Plant Cell Biotechnology and Molecular Biology 19(7&8):293-301; 2018 ISSN: 0972-2025

STUDY ON GENETIC DIVERSITY OF

Curculigo orchioides GAERTN POPULATIONS
FROM VIETNAM, AN ENDANGERED
MEDICINAL HERB
HOANG TAN QUANG, BUI LE THANH NHAN, TRAN MINH DUC,
TRAN VAN GIANG AND PHAM THANH*
Institute of Biotechnology, Hue University, Vietnam [HTQ]
College of Sciences, Hue University, Vietnam [BLTN]
University of Agriculture and Forestry, Hue University, Vietnam [TMD]
University of Education, Hue University, Vietnam [TVG, PT]
[*For Correspondence: E-mail: efphamthanh@gmail.com]

ARTICLE INFORMATION
Reviewers:
(1) Frédéric Ngezahayo, Ecole Normale Supérieure, Burundi, Africa.
(2) Sandeep Sharma, Kumaun University, Nainital, India.
(3) Ismail Poyraz, Bilecik Seyh Edebali University, Turkey.

Received: 27 July 2018

Accepted: 09 October 2018
Published: 23 October 2018 Original Research Article
_______________________________________________________________________________________________

ABSTRACT
Curculigo orchioides Gaertn is an endangered herb which is important ingredient of traditional
medicine of Vietnam. In this study, six random primers were used for genetic diversity analysis of C.
orchioides populations. Randomly Amplified Polymorphic DNA (RAPD) analysis of 92 samples
showed the 65 polymorphic DNA fragments. The genetic diversity is high within the studied
populations. Among these, Kon Tum has the highest diversity level (h=0.3889 and I=0.5636) while
Lam Dong is lowest (h=0.1991 and I=3064). The degree of genetic differentiation among populations
is also high (Gst=0.2505). Genetic identity between Quang Tri and Thua Thien Hue is highest
(0.9407) while between Quang Tri and Lam Dong is lowest (0.7439). The dendrogram also showed
that Quang Tri population is closely related to Thua Thien Hue population whereas Lam Dong
population and Quang Tri, Thua Thien Hue populations have large distance. Present study
indicated that C. orchioides genetic relationship and diversity levels are high. This is the first report
on genetic diversity of C. orchioides in Vietnam and RAPD is a good molecular marker to study the
genetic diversity of this species.

Keywords: Genetic diversity; Curculigo orchioides; polymorphic bands; RAPD; Vietnam.

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INTRODUCTION MATERIALS AND METHODS

In the genus Curculigo, C. orchioides Gaertn is Sample Collection

often used for traditional medicine in China, India
and Southeast Asia, including Vietnam. C.
orchioides was used for the treatment of
impotence, limb limpness, arthritis of the lumbar
and knee joints, and watery diarrhoea, and also
used as a potent immunomodulatory and
aphrodisiac [1,2]. Due to its important medicinal
valuable activity, C. orchioides demand is
dramatically increasing. As pharmaceutical
companies depended heavily upon material
procured from naturally occurring stands, C.
orchioides plants are being depleted rapidly; as a
result, it has raised concerns about possible
extinction [3]. Thus, restoration and conservation
of the natural population of this potential herb is
necessary.
Sample collection: Ninety-two individual trees
were captured from provinces of Vietnam
including Lai Chau, Quang Binh, Quang Tri, Thua
Thien Hue, Quang Nam, Kon Tum and Lam
Dong. The number of samples from each province
is not the same because of its origin, distribution
and the number of collected regions (Table 1 and
Fig. 2). The fresh leaves were stored on ice in
nylon bags (less than three days) and used directly
for DNA extraction. This study was conducted
during May to July, 2018.

