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Association between Chemical and Genetic Variation of Wild and Cultivated

Populations of Scrophularia ningpoensis Hemsl.

Article  in  Planta Medica · December 2010

DOI: 10.1055/s-0030-1250601 · Source: PubMed

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 Original Papers

Association between Chemical and Genetic

Variation of Wild and Cultivated Populations
of Scrophularia ningpoensis Hemsl.

Authors Shuting Yang 1, 2*, Chuan Chen 1*, Yunpeng Zhao 1, Wang Xi 1, Xiaolong Zhou 3, Binlong Chen 3, Chengxin Fu 2

1
Affiliations The Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education,
College of Life Sciences, Zhejiang University, Hangzhou, P. R. China
2
Laboratory of Systematic & Evolutionary Botany and Biodiversity, Institute of Plant Sciences
and Conservation Center for Gene Resources of Endangered Wildlife, Zhejiang University, Hangzhou, P. R. China
3
Panʼan Institute of Chinese Materia Medica, Panʼan, P. R. China

Key words Abstract tween chemical and genetic variation of S. ning-

l
" Scrophularia ningpoensis
! poensis. Based on both sets of data, suggestions

Downloaded by: Zhejiang University. Copyrighted material.

l
" Scrophulariaceae
Scrophularia ningpoensis Hemsl. is an important are proposed for the conservation of genetic di-
l
" Radix Scrophulariae
Chinese medicinal herb with a domestication his- versity, crop improvement, and good agricultural
l
" chemical diversity

tory of more than one thousand years. Although a practice. The present results will also facilitate
l
" genetic diversity

l
" ISSR fingerprinting number of studies have focused on either chemi- our theoretical understanding of the selective
l
" HPLC fingerprinting cal or genetic variation, none have dealt with and adaptive evolutionary processes of medicinal
their association to discuss the formation of plant species impacted by domestication and a
chemical diversity. We applied HPLC fingerprint- changing environment.
ing with identification of four predominant bioac-
tive compounds using LC‑ESI‑MS to assess chemi-
cal variation among 6 cultivated and 5 wild popu- Abbreviations
lations of S. ningpoensis. Significant chemical dif- !
ferences were revealed between wild and culti- FST: genetic differentiation estimated by
received April 30, 2010 vated populations in terms of chromatographic ANOVA
revised October 4, 2010 profiles, principal component analysis (PCA) ΦST: inter-population genetic differentia-
accepted Nov. 11, 2010 plots, and bioactive compounds contents. Com- tion estimated by AMOVA
pared to cultivated populations, the chemical pro- ΦCT: among-group genetic differentiation
Bibliography
DOI http://dx.doi.org/ files varied considerably among wild populations, estimated by AMOVA
10.1055/s-0030-1250601 of which some were remarkably similar to culti-
Published online vated populations. Inter simple sequence repeats Supporting information available online at
Planta Med © Georg Thieme
(ISSR) fingerprinting indicated a genetic differen- http://www.thieme-connect.de/ejournals/toc/
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943 tiation pattern parallel to chemical variation. Evi- plantamedica
dence strongly supported the association be-
Correspondence
Dr. Yunpeng Zhao
Department of Biology
College of Life Sciences, Introduction plasticity with respect to compositional quality
Zhejiang University ! as well as responses to biotic and abiotic stresses
388 Yukangtang Road
Hangzhou 310058 Phytochemical diversity, on which the pharma- [1, 4]. Therefore, wild ancestors and landraces,
P. R. China ceutical quality of medicinal plants relies, results which are likely to bear higher genetic diversity,
Phone: + 86 5 71 88 20 64 63 from plasticity of plant secondary metabolism attract an increasing interest for their potential
Fax: + 86 5 71 86 43 22 73
ypzhao@zju.edu.cn which has evolved to respond to stresses and in- role in improving chemical composition and oth-
teractions with continuously changing environ- er traits in crop plants [4].
Correspondence ments [1]. Genetic diversity underlies the plas- Radix Scrophulariae, the root of Scrophularia
Prof. Chengxin Fu
Laboratory of Systematic ticity of secondary metabolism [2]. However, do- ningpoensis Hemsl. (Scrophulariaceae), is a fa-
and Evolutionary Botany mestication has led to an overall reduction of ge- mous Chinese traditional medicinal herb with a
Institute of Plant Sciences, netic diversity of crop plants, including medicinal domestication history of over 1000 years. Iridoids,
Zhejiang University
388 Yukangtang Road plants, despite increased productivity [3]. The like harpagoside, phenylpropanoid glycosides,
Hangzhou 310058 narrowing genetic variability may weaken crop like angroside C and acteoside, and cinnamic acid
P. R. China were revealed to be its main bioactive compo-
Phone: + 86 5 71 88 20 66 07
Fax: + 86 5 71 86 43 22 73 nents with anti-inflammatory, antimicrobial, and
cxfu@zju.edu.cn * These two authors contributed to this work equally. antitumor activities [5–9]. Several methods based

