全 文 ：Journal of Systematics and Evolution 46 (4): 614–621 (2008) doi: 10.3724/SP.J.1002.2008.07123
(formerly Acta Phytotaxonomica Sinica) http://www.plantsystematics.com
Development of species-specific SCAR markers for identification of
three medicinal species of Phyllanthus
Piyada THEERAKULPISUT* Nantawan KANAWAPEE Duangkamol MAENSIRI
Sumontip BUNNAG Pranom CHANTARANOTHAI
(Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand)
Abstract Phyllanthus amarus Schum. & Thonn. has been widely used in traditional medicine in Thailand as an
antipyretic, a diuretic, to treat liver diseases and viral infections. Two closely related species, P. debilis L. and P.
urinaria Klein ex Willd., with different and less effective medicinal properties, are less commonly used. These
three species are similar in morphology and often occur in overlapping populations in nature. The latter two
species can easily be mistaken for P. amarus and collected for medicinal uses, which can lead to undesirable
results. DNA fingerprints of these species were obtained using RAPD-PCR techniques. RAPD markers specific
for each species were identified. Primers for highly specific sequence-characterized-amplified-regions (SCAR)
were then designed from nucleotide sequences of specific RAPD markers. These primers efficiently amplified
SCAR markers of 408, 501 and 319 bp unique to P. amarus, P. debilis and P. urinaria respectively. This method
of plant identification was rapid and highly specific when tested against DNA of several closely related species
and was able to amplify specific markers from mixed DNA samples.
Key words Phyllanthus amarus, Phyllanthus debilis, Phyllanthus urinaria, RAPD-PCR, sequence-charac-
terized-amplified-region (SCAR) marker.
The genus Phyllanthus L. (Phyllanthaceae, for-
merly Euphorbiaceae) consists of about 800 species of
trees, shrubs, and annual or biennial herbs distributed
throughout the tropical and subtropical regions of both
hemispheres (Govaerts et al., 2000). Among the small
herbs in this genus, P. amarus Schum. & Thonn., P.
debilis Klein ex Willd., P. fraternus Webster, P. niruri
L. and P. urinaria L. have been used in Ayurvedic
medicine for over 2000 years and have an enormous
number of traditional uses in many countries (Unander
et al., 1990). P. amarus has been extensively used in
pharmacological research due to its anti-HBV (hepati-
tis B virus, Liu et al., 2001), anti-HIV (Notka et al.,
2004), anti-mutagenic (Raphael et al., 2002), anti-
inflammatory (Raphael & Kuttan, 2003) and antioxi-
dant (Hari Kumar & Kuttan, 2004) properties. Less
pharmacological information was available for P.
urinaria, in terms of its anti-HBV (Chan et al., 2003),
anticancer (Huang et al., 2004) and anti-nociceptive
(Santos et al., 1995) properties. Limited research has
been done on P. debilis, for example, its effect against
CCl[4]-induced rat liver dysfunction (Shah et al.,
2002). Recent pharmacological research has been
focused on the inhibitory effects of P. amarus and
related species on HBV, which has been found to be
associated with chronic liver disease and primary liver
cancer. Unfortunately, a great deal of confusion exists
among scientists regarding plant identification, which
makes evaluation of published information difficult.
P. amarus is considered by some authors as a variety
of P. niruri, while in several reports one name is
indicated to be synonymous of the other.
In Thailand, four species of herbaceous Phyllan-
thus (P. amarus, P. debilis, P. urinaria and P. virgatus
Forst.) are collectively called “Luk-tai-bai” or
“Ya-tai-bai” (Bansiddhi, 1991). P. amarus, P. debilis
and P. urinaria are similar in apparent vegetative
morphology. Plants are annual herbs, growing to a
similar height (60–70 cm) with a similar pattern of
branching, leaf size and arrangement, flowers and
fruits. Microscopic inspection of the number of sepals
and fruit surface is needed for correct identification of
plants (Chantaranothai, 2007). Moreover, these plant
species commonly grow together in the same open
habitat and wastelands. However, ethnomedical uses
and some aspects of pharmacological activities among
these species are different (Bunyaprapasara &
Chokechaichareon, 2000). Some “Luk-tai-bai” herbal
products in Thailand do not indicate the species name
on the packages. Therefore, there exists the possibility
of collecting the wrong plant species for medical uses.
Recently, various DNA-based methods have been
successfully used for pharmacognostic characterization

