Genetic Diversity and Population Structure of Ginseng in China Based on RAPD Analysis

Open Life Sci. 2016; 11: 387–390

Special Issue on CleanWAS 2015 Open Access

Shi-jie Wang, Xiao-lin Chen, Feng-bo Han, Ru-sheng Li, Gang Li, Yan Zhao, Yong-hua Xu*, Lian-xue Zhang* Genetic diversity and population structure of ginseng in China based on RAPD analysis

DOI 10.1515/biol-2016-0051 Received April 12, 2016; accepted August 24, 2016 1 Introduction

Abstract: Population genetic diversity was estimated from For thousands of years, ginseng (Panax ginseng C.A. forty-four individual ginseng (Panax ginseng C.A. Meyer) Meyer; Araliaceae) has been used as a traditional plants collected from seven geographical populations medicine in East Asia [1]. Ginseng is indigenous to located in Heilongjiang, Liaoning, and Jilin Provinces of northeastern China as well as Korea and other parts of China as well as the People’s Republic of Korea by using East Asia. It is a slow-growing, herbaceous perennial. randomly amplified polymorphic DNA (RAPD) markers. Some ginseng species have been reported to be Overall, 41 polymorphic loci were amplified using ten allotetraploids (i.e., 2n = 48 rather than 2n = 24) [2]. primer pairs. The polymorphism percentage ranged from In order to adapt to environmental changes, species 50% to 100% among seven local populations of ginseng, must contain sufficient genetic variability within and indicating that there is plentiful genetic diversity in among populations [3]. While there are many biological wild ginseng populations. The genetic diversity at the complexities that increase survival of populations, species level was higher than that at the population level. such as heterozygosity and cross-incompatibility, Variance analysis showed that there was a significant both of which maintain heterozygosity, more and difference among populations in genetic diversity. The more scholars are studying the genetic structure genetic differentiation coefficient (i.e., FST) indicates that and population structure of species in this context. 43% of the variation occurred among populations, which Zhuravlev [4] reported genetic variation of wild ginseng indicates that substantial genetic differentiation occurred populations, and Seo [5] analyzed diversity of Panax among populations. At the same time, the measured value ginseng collected in Korea, but there are fewer reports of gene flow (Nm) was 0.66 based on the observed genetic on genetic variation and population structure in differentiation coefficient among populations, suggesting ginseng populations from China. there was moderate gene flow among populations. Population genetic structure is inseparable from the biology of species, and standing genetic variation Keywords: Panax ginseng; RAPD; genetic diversity; represents the raw material upon which evolution population structure operates, making it an important prerequisite for evolution [6]. Randomly amplified polymorphic DNA (RAPD) analysis is a quick and effective way to detected genetic variations among numerous plant populations [7-9]. *Corresponding authors: Yong-hua Xu, Lian-xue Zhang, In this study, we evaluated seven populations in order College of Chinese Medicinal Materials, Jilin Agricultural to estimate the genetic diversity present within these University, Changchun 130118, Jilin Province, P.R.China, E-mail: populations as well as to characterize the distribution [email protected], [email protected] of genetic variation within and among the populations. Shi-jie Wang, Feng-bo Han, Ru-sheng Li, College of Traditional Chi- nese Medicine, Jilin Agricultural Science and Technology University, Additionally, we also estimated the gene flow between Jilin132101, Jilin Province, P.R.China populations. Further, we analyzed the genetic differentiation Yan Zhao, College of Chinese Medicinal Materials, Jilin Agricultural among populations in order to determine if they correspond University, Changchun 130118, Jilin Province, P.R.China to the geographical distances between these locations. Xiao-lin Chen, Jilin Ginseng & Pilose Antler Office, Changchun This study provides an analysis of the genetic variability 130022, Jilin Province, P.R.China Shi-jie Wang, Gang Li, Jilin Academy of Agricultural Sciences, of ginseng inbred lines, which is essential for germplasm 130033, Jilin Province, P.R.China conservation and long-term breeding plans.

2 Material and methods gel run in a 0.5× TBE buffer and stained with ethidium bromide. The gels were then observed and photographed under ultraviolet light. 2.1 Plant material

Forty-four ginseng samples were collected from seven 2.3 Data analysis different regions (Table 1) for subsequent RAPD analysis. Fresh leaves were randomly collected from adult RAPD products were recorded for presence (as 1) or individuals that were at least more than 2 meters apart in absence (as 0) of bands, with the exclusion of smeared order to prevent repeated sampling of the same individual and weak bands as determined by visual inspection. or clone. Depending on the availability of adult plants Genetic diversity indexes and parameters, including the in each location, 5–9 plants were collected from each proportion of polymorphic bands (PPB), the number of population. alleles observed (na), effective allele number (Ne), Nei’s gene diversity (H), and Shannon’s diversity index (I), were Table 1. The population names and geographic locations of study calculated for a diploid organism with POPGENE 32 [12], sites. which assumes of Hardy–Weinberg equilibrium. At the Population Sample size Place species level, total population heterozygosity (Ht) was pop1 5 Dongning also estimated. Genetic differentiation was also estimated pop2 9 Changbai based on an AMOVA procedure implemented in the Arlequin Software Package (Ver. 3.0), which estimate the pop3 6 Ji’an among population component of genetic variation (FST), pop4 9 Kuandian and statistical significance of the variance component pop5 5 Ji’an estimate was inferred using 3000 permutations of the data between the seven individual populations. F estimates pop6 5 Fusong ST were used for an indirect calculation of historical levels pop7 5 Korea of gene flow, in accordance with the related effective

migration rate equation Nm = 0.5 (1 - FST)/FST.