Yang S et al. Association between Chemical … Planta Med

 Original Papers

Table 1 Sample information of 16 populations of S. ningpoensis surveyed for ISSR variation and HPLC fingerprinting.

Population Locality Sample Sample Vouchers

Code size for ISSR size for HPLC
Cultivated populations
DP Dapan, Panan County, Zhejiang Province 15 3 C. Chen, 060601-060603
YC Yaochuan, Panan County, Zhejiang Province 15 3 C. Chen, 060604-060606
RC Renchuan, Panan County, Zhejiang Province 15 3 C. Chen, 060607-060609
HB Enshi City, Hubei Province 10 3 C. Chen, 060614-060616
SX Zhenping County, Shaanxi Province 10 3 C. Chen, 060617-060619
CQ Jinfo, Chongqing Municipality 19 3 C. Chen, 060620-060622
Wild populations
TM Tianmu Mountain, Zhejiang Province 10 3 C. Chen, 060623-060625
TW Dapan Mountain, Zhejiang Province 10 3 C. Chen, 060626-060628
(introduced to SH)
AH Tiantangzhai, Anhui Province – 4 P. Li, 091120-091122
HN Pingjiang County, Hunan Province – 3 P. Li, 091131-091133
JX Jiujiang County, Jiangxi Province – 3 P. Li, 091134-091136

Fig. 1 Distribution of studied populations of

S. ningpoensis.

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on HPLC or combined with LC‑ESI‑MS were developed to qualify
and quantify all or part of the foregoing four bioactive com-
pounds for the purposes of quality assessment on Radix Scrophu-
lariae [10–14]. HPLC fingerprinting with no or few identified
chromatographic peaks combined with multivariate analysis
was also applied for quality evaluation [15, 16]. The chemical dif-
ferences of Radix Scrophulariae among various production re-
gions were demonstrated to different extents. Compared to
chemical assessment, there are relatively fewer studies on the ge-
netic variation of S. ningpoensis. Zhao et al. developed ISSR finger-
printing for this species [17] and analyzed the genetic relation-
ships among one wild and five cultivated populations [18]. Chen
et al. analyzed the genetic differentiation among three cultivars
markers, and (iii) to probe the possible relationship between
chemical and genetic variation of S. ningpoensis. The results will
facilitate an exploration of the genetic basis of chemical variation
and the development of strategies for utilization and conserva-
tion of S. ningpoensis, as well as understanding evolution of plant
secondary metabolism.
Materials and Methods
!
Sampling
Young leaves of S. ningpoensis were collected from May to June of
2006 and dried in silica gel (l " Table 1; l
" Fig. 1). Only two wild