———————————
Received: 18 September 2007 Accepted: 5 November 2007
* Author for correspondence. Tel.: +66-43-342908; Fax: +66-43-364169;
E-mail: .
THEERAKULPISUT et al.: Development of SCAR markers for identification of Phyllanthus species

615
of medicinal plants and herbal medicine for the pur-
pose of quality control and standardization (Joshi et
al., 2004). The aim of this research is to develop
species-specific sequence-characterized-amplified-region
(SCAR) markers that can identify and differentiate
these morphologically similar Phyllanthus species.
1 Material and methods
1.1 Plant materials
Whole plants of Phyllanthus amarus, P. debilis,
P. urinaria, P. emblica L., P. acidus (L.) Skeels, P.
chamaepeuce Ridl., P. isanensis, P. chantaranothai
(unpublished), P. myrtifolius (Wight) Müll. Arg., P.
mirabilis Müll. Arg., P. reticulatus Poir., P. pulcher
Wall. ex Müll. Arg. and Sauropus androgynus (L.)
Merr. were collected from different parts of Thailand.
Plants were identified (Chantaranothai, 2007) and
deposited at KKU Herbarium, Khon Kaen University,
Thailand. Fresh leaf samples were collected, frozen in
liquid nitrogen and stored at –80 until used for ℃
DNA isolation.
1.2 DNA isolation
DNA was isolated from fresh or frozen leaves
using a modified CTAB method (cetyl trimethyl
ammonium bromide, Doyle & Doyle, 1987) or the
Plant DNeasy Mini Kits (QIAGEN, Germany).
Briefly, leaf samples (0.2–0.5 g) were ground to fine
powder in liquid nitrogen and transferred to a micro-
centrifuge tube containing freshly prepared equal
volume of extraction buffer (100 mmol/L Tris buffer
pH 8.0, 20 mmol/L Na2EDTA, 1.4 mol/L NaCl, 2%
CTAB, 1% polyvinyl pyrrolidone). The suspension
was gently mixed and incubated at 60 for 60 min ℃
with occasional mixing. The suspension was then
cooled to room temperature and an equal volume of
chloroform : isoamyl alcohol (24:1) was added. The
mixture was centrifuged at 13000 r/min for 10 min.
The clear upper aqueous phase was then transferred to
a new tube containing 0.5 mL ice-cooled isopropanol
and incubated at –20 for 30 min. The nucleic acid ℃
was collected by centrifuging at 13000 r/min for 10
min. The resulting pellet was washed twice with 70%
ethanol containing 10 mmol/L ammonium acetate.
The pellet was air-dried under a sterile laminar hood
and the nucleic acid was dissolved in TE (10 mmol/L
Tris buffer pH 8.0, 1 mmol/L Na2EDTA) at 4 . The ℃
contaminating RNA was eliminated by treating the
sample with RNase A (20 µg/µL) for 30 min at 37 . ℃
DNA concentration and purity were determined by
measuring the absorbance of diluted DNA solution at
260 nm and 280 nm. The quality of the DNA was
determined using agarose gel electrophoresis stained
with ethidium bromide. For most plant species, sati-
sfactory DNA was obtained from the above method.
However, for a few plant species which contained a
large amount of secondary plant metabolites, better
quality DNA was obtained using the Plant DNeasy
Mini Kits according to the manufacturer’s instruc-
tions.
1.3 RAPD amplification
For the evaluation of genetic relationships among
four common medicinal species of Phyllanthus in
Thailand, samples of P. amarus, P. debilis, P. uri-
naria and P. emblica were collected from different
localities mainly in north-eastern Thailand. PCR
reactions were carried out in 25 µL reaction tubes
using eight random decanucleotide primers, OPA2,