2.2 DNA extraction and RAPD analysis 3 Results

Whole genomic DNA was extracted from 0.5-g samples 3.1 RAPD polymorphisms of fresh leaves using the CTAB method [10]. In brief, extracted DNA was resuspended in Tris-EDTA buffer, The 44 ginseng individuals subjected to RAPD genotyping which includes Tris–HCl (10 mM) and EDTA (1 mM, pH yielded a total of 52 bands ranging between 100 and 1200 8.0), and the DNA concentration of each sample was then bp in size, and the 10 primers produced an average of 5.2 determined using a spectrophotometer (N60, Geneflow bands. Of these bands, 41 were polymorphic, and PPB was Ltd., Lichfield, UK). 78.8%. A total of 100 RAPD primers (produced by Sangon The number of polymorphic bands amplified per Biotech Ltd., Shanghai, China) were tested for RAPD. primer varied from 2 to 6, averaging 4.1 (Table 2). Each of Clear, polymorphic, and reproducible bands were the 44 individuals had a distinct RAPD genotype, which selected and amplified under conditions similar to those indicates that no wild plants were clones or otherwise used by Williams et al. [11]. A 25-µL PCR reaction mixture genetically identical. containing 200 ng of genomic DNA was amplified in a PTC200 thermalcycler (MJ Research, Watertown, MA, USA) under the following reaction conditions: initial 3.2 Within population genetic diversity denaturation for 4 min at 94°C, followed by 40 cycles of 1 min at 94°C (denaturation), 1 min at 36°C (annealing), The PPB within populations ranged from 29.4% (Pop1) to and 2 min at 72°C (extension), and finished with a final 79% (Pop4), with an average value of 50.1%. According to extension at 72°C for 10 min. Amplified DNA samples Hardy–Weinberg equilibrium, the average values of Nei’s were then subjected to electrophoresis on a 1.4% agarose gene diversity were 0.2918 at the whole species level and Genetic diversity and population structure of ginseng in China based on RAPD analysis 389

Table 2. Summary of polymorphic bands generated by randomly 3.3 Population genetic structure amplified polymorphic DNA (RAPD) applied to seven local populations of ginseng. According to the AMOVA procedure, genetic differentiation Primer Number of bands Number of polymorphic was significant (P < 0.01) among the seven populations, bands and the coefficient of genetic differentiation among

S45 5 4 populations (FST) was 0.43, indicating that 43% of the genetic variability occurred among populations, with 57% S46 6 4 occurring within populations (Table 4). Thus, the AMOVA S80 4 4 results corresponded with the estimates of Nei’s gene S1122 4 3 diversity, and Shannon’s diversity index, which indicates S1125 6 6 that variation existed mostly among groups. S1127 5 4 S1188 5 4 4 Discussion S2021 4 2 S2029 7 6 4.1 Genetic diversity S2031 6 4 The genetic diversity of plant species depends largely on Total 52 41 the life history of species (for example, seed dispersal Mean 5.2 4.1 mechanisms, breeding systems, geographical range, and life styles), but the impact of environmental factors 0.1497 for populations. The Shannon’s diversity index is also important. In this study, ginseng PPB was 78.9% varied between 0.1510 (Pop1) to 0.2642 (Pop4), with an at the species level based on RAPD data, and PPB at average of 0.2223 at the population level and 0.4486 at the population level was 50.1%, indicating that ginseng the species level. Estimates of Nei’s gene diversity for populations contain relatively high levels of genetic all bands in individual populations was highest in Pop6 variation. Ginseng is a perennial herb that can survive in a (0.1887) and lowest in Pop1 (0.1063; Table 3). Estimates broad range of biological conditions, which can help create of Shannon’s index had different values, but exhibited and maintain genetic diversity observed at a higher level. trends that corresponded with Nei’s gene diversity. Based on species-level estimates of genetic diversity, the

Table 3. Genetic diversity statistics for the seven ginseng populations and the entire species based on RAPD data.

Popultion Sample size No of PB a PPB(%)b Nac Ned He If

1 5 10 29.4 1.2381 1.2020 0.1063 0.1510 2 9 18 48.6 1.4524 1.2655 0.1542 0.2312 3 6 19 54.2 1.4524 1.2344 0.1390 0.2134 4 9 24 79.0 1.5714 1.2923 0.1726 0.2642 5 5 22 52.3 1.4762 1.3022 0.1720 0.2559 6 5 21 48.8 1.4762 1.3338 0.1887 0.2761 7 5 13 38.2 1.2619 1.2149 0.1152 0.1645 average 50.1 1.4183 1.2635 0.1497 0.2223 species 44 78.9 2.0000 1.4825 0.2918 0.4486 a. Polymorphic bands. b. Percentage of polymorphic bands. c. Observed number of alleles. d. Effective number of alleles. e. Nei’s gene diversity. f. Shannon’s diversity index.

