American Journal of Plant Sciences, 2010, 1, 32-37
doi:10.4236/ajps.2010.11005 Published Online September 2010 (http://www.SciRP.org/journal/ajps)
Phylogenic Study of Twelve Species of Phyllanthus
Originated from India through Molecular Markers
for Conservation
Gyana Ranjan Rout*, Subhashree Aparajita
Department of Agricultural Biotechnology, College of Agriculture, Orissa University of Agriculture & Technology, Bhubaneswar, India.
Received July 2nd, 2010; revised July 22nd, 2010; accepted September 8th, 2010
ABSTRACT
The objective of the study was to characterize the germplasm for identification and phylogeny study for conservation.
Identification and characterization of germplasm is an important link b etween the conservation and utilization o f plant
genetic resources. The present investigation was undertaken to draw the phylogenetic relationship between twelve spe-
cies from India belonging to genus Phyllanthus with the help of molecular markers. In total, 259 marker loci were as-
sessed, out of which 249 were polymorphic revealing 96.13% polymorphism. Nei’s similarity index varies from 0.23 to
0.76 for RAPD and 0.26 to 0.81 for ISSR marker systems. Cluster analysis by unweighted pair group method (UPGMA)
of Dice coefficient of similarity genera ted dendogram with more or less similar topo logy for both the analysis that gave
a better reflection of diversity and affinities between the species. The phylogenetic tree obtained from both RAPD and
ISSR marker has divided the 12 species in two groups: group I consisting of only one species Phyllanthus angu stifolius
and the group II with the rest 11 species. This molecular result is comparable to notable morpholog ical charac teristics.
The present study revealed th e distant variation within the species of Phyllan thus. This investigation will help for iden-
tification and conservation of Phyllanthus species.
Keywords: Genetic Variation, ISSR, Medicinal Plant, RAPD
1. Introduction
The genus Phyllanthus belonging to family Euphorbiac-
eae is an important group of medicinal plants used for
various purposes. In Phyllanthus emblica L. Syn: Embli-
ca officinalis Gaertn, the fruit is used for diverse applic-
ations in healthcare, food and cosmetic industry. It has
been well studied for immunomodulatory, anticancer, an-
tioxidant and antiulcer activities [1]. Phyllanthus amarus
is an important folk remedy used in the treatment of a
variety of ailments [2]. In India, it is predominantly used
as a cure for liver disorders [3,4]. The aqueous extract
from Phyllanthus amarus has been reported to inhibit
DNA polymerase of Hepatitis-B and woodchuck hepati-
tis virus. Proper identification of genotype, therefore, re-
mains important for protection of both the public health
and industry. Chemo profiling and morphological eva-
luation are routinely used for identification of genotype.
Chemical complexity and lack of therapeutic markers are
some of the limitations associated with the identification
of genotype. Molecular markers have provided a power-
ful new tool for breeders to search for new sources of va-
riation and to investigate genetic factors controlling qua-
ntitatively inherited traits. The molecular approach for id-
entification of plant varieties/genotypes seems to be more
effective than traditional morphological markers because
it allows direct access to the hereditary material and ma-
kes it possible to understand the relationships between
individuals [5,6]. Genetic polymorphism in medicinal pl-
ants has been widely studied which helps in distinguish-
ing plants at inter- and / or intra-species level. The most
important role of conservation is to preserve the genetic
variation and evolutionary process in viable populations
of ecologically and commercially viable varieties / geno-
types in order to prevent potential extinction. PCR- based
molecular markers are widely used in many plant species
for identification, Phylogenetic analysis, population stud-
ies and genetic linkage mapping [5]. Both RAPD and IS-
SR marker, based on PCR techniques have proven to be a
reliable, easy to generate, inexpensive and versatile set of
marker that rely on repeatable amplification of DNA se-

Phylogenic Study of Twelve Species of Phyllanthus Originated from India through
Molecular Markers for Conservation
33
quence using single primers. The RAPD and ISSR mark-
ers can be used in the study of the genetic variability of
species or natural populations and in the identification of
genotypes [7-14]. In this communication, we report the
feasibility of PCR-based DNA (RAPD and ISSR) marker
for phylogeny study and identification for conservation
of Phyllanthus species.
2. Materials and Methods
2.1. Plant Materials
Twelve species of Phyllanthus were collected from natu-
ral forest of Orissa, India and used for molecular analy-
sis.