OPA3, OPA9, OPA13, OPB7, OPB17, OPJ15 and
OPJ20 (Operon Technologies Inc., USA). Each
reaction tube contained 50 ng template DNA, 1.5
mmol/L MgCl2, 300 µmol/L of dNTPs, 1×Taq DNA
polymerase buffer, 25 pmol decanucleotide primer
and 2 units of Taq DNA polymerase (Promega, USA).
Amplification was performed in a DNA thermal
cycler (Corbett Research, Australia) using the follow-
ing conditions: 95 for 3 min; 40 cycles at 94 for ℃ ℃
1 min, 40 for 1 min and 72 for 2 min; final ℃ ℃
extension at 72 for 7 min. PCR products were ℃
resolved in 1.5% agarose gel in 1×TBE buffer. The
DNA was stained with 0.5 mg/mL ethidium bromide,
visualized and photographed under a UV transillumi-
nator. PCR products were scored as either present (1)
or absent (0), on the basis of size. The data were used
to calculate similarity coefficient (Nei & Li, 1979),
and a dendrogram was constructed by UPGMA cluster
analysis using the NTSYS program to analyze genetic
relationships within and among species. For identify-
cation of species-specific RAPD markers, DNA of P.
amarus, P. debilis and P. urinaria (five samples of
each species collected from different localities) was
performed in 25-µL reaction using five random deca-
nucleotide primers which gave clear and reproducible
fingerprint pattern (OPA9, OPB7, OPB17, OPJ17 and
OPJ19). The conditions for PCR amplification and
electrophoresis were as described above.
1.4 Transformation of species-specific RAPD
fragments into sequence-characterized-amplified-
region (SCAR) markers
The species-specific RAPD fragments that are
monomorphic in all samples of each of the three
species but absent from samples of the other two
species (P. amarus, P. debilis and P. urinaria) were
identified, excised from the gel and eluted using a
Journal of Systematics and Evolution Vol. 46 No. 4 2008 616
QIAquick Gel Extraction Kit (QIAGEN, Germany).
The eluted putative species-specific DNAs were
cloned into pGEM®-T easy vector (Promega, USA)
following the manufacturer’s instruction. The ligated
plasmid was introduced into Escherichia coli strain
JM109 following the protocols for preparing compe-
tent cells and transformation using the calcium chlo-
ride method (Sambrook & Russell, 2001). White
colonies were picked from LB-X-gal plates and grown
overnight in LB medium containing ampicillin. The
recombinant DNA was isolated from the bacterial
culture using a rapid phenol extraction method
(Serghini et al., 1989). Confirmation of successful
cloning was carried out by amplifying the plasmid
DNA using SP6 and T7 primer. The inserted frag-
ments were sequenced at the Department of Biochem-
istry, Faculty of Medicine, Khon Kaen University, on
an Applied Biosystems 373 automated sequencer,
using an ABI PRISM dye terminator cycle sequencing
kit. Specific SCAR primers were designed using the
program GeneFisher (Giegerich et al., 1996). Primers
were synthesized by Bioservice, Biotec, Thailand.
1.5 Specific PCR amplification
The species specificity of the SCAR primer pairs
were verified by PCR amplification. Firstly, the
SCAR primers specific for P. amarus (PA-B7), P.
debilis (PD-A9) and P. urinaria (PU-J19) were used
to amplify genomic DNA from the three species (three
samples of each species from different localities).
Secondly, the amplification reactions were carried out
using mixed DNA samples as templates. The mixed
DNA samples were prepared by combining equal