using sequence-related amplified polymorphism (SRAP) [19].
Although studies have focused on either chemical or genetic
analyses, no previous work has combined both sets of data to deal
with the association between the chemical and genetic variation
of S. ningpoensis. Therefore, in the present paper we aim (i) to es-
timate the chemical variation pattern of wild and cultivated Rad-
ix Scrophulariae from different regions in China using HPLC‑DAD
populations (TM and TW) were subjected to ISSR analyses due
to the material availability. All the materials were authenticated
by Professor Chengxin Fu, and voucher specimens were depos-
ited at the Herbarium of Zhejiang University (HZU). Samples of
Radix Scrophulariae were collected in December of 2006 from
the same populations from which leaf materials were sampled
(l" Table 1). The remaining wild materials were collected in No-

fingerprinting and LC‑ESI‑MS, (ii) to determine genetic diversity vember of 2009. All the sampled roots were gently washed and
and differentiation of these S. ningpoensis populations using ISSR dried at 50 °C followed by pulverization and 40-mesh sieving.

Yang S et al. Association between Chemical … Planta Med


HPLC fingerprinting
An accurately weighed root powder (1.0 g) was ultrasonically ex-
tracted with 50 mL 70 % methanol for 30 min, and the extract was
diluted to a volume of 50 mL following paper-filtration. The solu-
tions were filtered through a 0.45 µm membrane prior to HPLC
analysis. Chromatograms were generated on an Agilent 1100 se-
ries HPLC system equipped with a 5 µm HC‑C18 column
(250 mm × 4.6 mm) using a gradient of acetonitrile (A) and 1 %
aqueous acetic acid (B). The gradient program was performed as
follows: 0–5 min, 15 % A; 5–30 min, 15–50 % A; 30–50 min, 50–
65 % A; 50–60 min, 65 % A, with a flow rate of 0.8 mL · min−1, col-
umn temperature 35 °C, and a detection wavelength of 280 nm.
One analyte was randomly selected for 5 consecutive analyses to
validate precision. Stability examination was accomplished with
the same analyte in 48 h (n = 5). Peak area variation was esti-
mated in terms of relative standard deviation (RSD).
Identification of four main compounds
in chromatograms
The four main bioactive compounds of Radix Scrophulariae, in-
cluding harpagoside, angroside C, acetoeside, and cinnamic acid,
were identified in terms of retention time and mass spectrum by
comparison with reference compounds. The ESI MSn spectra were
acquired in both positive and negative ion modes in Thermo Fin-
nigan LCQDECA XP system equipped with an electrospray ioniza-
tion source (Thermo LC/MS Division). The mass spectrometry de-
tector (MSD) parameters were as follows: nebulizer sheath gas,
N2 (80 uit); nebulizer auxiliary gas, N2 (20 uit); capillary temper-
ature, 350 °C; spray voltage, 4500 V in negative ion ESI mode,
5000 V in positive ion ESI mode; capillary voltage, − 13 V in (−)
ESI, 25 V in (+) ESI; lens voltage, 18 V in (−) ESI, − 16 V in (+) ESI;
isolation width for the MSn experiments, 1.0 m/z; collision gas,
He; and collision energy, 35%.
HPLC fingerprinting data analyses
Characteristic peaks extracted from chromatogram data in Agi-
lent Chemstation software were used as variables for principal
component analysis (PCA). Difference significance of bioactive
compound contents was tested using one-way ANOVA or inde-
pendent t-test. The program SPSS 13.0 was employed for both
analyses.
DNA extraction and ISSR amplification
Total genomic DNA was extracted using the modified CTAB
method [20]. ISSR‑PCR amplifications were performed in a PTC-
200 thermal cycler (MJ Research) programmed for an initial 5-
min denaturation at 94 °C, followed by 45 cycles of 1 min denatu-
ration at 94 °C, 45 s annealing at 49.4–65 °C (depending on differ-
ent primers), and 1.5 min elongation at 72 °C, as well as a final
elongation step of 10 min at 72 °C. Twelve primers (UBC primer
set no. 9, Biotechnology Laboratory, University of British Colum-
bia) were screened for fingerprinting all 100 individuals. The fol-
lowing primers were used (annealing temperature in parenthe-
ses): UBC809 (60.5 °C), UBC810 (53 °C), UBC811 (52.7 °C),