2.2. DNA Isolation and Quantification
DNA was extracted from fresh leaves by using the Cetyl-
trimethyl ammonium bromide (CTAB) method [15]. Ap-
proximately, 20 mg of fresh leaves was ground to pow-
der in liquid nitrogen using a mortar and pestle. The gr-
ound powder was transferred to a 50 ml falcon tube with
10 ml of CTAB buffer [2% (w/v) CTAB, 1.4 M NaCl, 20
mM EDTA, 100 mM Tris (tris (hydroxymethyl) aminom-
ethane)-HCl, pH 8.0, and 0.2% (v/v) β-mercaptoethanol].
The homogenate was incubated at 60˚C for 2 h, extracted
with an equal volume of chloroform/isoamyl alcohol (24:
1 v/v) and centrifuged at 10,000 x g for 20 min. DNA
was precipitated from the aqueous phase by mixing with
an equal volume of isopropanol. After centrifugation at
10,000 x g for 10 min, the DNA pellet was washed with
70% (v/v) ethanol, air-dried and resuspended in TE (10
mM Tris-HCl, pH 8.0, and 0.1 mM EDTA) buffer. DNA
quantifications were performed by visualizing under UV
light, after electrophoresis on 0.8% (w/v) agarose gel at
50 V for 45 min and compared with a known amount of
lambda DNA marker (MBI, Fermentas, Richlands B.C.,
Old). The resuspended DNA was then diluted in TE bu-
ffer to 5 µg/µl concentration for use in polymerase chain
reaction (PCR).
2.3. Primer Screening
Thirty decamer primers, corresponding to kits A, D, and
N from Operon Technologies (Alameda, California, USA)
and twenty synthesized ISSR primer (M/S Bangalore Ge-
nei, Bangalore, India) were initially screened using one
species of Phyllanthus i.e., ‘Phyllanthus virgatus’ to de-
termine the suitability of each primer for the study. Pr-
imers were selected for further analysis based on their ab-
ility to detect distinct, clearly resolved and polymorph-
hic amplified products within the species. To ensure rep-
roducibility, the primers generating no, weak, or complex
patterns were discarded.
2.4. RAPD and ISSR Assay
Polymerase chain reactions (PCR) with single primer
were carried out in a final volume of 25 l containing 20
ng template DNA, 100 M of each deoxyribonucleotide
triphosphate, 20 ng of decanucleotide primer (M/S Ope-
ron Technology), 1.5 mM MgCl2, 1X Taq buffer [10 mM
Tris-HCl (pH 9.0), 50 mM KCl, 0.001% gelatin], and 0.5
U Taq DNA polymerase (M/S Bangalore Genei, India).
Amplification was performed in a PTC-100 thermal cy-
cler (M J Research Inc., Watertown, MA, USA) progr-
ammed for a preliminary 2 min denaturation step at 94˚C,
followed by 40 cycles of denaturation at 94˚C for 20 s.,
annealing at required temperature for 30 s and extension
at 72˚C for 1 min, finally at 72˚C for 10 min for amplifi-
cation. Amplification products were separated alongside
a molecular weight marker (1.0 kb plus ladder, M/S Ban-
galore Genei) by 1% and 1.5% (W/V) agarose gel for
RAPD and ISSR respectively. Electrophoresis was done
in 1X TAE (Tris acetate EDTA) buffer, stained with eth-
idium bromide and visualized under UV light. Gel photo-
graphs were scanned through a Gel Documentation Sys-
tem (Gel Doc. 2000, BioRad, California, USA) and the
amplification product sizes were evaluated using the sof-
tware Quantity one (BioRad, USA).
2.5. Data Analysis
Data were recorded as presence (1) or absence (0) of ba-
nd products from the photographic examination. Each
amplification fragment was named by the source of the
primer, the kit letter or number, the primer number and
its approximate size in base pairs. Bands with similar
mobility to those detected in the negative control, if any,
were not scored. A pair-wise matrix of distance between
landraces was determined for the RAPD and ISSR data
using Dice formula [16] in the program Free Tree [17].
The average of similarity matrices was used to generate a
tree by UPGMA (unweighted pair-group method arith-
metic average) using the program Tree view.