amounts of leaf samples (0.2 g each) of different
species before proceeding for DNA isolation as
described. Four types of mixed DNA samples in-
cluded P. amarus with P. debilis, P. amarus with P.
urinaria, P. debilis with P. urinaria and all three
species together. Thirdly, the amplification reactions
were carried out using genomic DNA from P. amarus,
P. debilis, P. urinaria, P. emblica, P. acidus, P.
chamaepeuce, P. isanensis, P. myrtifolius, P. mirabi-
lis, P. reticulatus, P. pulcher and Sauropus an-
drogynus. PCR reactions were performed in a 25-μL
reaction tube containing 50 ng template DNA, 1
μmol/L each of SCAR-forward and SCAR-reverse
primer, 2.5 mmol/L MgCl2, 0.2 mmol/L dNTPs and
1.25 units Taq DNA polymerase. The parameters used
were 94 for 3 min; 40 cycles at 94℃ for 30 s, 62 ℃
or 64℃ for 30 s and 72℃ for 1 min, and final ℃
extension at 72 for 7 min. Annealing temperatures ℃
of either 62 or 64 were used depending on the ℃ ℃
SCAR primers used.
2 Results
2.1 Genetic relationships between four medicinal
species of Phyllanthus
Preliminary screening of 78 random decanucleo-
tide primers showed that 56 primers were able to
prime genomic DNA of P. amarus and resulted in
amplified PCR products of a variable number of DNA
bands from only one band to 17 bands per primer. In a
subsequent study, it was found that only eight primers
could be used to amplify DNA from all four medicinal
species of Phyllanthus. The use of eight random
primers to amplify DNA of P. amarus, P. debilis, P.
urinaria and P. emblica collected from 7–8 localities
in Thailand resulted in 142 polymorphic bands rang-
ing in size from 200–2100 bp. The dendrogram con-
structed based on RAPD polymorphisms (Fig. 1)
showed that P. urinaria, P. debilis and P. emblica are
more closely related to each other and clustered in one
major group, whereas P. amarus was separated in
another group. The results from the dendrogram
showed that different plant species showed different
degrees of genetic diversity among plants from dif-
ferent populations. However, RAPD polymorhisms
can differentiate species of Phyllanthus from one
another.
2.2 Identification of RAPD markers for P. ama-
rus, P. debilis and P. urinaria
Amplification of three morphologically similar
herbaceous Phyllanthus species using five selected
primers, namely OPA9, OPB7, OPB17, OPJ17 and
OPJ19, produced good quality, reproducible finger-
print patterns and showed a high level of consistency
of fingerprints among samples of the same species
collected from different localities. Several spe-
cies-specific RAPD markers were identified from
PCR amplification products using these primers (Fig.
2). For example, PCR amplification using primer
OPB7 (Fig. 2: A) produced three clear fragments
(approximately 740, 620 and 500 bp) which are
monomorphic for all five DNA samples of P. amarus
but absent from all DNA samples of P. debilis and P.
urinaria. Five fragments (900, 650, 550, 510 and 370
bp) were specific for P. debilis. When the primer
OPA9 was used (Fig. 2: B), four (approximately 1000,
900, 620 and 310 bp) and two (approximately 1500
and 550 bp) specific fragments were amplified from
P. amarus and P. debilis DNA respectively. When the
primer OPJ19 was used, a 500 bp band and a 400 bp
band specific to P. amarus and P. urinaria respec-
tively were detected (Fig. 2: C).
THEERAKULPISUT et al.: Development of SCAR markers for identification of Phyllanthus species