UBC812 (50.8 °C), UBC827 (60.5 °C), UBC834 (49.4 °C), UBC855
(62 °C), UBC859 (57 °C), UBC874 (65 °C), UBC881 (60.5 °C),
UBC887 (50.8 °C), and UBC889 (50.8 °C).
ISSR data analyses
The following parameters of genetic diversity were calculated us-
ing POPGENE version 1.31 [21]: (i) the percentage of polymorphic
fragments (PPF); (ii) Neiʼs expected heterozygosity (HPOP); and
Original Papers
(iii) Neiʼs coefficient of population differentiation (GST). Consider-
ing a possible bias of HPOP resulting from the assumption of the
Hardy-Weinberg equilibrium, Bayesian gene diversity (HB) was
also computed using HICKORY version 1.0 [22]. A dendrogram of
the unweighted pair-group method with arithmetic means
(UPGMA) was generated by TFPGA version 1.3 [23], and bootstrap
values were computed by resampling with replacement over loci
(1000 replicates). Hierarchical structuring of genetic variation
and pairwise ΦST distances among populations were also de-
termined by an analysis of molecular variance (AMOVA) with
WINAMOVA version 1.55 [24]. Significance levels of the variance
components were based on 1000 permutations. Furthermore, an
estimator of FST under a free model of population sampling, θB,
and GST‑B, a Bayesian analogue of Neiʼs GST, was produced using
HICKORY version 1.0. Principal coordinates analysis (PCoA) was
executed using MVSP version 1.3 [25].
Supporting information
Information on the peak areas of all analyzed samples, the chro-
matogram of wild and cultivated Radix Scrophulariae, the PCA
analysis of Radix Scrophulariae and the PCoA analysis of ISSR
phenotypes are available as Supporting Information.
Downloaded by: Zhejiang University. Copyrighted material.
Results
!
HPLC validation presented satisfactory precision, reproducibility,
stability, and recovery with RSD of peak areas less than 5%. In to-
tal, 29 characteristic peaks were extracted as fingerprint markers
from all 34 analyzed samples of Radix Scrophulariae (refer to
Fig. 1S for representative chromatograms in Supporting Informa-
tion). Harpagoside, angroside C, acteoside, and cinnamic acid
were characterized as the four predominant common peaks.
Eight peaks including the four compounds mentioned above
were shared by all samples (monomorphic) with varying inten-
sities, while the other 21 compounds were polymorphic
(72.41 %). Most wild samples exhibited two- to three-fold greater
intensities of chromatographic peaks than cultivated samples
(see Table 1S in Supporting Information). Compared to wild Rad-
ix Scrophulariae, slighter differences of peak amount and peak
area existed to different extents among cultivated ones.
In the PCA scatter plot of all samples generated by 29 peak areas,
cultivated populations approximately fell into a big cluster, while
the wild populations appeared more scattered, especially the dis-
tant TW population. However, wild populations of JX, TM (except
one sample), and HN distributed considerably close to the cluster
of cultivated populations (l " Fig. 2 a). Further PCA excluding wild
samples produced four clustered groups in coincidence with cul-
tivated populations (l " Fig. 2 b). We ran additional PCAs using the
four main bioactive compounds as variables, and the projections
showed similar trends (see Fig. 2S in Supporting Information), in-
dicating their potency as markers for quality assessment of Radix
Scrophulariae. The mean contents of the two compounds, angro-
side C and harpagoside, of wild populations were significantly
higher than those of cultivated ones (l " Table 2). Generally, one
of the wild populations, TW, produced the highest contents of
bioactive compounds. There were also significant differences of
angroside C and cinnamic acid contents among cultivated popu-
lations. Besides these two compounds, the wild populations
showed further difference of acetoside content, implying more
chemical variation among wild populations.
Yang S et al. Association between Chemical … Planta Med
 Original Papers

Fig. 2 a Scatter plot of principal component anal-

ysis (PCA) for all samples of Radix Scrophulariae.
PC1 and PC2 are the first principal components us-
ing 29 chromatographic peak areas as variables.

