Analysis on the difference of genetic factors between wild and cultivated population
SSR results suggested that wild and cultivated populations all maintained higher genetic polymorphism in P.odoratum, D.nipponica and A.sessiliflorus, with an average Ht index of 0.2952 and a fragrance I index of 0.4291, moreover, the average polymorphic loci were all more than 3.50%. Studies have proved that the perennial plants or plants with multiple reproduction generally remaining higher genetic diversity, while the genetic diversity of species with close genetic distance is easily affected by, for example, reproduction, natural selection and genetic drift. [37, 38]. P.odoratum, A.sessiliflorus and D.nipponica are perennials, therefore the wild populations of three medicinal plants maintain higher genetic diversity, which is also an embodiment of adapting to the complex and changeable environment in Changbai Mountain. But the cultivated populations of three medicinal plants also maintain high genetic diversity, which is contrary to the results with low genetic diversity of cultivated species. Tracing back to the provenance of cultivated populations, it was found that the provenances were mainly seedlings and shoots in Changbai Mountain, which were introduced randomly on a large scale, so abundant provenance types were introduced at the initial stage of cultivation, which also greatly improved the genetic diversity of the cultivated population. Moreover, the cultivated population has been excellently protection by humans for long times, which effectively suppresses the reduction of genetic diversity caused by gene loss. Finally, cultivated populations have been established for the shorter time, compared with wild populations, the risks such as natural selection and inbreeding decline have not been reflected. The stability of population genetic structure is largely determined by the level of genetic diversity [8]. Therefore, we considered that different populations of three medicinal plants maintain higher genetic diversity during the cultivation years, which also give rise to the population genetic structure remained stable. What’more, the results of population genetic structure analysis and PCoA analysis corroboration each other, indicating that except A.sessiliflorus, the the genetic structure among wild populations of P.odoratum and D.nipponica is more similar, and the the genetic distance is closer(F3).
The genetic differentiation is extremely weak when the GST is less than 0.0500, and greater than 0.2500, it is extremely large, whereas only the gene flow(Nm) is greater than 1 migration, can it begin to offset the genetic differentiation between populations [39, 40]. According to the results above, the GST index of P.odoratum and D.nipponica are both around 0.2500 and the gene flow (Nm) is not enough to offset genetic differentiation, so there has been obvious genetic differentiation between wild and cultivated populations of the two species. However, A.sessiliflorus showing a greatly weak genetic differentiation was opposite to other species, its GST index was close to 0.0500 and the gene flow(Nm) among populations is greatly greater than 1 migration, which partially offset the genetic differentiation among populations. There should be only the smaller gene flow(Nm) between the cultivated and wild populations of three medicinal plants due to geographical isolation. But A.sessiliflorus belongs to perennial shrubs and population size is greater, also studies have shown that the slow gene flow(Nm) continues among individuals for maintaining the relative stability of genetic structure in the population with large habitat scale, and the fluctuation after multi-generation reproduction will not generate significant impact on its gene frequency [41]. In the longer life history, slow gene flow(Nm) has been used to maintain population stability in wild population of A.sessiliflorus, while cultivated population of A.sessiliflorus still maintains the past gene flow(Nm) which has accumulated in the natural habitat for short times after cultivation, therefore, it still has a large gene flow(Nm) with the wild population. Combined with the analysis of population genetic structure, we indicated that during the cultivation years of this experiment, the higher genetic diversity of the wild population was transfered to the cultivated population in A.sessiliflorus.
AMOVA analysis of three medicinal plant revealed that the genetic differentiation within the populations is the dominant position. For grow in the original habitat for long time-scale,the wild populations of three perennial medicinal plants have produced various genetic variation, which have reflected after natural selection. Due to provenances of various genotypes were imported at the initial stage, the genetic variation within the cultivated medicinal plant populations is also more plentiful. While during the cultivation years, the gametes of cultivated populations of P.odoratum and D.nipponica may deviate from Hardy-Weinberg equilibrium due to the influence of genetic adaptability and linkage disequilibrium[42, 43], and after multiple generations of cultivation, it further intensifies the trend of variation within the populations. The number of genetic variation types of individuals in the population represents the level with genetic diversity[44], therefore, this molecular variation pattern also evidenced that wild and cultivated populations still maintain higher genetic diversity.