617
Coefficient
0.54 0.64 0.74 0.84 0.94
PA1
PA1
PA4
PA6
PA2
PA5
PA3
PA7
PA8
PU1
PU2
PU3
PU4
PU5
PU7
PU6
PD1
PD2
PD3
PD4
PD5
PD6
PD7
PE1
PE4
PE6
PE7
PE8
PE2
PE3
PE5

Fig. 1. A dendrogram showing genetic relationship among four species of Phyllanthus based on RAPD analysis. PA1–PA8=P. amarus collected
from 8 localities; PU1–PU7=P. urinaria from 7 localities; PD1–PD7=P. debilis from 7 localities; PE1–PE8=P. debilis from 8 localities.

2.3 Cloning, sequencing and design of SCAR
primers
Several specific RAPD fragments of high inten-
sity and reproducibility were eluted, cloned and
sequenced. A number of DNA fragments were suc-
cessfully cloned and sequenced. Nucleotide sequences
of three RAPD amplicons specific for P. amarus, P.
debilis and P. urinaria, amplified using primer OPB7,
OPA9 and OPJ17 respectively, are reported in Fig. 3.
Comparison of the nucleotide sequences of these three
RAPD fragments did not significantly match any
known sequences in the database. Three SCAR primer
pairs, generated from nucleotide sequences (Fig. 3) of
those three RAPD markers, are reported in Table 1.
2.4 Amplification of Phyllanthus species using
SCAR primers
The efficacy of each SCAR primer set for ampli-
fication of specific PCR product size in particular
species was assessed (Fig. 4) using DNA from three
samples (from three localities) of each of the three
species. Fig. 4A shows that SCAR primers PA-B7F/
PA-B7R were able to amplify only P. amarus DNA,
and not that of P. debilis and P. urinaria, producing a
specific band of 408 bp. Likewise, SCAR primers
PD-A9F/PD-A9R and PU-J19F/PU-J19R produced
specific bands of 549 and 321 bp in P. debilis (Fig. 4:
B) and P. urinaria (Fig. 4: C) respectively. The integ-
rity of all nine DNA samples was assessed by ampli-
fication of partial 18S rDNA from all samples (Fig. 4:
D).
To further confirm the effectiveness of each
SCAR primer set, four types of mixed DNA samples
were used as templates for PCR reactions. An equal
amount of DNA from each species was mixed for
each combination as follows: (1) P. amarus with P.
debilis, (2) P. amarus with P. urinaria, (3) P. debilis
with P. urinaria and (4) mixture of DNA from all
three species. As shown in Fig. 5, SCAR primers
PA-B7F/PA-B7R, PD-A9F/PD-A9R and PU-J19R/
PU-J19R were able to amplify specific DNA bands
arising from specific annealing between the SCAR
primers and DNA of P. amarus, P. debilis and P.
urinaria, respectively, despite the presence of con-
taminating DNA from closely related species. In Fig.
6 the specificity of each SCAR primer pair was veri-
fied against 11 species of Phyllanthus and Sauropus
androgynus (Phyllanthaceae). The SCAR primers
PA-B7F/PA-B7R, PD-A9F/PD-A9R and PU-J19R/
PU-J19R clearly amplified SCAR markers from P.
amarus, P. debilis and P. urinaria, respectively, but
Journal of Systematics and Evolution Vol. 46 No. 4 2008 618
Table 1 Species-specific SCAR primers for Phyllanthus amarus, P. debilis and P. urinaria derived from nucleotide sequences of RAPD amplicons
Plant species RAPD primer SCAR primer Sequence (5′ →3′) Product size Annealing temperature
P. amarus Schum. &
Thonn.
OPB7 PA-B7F
PA-B7R
AAACGAGTCCTCCCGGTA
CGCAGGGAAGGTGAAGGA
408 64
P. debilis Klein ex
Willd.
OPA9 PD-A9F
PD-A9R
AACGCCCAATATGCTCGA
GTAACGCCCAAAGCCTCA
549 62
P. urinaria Klein ex
Willd.
OPJ19 PU-J19F
PU-J19R
TGTCACTCCTCACCGTCA
CATCGGTTCCAGCCACCA
321 64




























Fig. 2. RAPD fingerprints resulted from PCR amplification of five
DNA samples each of P. amarus (lanes 1–5), P. debilis (lanes 6–10)
and P. urinaria (lanes 11–15) using primers OPB7 (A), OPA9 (B) and
OPJ19 (C). Fragments specified by the arrows were subsequently
cloned and sequenced. M, DNA size markers.