3. Results and Discussion
The present study offers an optimization of primer scree-
ning for evaluation of genetic relationship between twel-
ve Phyllanthus species collected from Indian origin. DNA
extraction of Phyllanthus proved difficult due to presence
of secondary metabolites and essential oil content. A mo-
dified CTAB method by Doyle and Doyle proved to be
fruitful. The modified method included higher concentra-
tion of CTAB (4%), EDTA (50mM) and 1% 2-Mercapt-
oethanol. Importantly purification by Choloform: Isoa-
myl alcohol (24:1) was performed twice. Significant qua-
ntities of DNA were always successfully extracted by

Phylogenic Study of Twelve Species of Phyllanthus Originated from India through
Molecular Markers for Conservation
34
this modified method that varied from 200 to 1000ng in
different Phyllanthus species. The reproducibility of both
RAPD and ISSR primer amplification were detected by
performing separate runs of PCR with DNA extraction
from different preparation. No significant differences
were observed in different experiments although occa-
sional variation in the intensities of individual bands was
detected. Bands with same mobility were considered as
identical fragments receiving equal values regardless of
their staining ability. When multiple bands in a region
were difficult to resolve, data of that region were not inc-
luded for the analysis. As a result ten RAPD and eight
ISSR primers were selected out of thirty RAPD and tw-
enty ISSR primers screened, as they generated clear and
scorable bands with considerable polymorphism.
Using ten RAPD primers, 157 bands were produced
with an average of ~ 16 bands per primer out of which
150 were polymorphic revealing 95.54% polymorphism.
The size of the RAPD fragments ranged from 0.2 to 2.4
Kilo base pairs (Table 1). The banding profile by RAPD
primer OPA-01 and OPD-18 has been shown in the Fig-
ure 1. The primer OPA-01 amplified a maximum of 24
fragments whereas OPD-02 produced least number of am-
plified bands (08). Similarly 102 amplified ISSR produ-
cts were scored across 12 species of Phyllanthus by eight
selected custom synthesized ISSR primers with 97.05%
polymorphism. The average number of amplification pr-
oducts per ISSR primer was ~ 13. The size of ISSR amp-
lified fragments varied from 0.3-2.5 Kilo base pair (Ta-
ble 2).
The banding pattern by ISSR primer IG-10 and IG-14
are presented in Figure 2. The genetic variation through
RAPD and ISSR markers has been highlighted in a nu-
mber of medicinal plants [18-21]. The result shows that
both the marker systems are efficient enough to distin-
guish 12 species of Phyllanthus and in revealing mo-
lecular relationship among them. The resolution of ISSR
markers (97.08%) is high in comparison to RAPD mark-
ers (95.54%). The similarity matrix of RAPD and ISSR
data after multivariant analysis using Nei and Li’s coef-
ficient has been presented in Tables 3 & 4 respectively.
The similarity value ranged from 0.23 to 0.76 in case of
RAPD and from 0.26 to 0.81 for ISSR. The similarity
matrix obtained in the present study was used to con-
struct a dendrogram with the UPGMA method by both
RAPD and ISSR data (Figures 3 & 4). The dendograms
generated by both the approaches (RAPD and ISSR)
were with broad agreement with each other and also with
accepted taxonomy; two major groups were obtained and
most of the related species were found to be grouped
together. Phyllanthus angustifolia, morphologically dis-
tinct from the rest 11 species had been grouped isolated
in group-I by both the molecular approaches. At the mo-
lecular level Phyllanthus angustifolia is having six un-
ique RAPD bands and five unique ISSR bands.
The remaining eleven species positioned in group II
are differentiated into two clad by both the marker sys-
tem. The first clad having six species (Phyllanthus spp
“Acc No-1”, Phyllanthus reticulus, Phyllanthus nivosus,
Phyllanthus nivosus “varigata”, Phyllanthus acidus, Phy-
llanthus emblica ) and other clad having five species (Ph-
yllanthus flatarnus, Phyllanthus urinaria, Phyllanthus ro-
tundifolius, Phyllanthus virgatus and Phyllanthus ama-
rus). Phyllanthus acidus and Phyllanthus emblica as well
as Phyllanthus nivosus and Phyllanthus nivosus “vari-
gata” are grouped together by both the approaches, where
Table 1. Total number of amplified fragments and number of polymorphic fragments generated by PCR using selected
RAPD primers.