Randomly Amplified Polymorphic DNA

(RAPD) is a simple technique that can be applied
where no knowledge of the target
DNA is available, and no information about the
region amplified is possible, but it is likely
that middle or highly repetitive DNA is primarily
targeted [4]. Since the introduction of
RAPD markers in 1990 [5], application in
plant genetic analysis has increased rapidly.
Molecular diversity studies in some threatened
and rare plants using RAPD polymorphism have
also reported [6]. In Vietnam, genetic diversity of
some medicinal plants also investigated, such as
Paris polyphylla Smith [7], Flemingia
macrophylla (Willd.) Kuntze ex Merr.
[8], Momordica cochinchinensis (Lour.) Spreng
[9].
Fig. 1. Curculigo orchioides herb
In C. orchioides, a study on genetic diversity was
reported. Patel et al. [10] assessed the genetic DNA Isolation
fidelity of somatic embryogenesis derived
regenerate by using RAPD technique, whereas Li Genomic DNA of C. orchioides leaf was isolated
et al. [11] analysed the genetic relationship of 7 as described by Babu et al. [13] with several
species in Curculigo genus. Li et al. [12] also modifications. Briefly, leaf pieces were washed
analysed the genetic diversity of C. orchioides with tap and double distilled water (DDW), cut
from different habitats at China by Inter Simple into small pieces (5×5 mm) and ground to powder
Sequence Repeats (ISSR) technique. However, in liquid nitrogen. The powder (50 mg) were
these studies are limited within C. orchioides homogenised with 1 ml 2× CTAB
populations in China or India. Therefore, this (hexadecyltrimethylammonium bromide) isolation
study aimed to investigate the genetic buffer (200 mM Tris.HCl, 50 mM
variability of C. orchioides populations in ethylenediaminetetraacetic acid (EDTA), 2%
Vietnam. CTAB, 2% β-mecaptoethanol, 2.5% polyvinyl

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pyrrolidone (PVP) and 1.4 M NaCl, pH 8) in a 1.5 UV light. Samples with lack of bands in agarose
mL tube. Twenty microliters of β-mecaptoethanol gel were replicated in twice to confirm. Six
were added and mixed thoroughly for 1-2 min, random decamer oligonucleotides primers
then incubated at 55°C for overnight. Genomic (Operon Technologies, USA) were selected for
DNA was purified by one volume of phenol: evaluation (Table 2).
chloroform: isoamine alcohol solution (25: 24: 1)
and precipitated with one volume of cold Data Analysis
isopropanol 100%. DNA pellets were dried for
overnight at room temperature and resuspended in PCR-RAPD amplicons were scored as presence
30 µL DDW. Total DNA concentration was (1) or absence (0) of a band in each sample. These
determined by using a photo spectrometer at data were used to compile a binary matrix for
260/280 nm. Genomic DNA was diluted to a final cluster analysis using the NTSYS-pc software
concentration of 50 ng/µL for PCR amplification. (numerical taxonomy system, Exeter software,
USA) ver. 2.1. The sizes of the RAPD markers
PCR-RAPD were estimated by using the DNA size standard
(Gene Ruler 1kb DNA Ladder, Thermo
Genomic DNA was used as a template for RAPD Scientific).
analysis (Table 1). PCR reactions were carried out
according to Quang et al. [7]. Each reaction was The genetic identity and genetic distance between
contained 10 µl 2× PCR master mix (M7502, populations were expressed using Nei’s genetic
Promega), 20 pmol primer and 50 ng genomic distance [14]. Genetic parameters were calculated
DNA in a 20 µL final volume. Amplification was as the number of polymorphic bands, percentage
performed in a thermocycler (AerisTM thermal of polymorphic bands (PPB), observed number of
cycler, ESCO) under the following conditions: 3 alleles (na), effective number of alleles (ne), Nei's
min at 95°C; followed by 42 cycles of denaturing (1973) gene diversity (h), Shannon's information
at 92°C for 1 min; annealing at 36°C for 1 min and Index (I), total genotype diversity in populations
an extension at 72°C for 2 min; a final extension at (Ht), total genotype diversity within populations
72°C for 10 min. PCR products were separated on (Hs), mean coefficient of gene differentiation
a 1.4% agarose gel for 6 hr at 40 V [7]. Agarose (Gst), and estimated the gene flow (Nm) for
gels were stained by 6× GelRedTM loading buffer RAPD data using the POPGENE software (ver.
with tricolour (ABT, Vietnam) and determined by 1.31) [15].
Table 1. Specimens for this study with locality and sample codes
Provinces Sample codes Latitudes and
longitudes
Lai Chau (n=12) BB1-1, BB1-2, BB1-3, BB1-4, BB1-5, BB1-6, BB2-1, BB2-2, BB2-3, BB2- 22°22'027N
4, BB2-5, BB2-7 108°11'485E
Quang Binh (n=6) QB2-1, QB2-2, QB2-3, QB2-4, QB2-5, QB2-6 17°23'412N
106°36'290E
Quang Tri (n=11) QT1-1, QT1-2, QT1-3, QT1-4, QT1-8, QT2-2, QT2-3, QT2-4, QT2-5, QT2- 17°03'616N
7, QT2-8 106°56'089E
Thua Thien Hue PB1-1, PB1-2, PB1-4, PB1-5, TP-2, TP-5, TP-6, TP-7, TB1-1, TB1-2, TB1- CH: 16°24'223N
(n=39) 3, TB1-6, TB1-8, CH1-1, CH1-2, CH1-3, CH1-4, CH1-5, PB2-2, PB2-3, 107°35'203E
PB2-4, PB2-5, PB2-7, PB2-8, TB2-1, TB2-2, TB2-3, TB2-4, TB2-5, CH2-1, PB: 16°22'560N
CH2-3, CH2-4, CH2-5, CH2-6, GL1-1, GL1-4, GL1-5, GL2-1, GL2-2, 107°40'563E
GL2-5 Others:
16°29'489N
107°26'509E
Quang Nam (n=8) QN1-2, QN1-3, QN2-1, QN2-2, QN2-3, QN2-4, QN25, QN2-6 15°30'406N
108°28'110E
Kon Tum (n=10) KT1-1, KT1-2, KT1-3, KT1-8, KT2-1, KT2-4, KT2-6, KT2-7, KT2-8, KT2- 14°29'596N
10 107°55'505E
Lam Dong (n=6) DL2-2, DL2-3, DL2-5, DL2-6, DL2-8, DL2-9 12°22'497N
108°25'391E