not from any other species of Phyllanthus or S. an-
drogynus.
3 Discussion
Based on the present investigation, polymor-
phisms among four common medicinal species of
Phyllanthus were detected effectively by RAPD
analysis, despite polymorphism among different
specimens of the same species (Fig. 1). Intraspecific
genetic variation can arise in natural populations as a
result of free pollen flow and cross-fertilization (Jain
et al., 2003). Moreover, further variation could have
arisen through dissemination of cross-hybridized
seeds by wind, animal and human activities due to the
weedy nature of these species. As shown in Fig. 1, P.
amarus, the most common and widely used medicinal
Phyllanthus species in Thailand, was genetically most
distant from the other three species, which are more
closely related to one another. Therefore, RAPD
polymorpisms can potentially be used to discriminate
P. amarus from its close relatives, P. debilis and P.
urinaria. RAPD analysis has been widely used for
differentiation of a large number of medicinal species
from their close relatives or adulterants, including
Panax species (Shaw & But, 1995), Coptis species
(Cheng et al., 1997), Astragalus species (Cheng et al.,
2000) and turmeric (Sasikumar et al., 2004). The
advantages of RAPD techniques include their simplic-
ity, rapidity, the low amount of genomic DNA re-
quired and the fact that isotopes and prior genetic
information are not required (Williams et al., 1990).
Many technical disadvantages associated with
RAPD have, however, raised questions on its fidelity
as a genetic marker technique and prevented its wide-
spread use in recent years. The reproducibility of
RAPD is affected by DNA quality, primer and tem-
plate concentration, different thermocyclers and even
different sources of DNA polymerase (Ellsworth et
al., 1993; Muralidharan & Wakeland, 1993). Subse-
quently, conversion of RAPD to SCAR markers
(Paran & Michelmore, 1993), by developing longer,
hence more specific primers from RAPD sequences,
has significantly improved the reproducibility and
reliability of PCR assays. In this study, we focused on
the identification of specific markers for three me-
dicinal herbaceous species of Phyllanthus. By using
five random decanucleotide primers, several RAPD
markers specific to different samples of the interested
Phyllanthus species, but absent from all samples of
the other two species, were recognized (Fig. 2).
However, only strong, reproducible RAPD bands of
appropriate sizes, from two repeating PCR reactions
for each primer, were selected for cloning and
sequencing. Three SCAR primers designed from three
specific RAPD sequences for each species were
THEERAKULPISUT et al.: Development of SCAR markers for identification of Phyllanthus species

619
obtained (Table 1; Fig. 2). The most suitable anneal-
ing temperature, which gave sharpest and most intense
products, was empirically determined for each primer
(Fig. 3). Performing PCR reactions using annealing
temperatures within a range of four degrees against
those reported in Table 1 also resulted in spe-
cies-specific SCAR marker bands, although with
lower intensity. In Thailand, P. amarus, P. debilis and
P. urinaria are common weed species usually growing
in open places, waste grounds and secondary forests
(Chantaranothai, 2007). Moreover, these species are
very similar and often found growing together in the
same habitat. Two or more species of “Luk-tai-bai”
can inadvertantly be harvested for production of
medicinal products. The SCAR markers developed in
this study can be used to verify the presence of spe-
cific species using PCR reactions even when the DNA
samples were contaminated with that of closely
related species (Fig. 4). The markers can also differ-
entiate P. amarus, P. debilis and P. urinaria from
several closely related species, some of which are
commonly used as food or traditional medicine,
namely, P. acidus, P. emblica, P. chamaepeuce, P.
mirabilis, P. myrtifolius, P. pulcher and S. an-
drogynus.
Species-specific SCAR markers have been used
in several studies to differentiate important medicinal
herbs from their close relatives or adulterants. In Hong
Kong, cheaper Panax ginseng (Ginseng) has been
substituted for the more expensive P. quinquefolius
(American ginseng) for export. An SCAR marker was
developed which can differentiate these two species as
well as four other species of Panax (Wang et al.,
2001). In Korea, six species of Artemisia herbs have
been used as medicine, food, spices and ornamentals.
However, only A. princeps and A. argyi have been
used in traditional medicine to treat colic pain, vomit-
ing and diarrhoea, and irregular bleeding from the
uterus. A pair of SCAR primers was developed which
efficiently amplified a 254-bp sized SCAR marker