Primer Primer sequence
Total no.
of bands
No. of Polymorphic
bands
Polymorphism
percentage
No. of Unique
bands
Band range
(kbp)
OPA-01 5’-TGCCGAGCTG-3’ 24 24 100 3 0.4-2.1
OPA-04 5’-AATCGGGCTG-3’ 18 18 100 2 0.25-2.4
OPA-10 5’-GTGATCGCAG-3’ 20 20 100 3 0.3-2.3
OPD-02 5’-GGACCCAACC-3’ 8 7 87.4 2 0.5-1.8
OPD-11 5’-AGCGCCATTG-3’ 12 10 83.3 1 0.3-2.3
OPD-18 5’-GAGAGCCAAC-3’ 15 15 100 1 0.2-2.1
OPD-20 5’-ACCCGGTCAC-3’ 11 11 100 0 0.3-2.2
OPN-06 5’-GAGACGCACA-3’ 20 20 100 3 0.3-2.5
OPN-15 5’-GGTGAGGTCA-3’ 14 11 78.5 2 0.4-2.4
OPN-16 5’-AAGCGACCTG-3’ 15 14 93 2 0.2-3.0
TOTAL ---------------- 157 150 95.5 11 0.2-3.0

Phylogenic Study of Twelve Species of Phyllanthus Originated from India through
Molecular Markers for Conservation
35
Figure 1. RAPD banding patterns of twelve species of Phyl-
lanthus generated by the primer s OPA- 01 (A) and OPD-18
(B) M – Molecular weight ladder (kb). 1-Phyllanthus nivo-
sus, 2-Phyllanthus flaternus, 3-Phyllanthus reticulus, 4-Ph-
yllanthus acidus, 5-Phyllanthus nivosus “Varigata”, 6-Phy-
llanthus spp “Àcc No.1”, 7-Phyllanthus rotundifolius, 8-Phy-
llanthus angustifolius, 9-Phyllanthus emblica, 10-Phyllant-
hus uninaria, 11-Phyllanthus virgatus, 12-Phyllanthus ama-
rus.
Figure 2. ISSR banding pattern in 12 species of Phyllanthus
obtained from PCR amplification by ISSR primer IG-10(A)
and IG-14(B). M indicates DNA size marker; 1-Phyllanthus
nivosus, 2-Phyllanthus flaternus, 3-Phyllanthus reticulus, 4-
Phyllanthus acidus, 5-Phyllanthus nivosus “Varigata”, 6-Ph-
yllanthus spp “Àcc No.1”, 7-Phyllanthus rotundifolius, 8-Ph-
yllanthus angustifolius, 9-Phyllanthus emblica, 10-Phyllan-
thus uninaria, 11-Phyllanthus virgatus, 12-Phyllanthus am-
arus.
Table 2. Total number of amplified fragments and number of polymorphic fragments ge nerated by PCR using selected ISSR
Primers.
Primer Primer sequence
Total no. of
bands
No. of Polymorphic
bands
Polymorphism
percentage
No. of
Unique bands
Band range
(kbp)
IG-01 5’AGGGCTGAGGAGGGC-3’ 12 12 100 1 0.5-1.6
IG-03 5’GAGGGTGGAGGATCT-3’ 8 08 100 1 0.5-1.6
IG-10 3’- (AG)8T-5’ 12 12 100 0 0.3-1.8
IG-11 3’- (AG) 8C-5’ 12 12 100 1 0.3-1.6
IG-13 3’- (AC) 8G-5’ 11 11 100 1 0.4-2.2
IG-14 3’- (GA) 88A-5’ 18 17 94.4 2 0.3-2.5
IG-15 3’- (GA) 8T-5’ 15 14 93.33 0 0.4-2.0
IG-23 3’- (GA) 8C-5’ 14 13 92.85 1 0.3-2.1
TOTAL -------------- 102 99 97.05 7 0.3-2.5
Table 3. Similarity matrix of 12 species of Phyllanthus generated by RAPD markers.
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12
P1 1.00
P2 0.31 1.00
P3 0.53 0.40 1.00
P4 0.57 0.43 0.50 1.00
P5 0.74 0.25 0.52 0.54 1.00
P6 0.45 0.49 0.51 0.48 0.50 1.00
P7 0.55 0.54 0.43 0.52 0.44 0.41 1.00
P8 0.41 0.23 0.41 0.33 0.39 0.29 0.36 1.00
P9 0.58 0.50 0.56 0.76 0.50 0.56 0.56 0.40 1.00
P10 0.38 0.37 0.43 0.46 0.38 0.46 0.54 0.35 0.45 1.00
P11 0.45 0.54 0.42 0.59 0.46 0.51 0.63 0.35 0.58 0.51 1.00
P12 0.41 0.41 0.35 0.47 0.45 0.44 0.52 0.36 0.45 0.55 0.72 1.00
P1-Phyllanthus nivosus, P2-Phyllanthus flaternus, P3-Phyllanthus reticulus, P4-Phyllanthus acidus, P5-Phyllanthus nivosus “Varigata”, P6-Phyllanthus spp
“Àcc No.1”, P7-Phyllanthus rotundifolius, P8-Phyllanthus angustifolius, P9-Phyllanthus emblica, P10-Phyllanthus uninaria, P11-Phyllanthus virgatus, P12-
Phyllanthus amarus.