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Fig. 2. Map of selected C. orchioides populations in Vietnam

RESULTS AND DISCUSSION (OPA-01) (Table 2), and the number of PCR
products of each primer varied from 10 to 12. All
RAPD Analysis of the bands were polymorphic (with a mean of
10.83 bands per primer). This study is in
Among twenty tested random primers, six accordance with the report of Susindran et al. [16]
primers showed high numerous PCR on amplified bands per RAPD primer in
products (data not shown). These selected primers C. orchioides from India which represent 12 bands
generated a total of 65 amplified bands, ranging in for OPA-01 primer and 11 bands for OPA-03
size from approximately 200 (OPN-06) to 2000 bp primer.

Table 2. Sequence of RAPD primer, band sizes, and number of amplified bands

Primer Nucleotide sequence Number of Size range of amplified Number of

code (5’-3’) amplification bands (bp) amplified bands
genotypes
OPA-01 CAGGCCCTTC 90 250-2000 12
OPA-03 AGTCAGCCAC 87 250-1500 10
OPB-18 CCACAGCAGT 87 200-1450 10
OPF-04 GGTGATCAGG 86 250-1750 12
OPG-17 ACGACCGACA 90 280-1200 10
OPN-06 GAGACGCACA 90 200-1500 11
Overall 200-2000 65
Mean 10.83