Downloaded by: Zhejiang University. Copyrighted material.

Fig. 2 b Scatter plot of principal component anal-
ysis (PCA) for the cultivated samples of Radix
Scrophulariae. PC1 and PC2 are the first principal
components using 29 chromatographic peak areas
as variables.

Yang S et al. Association between Chemical … Planta Med

 Original Papers

Table 2 Content comparison of the four bioactive compounds among different populations of S. ningpoensis (µg/mg).

Population Acteoside Angroside C Harpagoside Cinammic acid

Cultivated populations
DP 0.104 ± 0.002c 0.152 ± 0.017cd 1.823 ± 0.613cde 0.596 ± 0.127bc
YC 0.122 ± 0.033bc 0.138 ± 0.020cd 1.557 ± 0.495de 0.702 ± 0.097b
RC 0.086 ± 0.029c 0.138 ± 0.019cd 1.465 ± 0.440de 0.445 ± 0.109bcd
HB 0.064 ± 0.017c 0.127 ± 0.022d 0.685 ± 0.389e 0.310 ± 0.073cd
SX 0.063 ± 0.008c 0.251 ± 0.055b 2.236 ± 0.822cde 0.289 ± 0.040d
CQ 0.059 ± 0.008c 0.159 ± 0.025bcd 1.521 ± 0.280de 0.287 ± 0.016d
Mean 0.083 ± 0.029 0.161 ± 0.049 1.548 ± 0.657 0.438 ± 0.181
Wild populations
TM 0.178 ± 0.081ab 0.369 ± 0.128a 5.732 ± 3.739b 0.475 ± 0.104bcd
TW 0.226 ± 0.061a 0.418 ± 0.049a 3.506 ± 1.119bcd 1.918 ± 0.417a
JX 0.077 ± 0.020c 0.227 ± 0.062bc 4.077 ± 0.632bc 0.272 ± 0.036d
HN 0.071 ± 0.004c 0.195 ± 0.030bcd 5.369 ± 1.253b 0.361 ± 0.145cd
AH 0.069 ± 0.028c 0.233 ± 0.027bc 11.434 ± 0.959a 0.204 ± 0.141d
Mean 0.121 ± 0.078 0.285 ± 0.106 6.362 ± 3.506 0.618 ± 0.676

Lowercases represent significant differences at the level of 95 % revealed by post hoc multiple comparison

The survey of 100 individuals from 8 populations of S. ningpoen-

Table 3 Genetic diversity indices of the ten populations of S. ningpoensis.
sis with 12 ISSR primers produced a total of 124 reproducible

Downloaded by: Zhejiang University. Copyrighted material.

fragments, of which 108 were polymorphic (87.10 %). The highest Population PPF (%) HPOP HB
genetic diversity was observed in the wild population TM, with Cultivated populations
PPF, HPOP, and HB up to 48.39 %, 0.166, and 0.208, respectively. In DP 12.90 0.052 0.073
contrast, the level of genetic diversity was lowest in the culti- YC 20.97 0.086 0.094
vated population DP (PPF = 12.90 %, HPOP = 0.052, HB = 0.073). RC 13.71 0.042 0.058
HB 22.58 0.085 0.103
Considering groups of populations, those from the wild popula-
SX 16.94 0.068 0.092
tions (TM and TW) possessed on average higher levels of diversity
CQ 13.71 0.048 0.060
(PPF = 41.94 %, HPOP = 0.150, HB = 0.186) than those from cultivated
Mean 16.80 ± 4.13 0.064 ± 0.019 0.080 ± 0.019
populations (PPF = 16.80 %, HPOP = 0.064, HB = 0.80) (l " Table 3).
Wild populations
Neiʼs estimator of population substructure (GST) indicated a TM 48.39 0.166 0.208
strong differentiation among cultivated populations (GST = TW 35.48 0.134 0.164
0.706) while a greatly lower value among wild ones (GST = Mean 41.94 ± 9.13 0.150 ± 0.023 0.186 ± 0.031
0.289). The analysis of molecular variance (AMOVA) produced a
PPF, percentage of polymorphic fragments; HPOP, Neiʼs expected heterozygosity; HB,
parallel result of population differentiation within both groups expected Bayesian heterozygosity
(ΦST = 0.801 vs. ΦST = 0.399) (l " Table 4). As the hierarchical esti-

