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
Abstract: Population genetic diversity was estimated from
forty-four individual ginseng (Panax ginseng C.A. Meyer)
plants collected from seven geographical populations
located in Heilongjiang, Liaoning, and Jilin Provinces of
China as well as the People’s Republic of Korea by using
randomly amplified polymorphic DNA (RAPD) markers.
Overall, 41 polymorphic loci were amplified using ten
primer pairs. The polymorphism percentage ranged from
50% to 100% among seven local populations of ginseng,
indicating that there is plentiful genetic diversity in
wild ginseng populations. The genetic diversity at the
species level was higher than that at the population level.
Variance analysis showed that there was a significant
difference among populations in genetic diversity. The
genetic differentiation coefficient (i.e., FST) indicates that
43% of the variation occurred among populations, which
indicates that substantial genetic differentiation occurred
among populations. At the same time, the measured value
of gene flow (Nm) was 0.66 based on the observed genetic
differentiation coefficient among populations, suggesting
there was moderate gene flow among populations.
Keywords: Panax ginseng; RAPD; genetic diversity;
population structure
*Corresponding authors: Yong-hua Xu, Lian-xue Zhang,
College of Chinese Medicinal Materials, Jilin Agricultural
University, Changchun 130118, Jilin Province, P.R.China, E-mail:
[email protected], [email protected]
Shi-jie Wang, Feng-bo Han, Ru-sheng Li, College of Traditional Chinese Medicine, Jilin Agricultural Science and Technology University,
Jilin132101, Jilin Province, P.R.China
Yan Zhao, College of Chinese Medicinal Materials, Jilin Agricultural
University, Changchun 130118, Jilin Province, P.R.China
Xiao-lin Chen, Jilin Ginseng & Pilose Antler Office, Changchun
130022, Jilin Province, P.R.China
Shi-jie Wang, Gang Li, Jilin Academy of Agricultural Sciences,
130033, Jilin Province, P.R.China
1 Introduction
For thousands of years, ginseng (Panax ginseng C.A.
Meyer; Araliaceae) has been used as a traditional
medicine in East Asia [1]. Ginseng is indigenous to
northeastern China as well as Korea and other parts of
East Asia. It is a slow-growing, herbaceous perennial.
Some ginseng species have been reported to be
allotetraploids (i.e., 2n = 48 rather than 2n = 24) [2].
In order to adapt to environmental changes, species
must contain sufficient genetic variability within and
among populations [3]. While there are many biological
complexities that increase survival of populations,
such as heterozygosity and cross-incompatibility,
both of which maintain heterozygosity, more and
more scholars are studying the genetic structure
and population structure of species in this context.
Zhuravlev [4] reported genetic variation of wild ginseng
populations, and Seo [5] analyzed diversity of Panax
ginseng collected in Korea, but there are fewer reports
on genetic variation and population structure in
ginseng populations from China.
Population genetic structure is inseparable from
the biology of species, and standing genetic variation
represents the raw material upon which evolution
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].
In this study, we evaluated seven populations in order
to estimate the genetic diversity present within these
populations as well as to characterize the distribution
of genetic variation within and among the populations.
Additionally, we also estimated the gene flow between
populations. Further, we analyzed the genetic differentiation
among populations in order to determine if they correspond
to the geographical distances between these locations.
This study provides an analysis of the genetic variability
of ginseng inbred lines, which is essential for germplasm
conservation and long-term breeding plans.
© 2016 Shi-jie Wang et al., published by De Gruyter Open.
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388 Shi-jie Wang, et al.
2 Material and methods
2.1 Plant material
Forty-four ginseng samples were collected from seven
different regions (Table 1) for subsequent RAPD analysis.
Fresh leaves were randomly collected from adult
individuals that were at least more than 2 meters apart in
order to prevent repeated sampling of the same individual
or clone. Depending on the availability of adult plants
in each location, 5–9 plants were collected from each
population.
Table 1. The population names and geographic locations of study
sites.
Population
Sample size
Place
pop1
5
Dongning
pop2
9
Changbai
pop3
6
Ji’an
pop4
9
Kuandian
pop5
5
Ji’an
pop6
5
Fusong
pop7
5
Korea
2.2 DNA extraction and RAPD analysis
Whole genomic DNA was extracted from 0.5-g samples
of fresh leaves using the CTAB method [10]. In brief,
extracted DNA was resuspended in Tris-EDTA buffer,
which includes Tris–HCl (10 mM) and EDTA (1 mM, pH
8.0), and the DNA concentration of each sample was then
determined using a spectrophotometer (N60, Geneflow
Ltd., Lichfield, UK).
A total of 100 RAPD primers (produced by Sangon
Biotech Ltd., Shanghai, China) were tested for RAPD.
Clear, polymorphic, and reproducible bands were
selected and amplified under conditions similar to those
used by Williams et al. [11]. A 25-µL PCR reaction mixture
containing 200 ng of genomic DNA was amplified in a
PTC200 thermalcycler (MJ Research, Watertown, MA,
USA) under the following reaction conditions: initial
denaturation for 4 min at 94°C, followed by 40 cycles of
1 min at 94°C (denaturation), 1 min at 36°C (annealing),
and 2 min at 72°C (extension), and finished with a final
extension at 72°C for 10 min. Amplified DNA samples
were then subjected to electrophoresis on a 1.4% agarose
gel run in a 0.5× TBE buffer and stained with ethidium
bromide. The gels were then observed and photographed
under ultraviolet light.
2.3 Data analysis
RAPD products were recorded for presence (as 1) or
absence (as 0) of bands, with the exclusion of smeared
and weak bands as determined by visual inspection.