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In this study, the percentage of polymorphic bands
(PPB) of each population was ranged from 60%
(Lam Dong) to 100% (Thua Thien Hue) (Table 3).
The PPB of all samples was 100%, higher than
PPB value from other reports. In the study about 7
species of Curculigo genus in China, Li et al. [11]
reported that the PPB was 92.36%. Li et al. [12]
reported the genetic diversity of C. orchioides
from different habitats in China implies that 25
germplasm resources by ISSR-PCR. The results
showed that the PPB was 85.80% and genetic
distances were changed from 0.1772 to 0.4030
[12]. The result is higher in compared to the
present study indicates PPB value of 80.66% and
genetic distance ranges from 0.0611 to 0.2958.
The total amplicon among of seven C. orchioides
populations varied from 45 (Lam Dong) to 65
(Thua Thien Hue and Quang Tri). The population
from Lam Dong showed the lowest percentage of
polymorphic bands (60%), while Thua Thien Hue
had the highest polymorphism (100%). These
results indicated that C. orchioides populations in
Viet Nam have a high level of genetic diversity.
Genetic Diversity
Data for observed number of alleles (na), effective
number of alleles (ne), Nei’s (1973) genetic
diversity (h), Shannon’s information index (I), for
all the 7 populations were analysed by using six
RAPD primers and their respective values were
found as 1.8066, 1.5244, 0.3025, and 0.4481,
respectively (Table 4). This study is in accordance
with the previous findings of Li et al. [11],
reported that the effective number of alleles (Ne),
Table 3. Summary of PCR amplicons of C. orchioides in Vietnam using RAPD markers
Populations Number of PCR
amplicons
Lai Chau 61
Quang Binh 54
Quang Tri 65
Thua Thien Hue 65
Quang Nam 60
Kon Tum 61
Lam Dong 45
Mean 58.57
Overall 65
Quang et al.
Nei's gene diversity (h), and Shannon's
information index (I) for 7 species of Curculigo
genus in China were 1.4937, 0.3001, and 0.4577,
respectively.
Lam Dong displayed the lowest value of Nei's
(1973) gene diversity (h=0.1991) and Shannon's
information index (I=3064), whereas the highest
population was recorded for Kon Tum (h=0.3889
and I=0.5636) (Table 4). The average diversity
within the populations (Hs) was 0.3025, which
accounted for 74.93% of the total diversity
recorded in the populations (Ht = 0.4037). The
mean coefficient of gene differentiation (Gst)
value of 0.2505 indicated a high degree of genetic
differentiation among populations. The gene flow
(Nm) was 1.4957, asserted that it was large among
the populations (Table 5).
The values of genetic identity and genetic distance
between 7 C. orchioides populations from Lai
Chau to Lam Dong were given in Table 6. The
analysed data indicated that the values of genetic
identity between populations were high, ranging
from 0.7439 to 0.9407. The populations originate
from Quang Tri and Thua Thien Hue had the
highest genetic identity (0.9407). The values of
genetic distance between populations were low,
varying from 0.0611 to 0.2958 and the population
pairs Quang Tri-Lam Dong have the greatest
genetic distance (0.2958) (Table 6). Genetic
identity between populations was also an indicator
of genetic distance. Genetic identity between
Quang Tri and Thua Thien Hue was the highest
(0.9407) while between Quang Tri and Lam Dong
recorded lowest (0.7439) (Table 6 and Fig. 2).
Number of polymorphic loci Percentage of polymorphic
bands
57 87.69
43 66.15
56 86.15
65 100
46 70.77
61 93.85
39 60
52.43 80.66
65 100
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 M TB1-1 QT1-1
M
QB2-1 M
TB2-1 QN2-1 TB1-2
QB2-2 QT1-2
QN2-2 M
TB2-2
M TB1-3 QT1-3
TB2-3 QN2-3
QB2-3 TB1-6 QT1-4
QN2-4
TB2-4
QB2-4 TB1-8 QT1-8
TB2-5 QN2-5
QB2-5 GL1-1 PB1-1
GL2-1 QN2-6
QB2-6 GL1-4 PB1-2
GL2-2 KT2-1

GL2-5 QT2-2 GL1-5 PB1-4

KT2-4

CH2-1 QT2-3 CH1-1 PB1-5

KT2-6

298
QT2-4 CH1-2

of lane.
CH2-3 QN1-2
KT2-7
CH2-4 QT2-5 CH1-3 QN1-3
KT2-8
QT2-7 CH1-4 KT1-1
CH2-5
KT2-
CH2-6 10 QT2-8 CH1-5
KT1-2

BB2-1 DL2-2 PB2-2 BB1-1

KT1-3

BB2-2 DL2-3 PB2-3 BB1-2

KT1-8

DL2-5 PB2-4 BB1-3

BB2-3 TP-2
BB2-4 DL2-6 PB2-5 BB1-4
TP-5
BB2-5 DL2-8 BB1-5
PB2-7 TP-6

Fig. 3. PCR products of OPG-17 primer of 92 samples. M. DNA molecular size marker
DL-2
BB2-7 PB2-8 BB1-6
TP-7

(GeneRulerTM DNA 1 kb ladder, Thermo Fisher Scientific). Sample codes were located at the top
Quang et al.
 Quang et al.