Table 4. Analysis of molecular variance (AMOVA) for 44 individuals from the seven natural populations of ginseng using RAPD markers.

Source of variation Degrees of freedoms Sum of squares Variance components Percentage of variation P-value among populations 6 129.910 21.652 43 0.01 within populations 37 138.522 3.771 57 0.01 total 43 269.432 100 390 Shi-jie Wang, et al. genetic diversity of ginseng was higher than those of other of Science & Technology of Jilin Province (grant number species with similar life history characteristics. Because of 111042014010); the Seed Fund of Jilin Agricultural Science weak genetic differentiation, habitat loss hinders species and Technology University (grant number 119032014004) migration and gene flow; our findings suggest that this differentiation should be taken into when ginseng is bred Conflict id interest: Authors declare nothing to disclose. and cultivated. References

4.2 Genetic structure [1] Liu G.Y., Zhou H.I, Lu J., Zhu N., Gui M.Y., Determination of Saponins in Leaf of Panax Ginseng C. A. Mey. by High Performance Liquid Chromatography, Chem. Res. Chinese The analysis of the genetic structure of these ginseng Universities, 2009, 25, 3, 297-301 populations using AMOVA revealed that there was less [2] In D.S., Kim Y.C., Bang K.H., Kim O.T., Genetic Relationships of genetic variation shared among the populations than Panax Species by RAPD and ISSR Analyses, Kor. J. Med. Crop S, within. Mantel’s test showed that geographic distance 2005, 13, 5, 249-253 and gene flow did not correspond with genetic distance, [3] Barrett S.C.H., Kohn J.R., Falk D.A., Holsinger K.E., Genetic and evolutionary consequences of small population size in plants: suggesting that isolation by distance did not play an implications for conservation, Oxford University Press, New important role in the genetic structure of ginseng York, 1991 populations. However, other factor may have some impact [4] Zhuravlev Y.N., Reunova G.D., Artyukova E.V., Kozyrenko M., on the genetic structure of ginseng, including human- Genetic variation of wild ginseng populations (RAPD analysis), mediated gene flow and natural selection. Breeding Mol. Biol., 1998, 32, 6, 910-914 systems have been shown to be related to seed dispersal [5] Noh J.H., Kim Y.J.,, Choi K.J., Kim S.W., Kim S.K.., Characteristics of Seedling and Rhizome Propagation in Epimedium koreanum mechanisms, in particular, the among population NAKAI, Kor. J. Med. Crop Sci., 2003, 11, 2, 155-160 proportion of genetic variation. As an outcrossing species, [6] Przyborowski J.A., Sulima P., The analysis of genetic diversity of cultivated ginseng mainly relies on human activities, Salix viminalis genotypes as a potential source of biomass by so seeds are dispersed over much longer distances than RAPD markers, Ind. Crops Prod., 2010, 31, 2, 395 would be otherwise possible. Because of its biological [7] Kim C., Na H.R., Choi H.K., Genetic diversity and population characteristics, ginseng has maintained higher levels of structure of endangered Isoetes coreana in South Korea based on RAPD analysis, Aquatic Bot. 2008, 89, 1, 43-49 genetic diversity and lower population differentiation, [8] Fischer M., Prati D.P.M., Van K.M., Schmid B., Husi R., RAPD along with high rates of gene flow (i.e., the Nm estimate variation among and within small and large populations of the is 0.66) among populations at the same time. When the rare clonal plant Ranunculus reptans (Ranunculaceae), Am. J. Nm value reaches 1.0 there is no population divergence; Bot., 2000, 87, 8, 1128-1137 in this paper, we demonstrate that the Nm values of [9] Wendel J.F., Weeden N.F, The Interpretation of Mark, Dioscorides Press, Portland, 1989 ginseng populations cannot exceed 1.0 without very high [10] Doyle J., A rapid DNA isolation procedure for small quantities of migration rates, indicating that genetic drift did not likely fresh leaf tissue, Phytochem. Bull., 1987, 19, 11-15 play a significant role in determining the population [11] Williams J.G., Kubelik A.R., Livak K.J., Rafalski J.A., Tingey S.V., genetic structure of ginseng. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers, Nucleic Acids Res., 1990, 18, 22, 6531-6535 [12] Yeh F.C., Yang R.C., Boyle T., Ye Z. H., Mao J. X., The Acknowledgements: This research was supported by User-Friendly Shareware for Population Genetic Analysis. the Research Foundation of the Education Bureau of Jilin POPGENE, Edmonton: Molecular Biology and Biotechnology Province (grant number 111022013033); the Department Center, University of Alberta, 1999