mates of variance components displayed, the variance compo-

nent was large and significant among groups (48.04 %, p < 0.001;
l" Table 4). The Bayesian estimate of F
ST (θB) and Neiʼs GST (GST‑B) patterns. Both chemical and genetic diversity of wild populations
indicated a similar result: cultivated populations harbored higher were significantly higher than that of cultivated ones (l " Table 2;

levels of variance distributed among populations compared to l" Table 3; Table 1S in Supporting Information). Regarding the

wild populations (l " Table 4).
UPGMA clustering based on the genetic distance matrix resulted
in two clades in the studied populations of S. ningpoensis, culti-
vated and wild clades (l " Fig. 3). In the cultivated clade, popula-
tion CQ was clearly separated from the other cultivated popula-
tions, while all populations from Zhejiang (DP, RC, and YC) com-
posed a close clade parallel to HB and SX populations. PCoA indi-
cated four main plots approximately consistent with UPGMA
clustering (see Fig. 3S in Supporting Information).
currently unanalyzed wild populations of S. ningpoensis, our fur-
ther ITS-based maximum parsimony (MP) tree revealed a sub-
stantial differentiation within population TM with one clade par-
allel to clade AH and another relatively remote clade close to cul-
tivated populations. Populations JX and HN considerably re-
sembled all the cultivated populations in the ITS tree (C. Chen et
al., unpublished manuscript). All the above genetic relationships
indicated by the ITS tree unexpectedly coincided with the chem-
ical differentiation represented in l " Fig. 2. The correlation be-

tween chemical and genetic variation was further supported by

the detection of a specific chloroplast DNA haplotype in popula-
Discussion tion TW, which is phytochemically distinct, and by the existence
! of a shared haplotype between most cultivated populations and a
In many cases, chemical diversity has been largely attributed to a wild population, JX, which is chemically the closest to the culti-
variable environment. However, recent work demonstrated that vated ones (C. Chen et al., unpublished manuscript). Therefore,
chemical diversity was correlated with genetic variation in Ane- chemical variation was strongly indicated to be associated with
mopsis californica [26, 27], Pinus ponderosa [28], and Vitex rotun- genetic variation in S. ningpoensis.
difolia [29]. In our present research, PCA (l" Fig. 2) and UPGMA Compared to cultivated populations, wild populations of S. ning-
(l Fig. 3) showed that chemical and genetic differentiation
" poensis produced a couple of unique chromatographic peaks as
among S. ningpoensis populations demonstrated nearly identical well as much higher contents of most metabolites, especially in

Yang S et al. Association between Chemical … Planta Med

 Original Papers

Table 4 Analysis of molecular variance (AMOVA) and Bayesian FST estimators for ISSR data of wild and cultivated S. ningpoensis.