Table 4. Summary of genetic parameters estimate for 7 populations of C. orchioides in Vietnam

using RAPD markers

Populations na ne h I
Lai Chau 1.8769 1.5242 0.3060 0.4587
Quang Binh 1.6615 1.4570 0.2590 0.3807
Quang Tri 1.8615 1.5935 0.3362 0.4933
Thua Thien Hue 2.0000 1.6233 0.3608 0.5367
Quang Nam 1.7077 1.4584 0.2678 0.3970
Kon Tum 1.9385 1.7007 0.3889 0.5636
Lam Dong 1.6000 1.3136 0.1991 0.3064
Mean 1.8066 1.5244 0.3025 0.4481
Overall 2.0000 1.7092 0.3993 0.5835
Standard deviation (SD) 0 0.2647 0.1034 0.1174

Table 5. Summary analysis of genetic variability across all 7 populations of C. orchioides

Parameters Ht Hs Gst Nm
Across 7 populations 0.4037 0.3025 0.2505 1.4957
SD 0.0104 0.0066

Table 6. Nei’s (1978) genetic identity (above diagonal) and genetic distance (below diagonal)
between 7 populations of C. orchioides in Vietnam

Populations Lai Chau Quang Quang Tri Thua Thien Quang Kon Tum Lam Dong
Binh Hue Nam
Lai Chau *** 0.8402 0.8582 0.9124 0.8116 0.8662 0.7463
Quang Binh 0.1741 *** 0.8551 0.8226 0.7528 0.8238 0.8044
Quang Tri 0.1529 0.1565 *** 0.9407 0.8373 0.8630 0.7439
Thua Thien Hue 0.0917 0.1953 0.0611 *** 0.8990 0.8998 0.7795
Quang Nam 0.2088 0.2840 0.1776 0.1064 *** 0.8372 0.7693
Kon Tum 0.1436 0.1938 0.1473 0.1056 0.1777 *** 0.8716
Lam Dong 0.2926 0.2177 0.2958 0.2491 0.2623 0.1374 ***

In other Curculigo species, Babaei et al. [17]
reported that the observed and expected
heterozygosity of Curculigo latifolia in Malaysia
by using ISSR technique was ranged between
0.00-0.65 and 0.38-0.79, respectively, lower than
that of C. orchioides in the present study.
Meanwhile, Shannon’s information index ranged
from 0.82 to 1.57 indicates the higher value in this
study.
Cluster Analysis
In the genetic similarity dendrogram constructed
on the basis of comparative analysis of the total
loci obtained with the six RAPD primers, the
seven populations distribute based on their
location (Fig. 4). The dendrogram showed that
Quang Tri population was closely related to Thua
Thien Hue population whereas Lam Dong
population and Quang Tri, Thua Thien Hue
populations had large distance. The intraspecific
genetic variations might be due to a difference in
the population architecture and the geographical
conditions. Quang Tri, Thua Thien Hue, Quang
Nam, Quang Binh provinces located at central
Vietnam, all of them are coasted delta areas. Other
provinces are mountain or highland regions, Lai
Chau is a west-northern mountain province and
Kon Tum, Lam Dong are west highland in the
south (Fig. 2). The long geographical
distance indicates the large number of genetic
distances.

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Fig. 4. Dendrogram obtained with UPGMA method based on Nei's (1972) distance for 7 populations
of C. orchioides in Vietnam
CONCLUSION
Among seven regions, Kon Tum has the highest
diversity level while Lam Dong is lowest. It is
concluded that RAPD is a good molecular marker
to study the genetic diversity of C. orchioides.
ACKNOWLEDGEMENTS
This study was supported by the grant from
Vietnam Ministry of Education and Training
(Project No. B2017-DHH-40).
COMPETING INTERESTS
Authors have declared that no competing interests
exist.
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