Conservation Genetics Of Rhodiola Sachalinensis Endemic To Changbai Mountain, China

In this paper, from conservation genetics the point view, we selected as studysubjects different geographic populations of R. sachalinensis, including threenatural populations and one ex situ conservation population, and mainlyresearched the effect of environmental difference caused by elevation gradienton genetic characteristics and secondary metabolites of R. sachalinensis. On theone hand, we applied a genomic scan approach using RAPD and ISSR makersto analyze genetic diversity and genetic structure of different geographicpopulations; on the other hand, to reveal DNA methylation patterns andepigenetic differentiation in the different geographic populations, MSAPmarkers was then used to identify DNA methylation polymorphisms. Finally,we obtained chemical fingerprints of secondary metabolites using HPLC, andanalysed the relationship among altitude, genetic and epigenetic structure, andchemical fingerprints. The major results are list as follow:（1）We applied a genomic scan approach using RAPD and ISSR makers toanalyze genetic diversity and genetic structure of the four different geographicpopulations.7RAPD primers amplified a total of177bands, the percentage ofpolymorphic loci (PPL) and the Shannon’s information index (I) were94.92%and0.5013, respectively.12ISSR primers amplified a total of349bands, thepercentage of polymorphic loci (PPL) and the Shannon’s information index (I)were97.71%and0.4299respectively. Two molecular markers indicated that thedifferent geographic populations of R. sachalinensis existed in a high level ofgenetic diversity. Compared with the in situ conservation populations, the exsitu conservation population（ESP）has much higher level of genetic diversity.The analysis of Nei’s genetic structure revealed that the different geographical populations of R. sachalinensis showed obvious geneticdifferentiation (Gst,0.4784in RAPD and0.3490in ISSR); further the AMOVAresults suggested that the genetic differentiation among populations occupy acertain position (55.61%in RAPD and45.12％in ISSR).On the basis of genetic differentiation, the calculated gene flow were0.5451（RAPD）and0.9327migrants per generation. Two molecular markersindicated that the different geographic populations of R. sachalinensis are lackof gene exchange, which are a major reason caused by a distinct geneticdifferentiation among populations.Based on the Nei genetic distance matrix, a UPGMA dendrogram of allsampled R. sachalinensis individuals was reconstructed. In this dendrogram, allthe individuals in a given population formed a distinct cluster. Ex situconservation population (ESP) and natural population located at the altitude of2,000meter were always clustered together, and a principal coordinates (PCO)analysis revealed that there were several individuals with minimal overlapbetween TZ3and ESP; which showed that the ex situ conservation populationof R. sachalinensis is introduced from natural population located at the altitudeof2,000meter.（2）We used MSAP markers to detect the DNA methylation level andepigenetic structure of the different geographic populations of R. sachalinensis,which revealed the epigenetic variation of R. sachalinensis along steep altitudegradient. The results showed that natural populations located at high altitudeexisted in a higher level of DNA methylation, but the DNA methylation level ofex situ conservation located at low altitude is relatively lower. The results ofepigenetic structure implied that high levels of DNA methylation polymorphismwere uncovered in four R. sachalinensis populations, with PPL=85.99%, I=0.3778.DNA methylation patterns were specific among the different geographicpopulation, with distinctly epigenetic differentiation (Gst=0.5892). TheAMOVA analysis of the four populations revealed that63.87%of the epigenetic variation occurred among populations. A UPGMA dendrogram wasreconstructed from epigenetic distances. This dendrogram was similar to theRAPD and ISSR marker-based dendrogram, all the individuals in a givenpopulation formed a distinct cluster. In this dendrogram, TZ3and ESP werejoined together along a third major branch as well, but a little overlap betweenthem using principal coordinates (PCO) analysis, which implied a closerrealationship.（3）To characterize main chemical fingerprints on R. Sachalinensis, weanalyzed TCMF by HPLC. Analysis results showed that the of the mainmedicinal ingredients salidroside content from ex situ conservation populationwas significantly lower than the natural populations of R. Sachalinensis, itsmedicinal value has been reduced.The results of similarity based on TCMF showed an obvious difference ofchemical compositions between natural populations and ex situ conservationpopulation. Based on the similarity, the results of clustering indicated that ESPwas clustered a single branch. Those indicated that not only the main medicinalingredients of the ex situ conservation population of R. Sachalinensis but alsothe secondary metabolites composition have beeb changed.（4）Correlation analysis revealed close relationships among altitude,genetic structure, epigenetic structure, and TCMF patterns. The differences ofenvironmental factors caused by altitude gradient, which is the major reasonthat resulted in distinctly variation among the different geographic populationof R. Sachalinensis, besides reproductive strategy.