Genetic diversity indexes and parameters, including the
proportion of polymorphic bands (PPB), the number of
alleles observed (na), effective allele number (Ne), Nei’s
gene diversity (H), and Shannon’s diversity index (I), were
calculated for a diploid organism with POPGENE 32 [12],
which assumes of Hardy–Weinberg equilibrium. At the
species level, total population heterozygosity (Ht) was
also estimated. Genetic differentiation was also estimated
based on an AMOVA procedure implemented in the
Arlequin Software Package (Ver. 3.0), which estimate the
among population component of genetic variation (FST),
and statistical significance of the variance component
estimate was inferred using 3000 permutations of the data
between the seven individual populations. FST estimates
were used for an indirect calculation of historical levels
of gene flow, in accordance with the related effective
migration rate equation Nm = 0.5 (1 - FST)/FST.
3 Results
3.1 RAPD polymorphisms
The 44 ginseng individuals subjected to RAPD genotyping
yielded a total of 52 bands ranging between 100 and 1200
bp in size, and the 10 primers produced an average of 5.2
bands. Of these bands, 41 were polymorphic, and PPB was
78.8%.
The number of polymorphic bands amplified per
primer varied from 2 to 6, averaging 4.1 (Table 2). Each of
the 44 individuals had a distinct RAPD genotype, which
indicates that no wild plants were clones or otherwise
genetically identical.
3.2 Within population genetic diversity
The PPB within populations ranged from 29.4% (Pop1) to
79% (Pop4), with an average value of 50.1%. According to
Hardy–Weinberg equilibrium, the average values of Nei’s
gene diversity were 0.2918 at the whole species level and
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Genetic diversity and population structure of ginseng in China based on RAPD analysis Table 2. Summary of polymorphic bands generated by randomly
amplified polymorphic DNA (RAPD) applied to seven local populations of ginseng.
Primer
Number of bands
Number of polymorphic
bands
S45
5
4
S46
6
4
S80
4
4
S1122
4
3
S1125
6
6
S1127
5
4
S1188
5
4
S2021
4
2
S2029
7
6
S2031
6
4
Total
52
41
Mean
5.2
4.1
389
3.3 Population genetic structure
According to the AMOVA procedure, genetic differentiation
was significant (P < 0.01) among the seven populations,
and the coefficient of genetic differentiation among
populations (FST) was 0.43, indicating that 43% of the
genetic variability occurred among populations, with 57%
occurring within populations (Table 4). Thus, the AMOVA
results corresponded with the estimates of Nei’s gene
diversity, and Shannon’s diversity index, which indicates
that variation existed mostly among groups.
4 Discussion
4.1 Genetic diversity
0.1497 for populations. The Shannon’s diversity index
varied between 0.1510 (Pop1) to 0.2642 (Pop4), with an
average of 0.2223 at the population level and 0.4486 at
the species level. Estimates of Nei’s gene diversity for
all bands in individual populations was highest in Pop6
(0.1887) and lowest in Pop1 (0.1063; Table 3). Estimates
of Shannon’s index had different values, but exhibited
trends that corresponded with Nei’s gene diversity.
The genetic diversity of plant species depends largely on
the life history of species (for example, seed dispersal
mechanisms, breeding systems, geographical range,
and life styles), but the impact of environmental factors
is also important. In this study, ginseng PPB was 78.9%
at the species level based on RAPD data, and PPB at
the population level was 50.1%, indicating that ginseng
populations contain relatively high levels of genetic
variation. Ginseng is a perennial herb that can survive in a
broad range of biological conditions, which can help create
and maintain genetic diversity observed at a higher level.
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
average
species
44
38.2
1.2619
1.2149
0.1152
0.1645
50.1
1.4183
1.2635
0.1497
0.2223
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
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390 Shi-jie Wang, et al.
genetic diversity of ginseng was higher than those of other
species with similar life history characteristics. Because of
weak genetic differentiation, habitat loss hinders species
migration and gene flow; our findings suggest that this
differentiation should be taken into when ginseng is bred
and cultivated.
of Science & Technology of Jilin Province (grant number
111042014010); the Seed Fund of Jilin Agricultural Science
and Technology University (grant number 119032014004)
4.2 Genetic structure
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The analysis of the genetic structure of these ginseng
populations using AMOVA revealed that there was less
genetic variation shared among the populations than
within. Mantel’s test showed that geographic distance
and gene flow did not correspond with genetic distance,
suggesting that isolation by distance did not play an
important role in the genetic structure of ginseng
populations. However, other factor may have some impact
on the genetic structure of ginseng, including humanmediated gene flow and natural selection. Breeding
systems have been shown to be related to seed dispersal
mechanisms, in particular, the among population
proportion of genetic variation. As an outcrossing species,
cultivated ginseng mainly relies on human activities,
so seeds are dispersed over much longer distances than
would be otherwise possible. Because of its biological
characteristics, ginseng has maintained higher levels of
genetic diversity and lower population differentiation,
along with high rates of gene flow (i.e., the Nm estimate
is 0.66) among populations at the same time. When the
Nm value reaches 1.0 there is no population divergence;
in this paper, we demonstrate that the Nm values of
ginseng populations cannot exceed 1.0 without very high
migration rates, indicating that genetic drift did not likely
play a significant role in determining the population
genetic structure of ginseng.
Acknowledgements: This research was supported by
the Research Foundation of the Education Bureau of Jilin
Province (grant number 111022013033); the Department
Conflict id interest: Authors declare nothing to disclose.
References
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