OPB7
(a) 1 GGTGACGCAG CAGAGTAAGT TCAATCCTTA TGGCAACAAG TGTAAATGTA CGACTTCTTC
PA-B7F
61 ATGGAACGAG TACAGTAGCA AACGAGTCCT CCCGGTATCT CGTAATCGAT GAATAATCGG
121 TTGCATTGCA TTCTGTTACC TGTTTGTAAT TTGGTCCCAA GTTCAGGCAA GAACTGAGAA
181 GGTTCCTTCC ACTCGTATTT TTCTTTCACC CAAGTATCGA AGATATTAAG CACGTGTAGC
241 CATTGGCTCT GGTGAGTCGA TCACCGGACC CGTAACAAAA TACCGCCCAT CCATCTTCAT
301 TCGAATCGAA GCTGAGCAAA TTTATGATAT CTGACGCTAG TCTCTGTTGC AATCCTTTGT
361 CAATGCGTCC ATTTGAATAG AGAATACTCT TCAACCGGCT CCAAAATAGC CATATGGATG
421 ACTCGCTCCA GCTCTGGCTT ATCTTATTCA CATTTATCTT CGAAATGTTC TCCTTCACCT
OPB7 PA-B7R
481 TCCCTGCGTC ACC

OPA9 PD-A9F
(b) 1 GGGTAACGCC CAATATGCTC GATGCTCAAG CTCTGCCCAA AGATGACACG GCTTGCCATA
61 GACGAGCCGA AATGGGGACA TCCCAATAGG CATCTTATAA GCAGTTTGGT ATGCCCATAA
121 TGCATCATCA AGCTTCATAG CCCAATCTTT CCTAGTAGAA GATATCGTCT TTTCCAAGAT
181 CCTTTTCAAC TCTCTATTGG ACACTTCTAC TTGGCCACTT TTTTGAGGAT GATAAGAAGT
241 TGCCAAAAAA TGTTTGCGTC CATACTTCAA TAGAAGTGCA GTGAATTGGT GATTTGTAAA
301 ATGTACCCTG TTGTCATTTA TAATCACCCT AGGAACTCCG AATCTTGTAG AGATGTTTCT
361 CTTGAAAAA TTTCACTATC GAATGAGCCT TGTTATCCAG CAAAGCGACG GCTTCCACCC
421 ATTGCTCACA TAGTCGACCG CCACCAAAAT ATACTCGTTT CCAAAATATT TAAGAAACGG
481 TCCCATAAAA TCCAAACCCC AAACATCAAA TAGCTCGACT TTAAAGATCC CTGTTTGAGG
OPA9 PD-A9R
541 CTTTGGGCGT TACCC
OPJ19
(c) 1 GGACACCACT CTCCCCTCTC CTTTTCCTCT CTCATGTGTC TCTCCTCTCT TCTCTCTCTC
PU-J19F
61 GTTGACGTTT GTCACTCCTC ACCGTCAACG TCATGCACAG ACGTAAACCA TTTTCTTCTC
121 TTGTTCAGCT TTATCATTGA ACACAACTGA CTTTTCCTTC CTCTCATGTT CGATTCTCTC
181 TCCATCGTCT TCATGTCGTC ATTTGCTCTC TTCCTCCACT GTTTCTCCTT AGGTCATGT
241 CATGGTTAAG GTAGGGTCTT GGTTTGGTTA GGGTTTGTAC TTGGTTGGGT TGGGTTACAG
301 TTTTAGGGGC TGGTTTTGCG TGGATGTGGA GTTTTGGAGC TGGTGGGGTT CAAGTCAGGT
361 TGTGGTTGAG TTTGGTGGCT GGAACCGATG GAGTTCATGT AGTGGTGTCC
PU-J19R OPJ19