The influence of soil environmental factors on genetic structure
The Spearman correlation analysis attested that the change of soil environmental factors has no significant effect on the genetic structure with the cultivated populations of medicinal plants, so it is not the main reasons which affect the genetic variation of the cultivated populations within the cultivation period. Studies have concluded that when the geographical isolation exceeds a certain distance, the change of its habitat will only affect the genetic structure of the population 14% [45]. The cultivated population of A.sessiliflorus maintains stable and similar genetic structure with the wild population during the cultivation years, which also confirms this point. With wild populations live in natural habitat with complex environments for long times, their population genetic structure has already greatly stabilized. While cultivated populations introduced abundant types of genetic variation in the initial stage, which allowed the cultivated populations to maintain stable genetic structure during the cultivation period. However, for the much longer cultivation time scale, P.odoratum and D.nipponica did not fully comply with the Hardy-Weinberg equilibrium, which result in genetic variation among the cultivated populations, which made the cultivated populations form stable genetic structure distinguished the wild populations (F3a-b). On the contrary, due to the shorter cultivation time-scale and different life forms, cultivated population of A.sessiliflorus still largely degree maintained the genetic structure consistented with wild populations (F3c).
The Influence of Environmental Factors on the Secondary Metabolites
Plants produce secondary metabolites with adapt to the complex environment, which plays an important role in regulating plant physiology and environmental response[46]. Moreover, environmental factors are the main factors affecting the secondary metabolites of traditional Chinese medicine [47]. While based on our research also found that the secondary metabolites of three medicinal plants are significantly affected by environmental factors(F6, P < 0.05). Among them, the total flavonoids of P.odoratum significantly negatively correlated with soil environmental factors, for example total organic carbon, total nitrogen, soil invertase activity, and soil fungal community diversity (F6a, P < 0.05); The diosgenin is immensely affected by soil physicochemical properties and soil enzyme activity than disogluside in D.nipponica, and it is only positively correlated with nitrate nitrogen(F6b, P < 0.05); The responses of secondary metabolites in leaves and stems of A.sessiliflorus to soil environmental factors were also variant. In leaves, Eleutheroside E and isofraxidin which was positively correlated with most soil environmental factors were vastly impacted by soil physicochemical properties and soil enzyme activity, while syringin and chlorogenic acid were only significantly affected by soil microbial community diversity(F6c,P < 0.05). In stem, syringin and Eleutheroside E were significantly influenceted by soil environmental factors, while the other secondary metabolites are only significantly correlated with the diversity of soil microbial community (F5c, P < 0.05).
On the other hand, temperature with the main environmental factor regulating plant secondary metabolism has a significantly effect on plant secondary metabolites [48]. In the florescence (May to June) and fruit period (July to September) of P.odoratum [49], the influence of temperature to total polysaccharides and total flavonoids was significant (F6d, P < 0.05). D.nipponica in the time-scale with besides florescence (June to August) [50], only diosgenin has significant correlation with temperature than disogluside,(F6e, P < 0.05). The effect of temperature to the secondary metabolites of A.sessiliflorus covered the growing season [51], which had significant correlation with five secondary metabolites, and the response of metabolites in leaves for temperature changes was stronger than stems (F6f,P < 0.05). Moisture is a necessary condition for plant growth and development as well as one of the environmental factors affecting plant metabolism [52]. During the growing season of the three medicinal plants, monthly rainfall has no significant effect on their secondary metabolites, which may not reach the stress level of drought or flood.
In conclusion, the environmental factors, including soil environmental factors and temperature, are one of the reasons for the significant differences in secondary metabolism between the wild and cultivated populations of the three medicinal plants during the cultivation years. When the habitat changed, the wild and cultivated populations of medicinal plants all had the content of dominant secondary metabolites belonging to their own populations. Although the wild population generate more complex secondary metabolic composition, combined with the results of HPLC fingerprint, the similarity of HPLC fingerprint among wild populations is low for the different habitats and the influence of environmental factors to secondary metabolism for a long time. It is further indicated that the effect of environmental factors on secondary metabolites of medicinal plants is extremely significant.
The consistency between the changes of genetic structure and the differences of secondary metabolism
Based on the previous research, the consistency between the differences of secondary metabolism and the changes of genetic factors in medicinal plants has been further analyzed. The wild populations of P.odoratum and D.nipponica (JDYZ and JYYZ, CBCL-1 and CBCL-2) live in the natural habitat, and existed geographical isolation at a certain distance among populations. During the longer time-scale, the populations continuously maintain higher genetic diversity and its genetic structure has tended to be stable, furthermore our results also evidenced that the similarity of genetic structure between the wild populations of P.odoratum and D.nipponica is higher than cultivated population (F3a). However, HPLC fingerprints displayed that there were significant differences in secondary metabolism among wild populations of P.odoratum and D.nipponica (F4a-b,Table S5).More importantly, wild and cultivated populations of A.sessiliflorus maintained a higher genetic diversity and hugely similar genetic structure (F3c), but there were significant distinction to secondary metabolism between wild and cultivated populations in stem and leaf. In conclusion, we considered that there is no obvious consistency between the changes of genetic factors and the alteration of secondary metabolism during the cultivation years of this experiment, hence, there may still be significant discrepant in secondary metabolism among different populations of the identical medicinal plant with similar and stable genetic structure after the habitat changes.