Phylogenic Study of Twelve Species of Phyllanthus Originated from India through
Molecular Markers for Conservation
36
Table 4. Similarity matrix of 12 species of Phyllanthus generated by ISSR markers.
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12
P1 1.00
P2 0.32 1.00
P3 0.37 0.29 1.00
P4 0.56 0.50 0.42 1.00
P5 0.81 0.36 0.32 0.64 1.00
P6 0.46 0.50 0.53 0.57 0.43 1.00
P7 0.47 0.41 0.42 0.39 0.55 0.46 1.00
P8 0.29 0.44 0.26 0.44 0.36 0.33 0.32 1.00
P9 0.57 0.50 0.48 0.72 0.55 0.52 0.47 0.38 1.00
P10 0.36 0.69 0.29 0.54 0.48 0.46 0.57 0.47 0.43 1.00
P11 0.43 0.48 0.28 0.45 0.53 0.41 0.57 0.37 0.41 0.57 1.00
P12 0.44 0.46 0.27 0.38 0.56 0.32 0.49 0.33 0.42 0.52 0.64 1.00
P1-Phyllanthus nivosus, P2-Phyllanthus flaternus, P3-Phyllanthus reticulus, P4-Phyllanthus acidus, P5-Phyllanthus nivosus “Varigata”, P6-Phyllanthus spp
“Àcc No.1”, P7-Phyllanthus rotundifolius, P8-Phyllanthus angustifolius, P9-Phyllanthus emblica, P10-Phyllanthus uninaria, P11-Phyllanthus virgatus, P12-
Phyllanthus amarus.
Figure 3. Dendogram showing the cluster analysis of 12 spe-
cies of Phyllanthus using RAPD markers. 1-Phyllanthus ni-
vosus, 2-Phyllanthus flaternus, 3-Phyllanthus reticulus, 4-
Phyllanthus acidus, 5-Phyllanthus nivosus “Varigata”, 6-Ph-
yllanthus spp “Àcc No.1”, 7-Phyllanthus rotundifolius, 8-
Phyllanthus angustifolius, 9-Phyllanthus emblica, 10-Phyll-
anthus uninaria, 11-Phyllanthus virgatus, 12-Phyllanthus
amarus.
Figure 4. Dendrogram showing the cluster analysis of 12 sp-
ecies of Phyllanthus using ISSR markers. 1-Phyllanthus ni-
vosus, 2-Phyllanthus flaternus, 3-Phyllanthus reticulus, 4-
Phyllanthus acidus, 5-Phyllanthus nivosus “Varigata”, 6-Ph-
yllanthus spp “Àcc No.1”, 7-Phyllanthus rotundifolius, 8-Ph-
yllanthus angustifolius, 9-Phyllanthus emblica, 10-Phyllant-
hus uninaria, 11-Phyllanthus virgatus, 12-Phyllanthus ama-
rus.
as Phyllanthus spp “Acc No-1” and Phyllanthus reticulus
forming single cluster in case of ISSR are grouped sepa-
rately in RAPD approach. Phyllanthus amarus and Phy-
llanthus virgatus in clad II is always grouped together in
both the approaches. The differences in number of indi-
viduals estimated by RAPD markers in this study are
similar to the result obtained by Rajaseger et al. [22] in
RAPD studies of the Ixora coccinea and Ixora. javanica.
They also found that the taxa-specific RAPD and ISSR
bands could be utilized to define the identification.
The present findings include the identification and ge-
netic variation within twelve species of Phyllanthus. The
dendogram shows the distant variation within the species.
The genetic relation through RAPD and ISSR markers
provides a reliable method for identification of species
than morphological characters. This investigation as an
understanding of the level and partitioning of genetic
variation within the species would provide an important
input into determining efficient management strategies.
The genetic variability in a gene pool is normally con-
sidered as being the major resource available to breeders
for improvement program.
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