Fig. 3. Nucleotide sequence of RAPD amplicons specific to (a) P. amarus, (b) P. debilis, and (c) P. urinaria. Nucleotides in bold represent the
RAPD primers and those underlined indicate the designed SCAR primers.
Journal of Systematics and Evolution Vol. 46 No. 4 2008 620


















Fig. 4. PCR amplification of genomic DNA from P. amarus (lanes
1–3), P. debilis (lanes 4–6) and P. urinaria (lanes 7–9) using the
designed SCAR primers (A) PA-B7F/PA-B7R, (B) PD-A9F/PD-A9R,
(C) PU-J19F/PU-J19R, and (D) PCR amplification using 18S rDNA
primers. C, negative control with no genomic DNA template; M,
DNA size markers.




















Fig. 5. Test of specificity of SCAR primers using mixed genomic
DNA as templates, (A) PA-B7F/PA-B7R, (B) PD-A9F/PD-A9R and
(C) PU-J19F/PU-J19R. Lane 1, DNA of P. amarus and P. debilis; lane
2, DNA of P. amarus and P. urinaria; lane 3, DNA of P. debilis and P.
urinaria; lane 4, mixed DNA from all three species; M, DNA size
markers.

from A. princeps and A. argyi, not from other Ar-
temisia herb species (Lee et al., 2006). An SCAR
marker of 343 bp was previously found to be specific
for fresh and medicinal samples of Phyllanthus em-
blica (Warudee et al., 2006). This marker was absent
from six other species of Phyllanthus used in Ay-
urvedic medicine in India. An SCAR marker has also
been used to differentiate Sinocalycanthus chinensis,
an endangered shrub species with high ornamental
value in China, from Calycanthus and Chiminanthus
species which are close relatives and have a similar
morphology at the seedling stage (Ye et al., 2006).
Molecular marker technology proves to be valu-
able tools for genotyping medicinal plants and detect-
ing adulterations and substitutions in herbal medicine.
The SCAR markers obtained from this study are
useful for identifying three morphologically similar
herbaceous species of Phyllanthus. Using DNA
markers developed in this study, P. amarus, the most
widely used in traditional medicine can be efficiently
differentiated from P. urinaria, which is used to a
much lesser extent and also from the least known P.
debilis. The PCR-based identification is especially
useful when botanical identification based on mor-
phology becomes difficult, such as in the case of
incomplete or damaged samples, and in dried herbal
products.




















Fig. 6. PCR products of eleven species of Phyllanthus and Sauropus
androgynus using the designed SCAR primers. (A) PA-B7F/PA-B7R,
(B) PD-A9F/PD-A9R, (C) PU-J19F/PU-J19R and (D) PCR amplifica-
tion using 18S rDNA primers. Lane 1, P. amarus; 2, P. debilis; 3, P.
urinaria; 4, P. emblica; 5, P. acidus; 6, P. chamaepeuce; 7, P.
isanensis; 8, P. myrtifolius; 9, P. mirabilis; 10, P. reticulatus; 11, P.
pulcher; 12, P. welwitschianus; 13, Sauropus androgynus; C, negative
control; M, DNA size markers.
Acknowledgements Financial support from the
Khon Kaen University Research Fund is highly
appreciated. The authors also wish to thank Dr. David
SIMPSON for his help during the second author’s
THEERAKULPISUT et al.: Development of SCAR markers for identification of Phyllanthus species

621
training at the Royal Botanic Gardens, Kew and for
critically reading the manuscript.
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