significant proportion of genetic variation (91.64%; P <0.001) resided within groups (wild/cultivated) while only 8.36% (P >0.001) of the total genetic variation resided between the two groups.Secondly methylation sensitive amplification polymorphism (MSAP) with 28 selected primers was performed on the same plants. This generated a total of 1821 clear and reproducible sites, of which 379 (20.81%) were methylated while 79.19% were un-methylated. Similar results to AFLP markers were observed by using methylation insensitive polymorphisms (MIP). The combined AFLP and MIP results showed that wild and cultivated groups of P. ginseng were not genetically distinct. On the contrary, methylation sensitive polymorphisms (MSP) showed a distinct grouping of different plants, each sub-group forming a cluster although the natural wild (WLG) and the cultivated wild (WDM) remained clustered together. This was corroborated by principal coordinate analysis (PCOORDA) that showed the same grouping. Clear differences in methylation levels at both CG and CHG sites between wild and some cultivated plants were also observed, the lowest levels of methylation being observed in Yuanbangyuanlu, Changbo and Shizhu being almost a third of the methylation level in wild groups (WLG or WDM). Moreover, AMOVA showed that the inter-group epigenetic variation was higher than AFLP and MIP methods (8.36 and 9.17% respectively). Coefficients of epigenetic differentiation showed a proportion higher than genetic (Gst = 34.59%) resided between the two groups, i.e., wild and cultivated ginseng plants. This indicates that domestication of ginseng engendered clear global methylation polymorphisms between wild and cultivated plants of P. ginseng.Finally, bisulfite sequencing analysis validated the MSP results showing lower levels of methylation in the cultivated type (especially in Changbo and Shizhu) while higher levels were shown in the wild groups of P. ginseng. Mantel test based on Jaccard coefficients of similarity showed absence of correlation between genetic and epigenetic polymorphisms between the studied plants, suggesting that only the cytosine DNA methylation was affected by domestication of ginseng. Sequence analysis of a subset of genetic and epigenetic variable bands between different sub-groups of ginseng showed homology to known-functional and putative protein-coding genes while repetitive sequences such as retrotransposons or transposons were less-represented.