AMOVA Bayesian estimators

Source of variation d. f. Variance percent Φ-statistic P value θB ± SD GST‑B ± SD
Wild populations
Variation among populations 1 39.92% ΦST = 0.399 < 0.001 0.397 ± 0.041 0.236 ± 0.021
Variation within populations 18 60.08%
Cultivated populations
Variation among populations 5 80.13% ΦST = 0.801 < 0.001 0.720 ± 0.018 0.691 ± 0.016
Variation within populations 70 19.87%
Hierarchical analysis for all studied populations
Among groups of populations 1 48.04% ΦCT = 0.480 < 0.001
Among populations within the groups 6 36.80%
Within populations 88 15.16%

ΦST: inter-population genetic differentiation; ΦCT: genetic differentiation among groups; θB: FST analogues under a free model of population sampling; GST‑B: Bayesian analogue of
Neiʼs GST; d. f.: degree of freedom

Fig. 3 UPGMA dendrogram illustrating the genet-

ic relationships among eight populations of S. ning-
poensis. Numbers on branches indicate bootstrap
values from 1000 replicates.

Downloaded by: Zhejiang University. Copyrighted material.

terms of angroside C and harpagoside (l " Table 2; Fig. 1S and
Table 1S in Supporting Information). Secondary metabolites
function in response to stresses and changing environments
[1]. High genetic diversity was proposed to benefit the long-
term persistence of plant species through increasing their
adaptivity to ever changing environments [30]. The wild popu-
lations of S. ningpoensis (like population TW), harboring a much
higher level of genetic and chemical variation, would be highly
valuable for breeding programs promoting metabolite production
and stress resistance. Considering that variation was largely ap-
portioned within populations (60.08 %, see l " Table 4), a sampling
strategy using more individuals from one population was sug-
gested for both breeding program and germplasm conservation.
In contrast, the six cultivated populations of S. ningpoensis exhib-
ited a remarkably low level of average ISSR diversity within pop-
ulations (l" Table 3) and high differentiation (l" Table 4; l" Fig. 3).
These results were consistent with the previous report of S. ning-
poensis [18] as well as many other crop plants [31, 32]. This could
be explained by the long-term artificial selection, the mode of
clonal propagation, and limited among-population exchange of
genetic materials in cultivated populations of S. ningpoensis. The
cultivated populations with remarkable genetic differentiation
(landraces) displaying a high among-population portion of genet-
ic variation (80.13 %, see l" Table 4) should be included for ex situ
conservation. Although clonally propagated in cultivated popula-
tions, S. ningpoensis reproduces sexually in wild populations.
Therefore, to maintain and improve genetic diversity, sexual
propagation should be encouraged to a certain extent within
and among cultivated populations, even between cultivated and
wild populations of S. ningpoensis.
The following conclusions can be drawn from the present results.
(i) There is a strong association between chemical and genetic
variation of wild and cultivated populations of S. ningpoensis. (ii)
The wild and genetically differentiated cultivated populations
call for priority to in situ or ex situ conservation and utilization.
(iii) Sexual propagation should be partly carried out in practice
to maintain and improve genetic diversity. The present research
considerably promotes our theoretical understanding of medici-
nal plant species regarding the selective and adaptive evolution-
ary processes under the influence of both domestication and
their habitats. Our findings are also beneficial to genetic conser-
vation, crop improvement, and quality control of S. ningpoensis.

Yang S et al. Association between Chemical … Planta Med

 Original Papers

Acknowledgements
! and HPLC‑ESI/TOF MS analysis for predicting potential bioactive com-
This work was financially supported by the National Basic Re-
search Program of China (Grant No. 2007CB411600), the National
Science & Technology Pillar Program during the Eleventh Five-
Year Plan Period (Grant No. 2006BAI21B07), Zhejiang Provincial
Natural Science Foundation of China (Grant No. Y3080087), and
the Fundamental Research Funds for the Central Universities.
The authors are grateful to Pan Li for collecting part of wild pop-
ulations, the local producers for their kindness of facilitating
sampling, and Xinhang Jiang and Dr. Zhican Wang for LC‑MS
analysis. We also thank Dr. Yingxiong Qiu for his assistance in ge-
netic data analyses and crucial suggestions and Dr. Jimmy Trip-
lette for manuscript improvement.
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