Development of ITS based SCAR Markers for Some Medicinally Important Species of Phyllanthus
Internal Transcribed Spacer (ITS) 1 and 2 were sequenced for five species of the genus Phyllanthus namely P. emblica, P. reticulatus, P. amarus, P. fraternus and P. urinaria, with a view to identify accurately herbaceous species. All the five species used in the study are medicinally important Affinities among these species were deduced through the help of phylograms generated from their ITS sequences. It was found that internal transcribed spacer 1 showed more potential than internal transcribed spacer 2 in assessing their relationship at molecular level. SCAR markers based on ITS 1 sequences of P. urinaria, P. amarus and P. emblica were designed. All three markers successfully distinguished these species from other closely related species. This is an important tool for identifying plant parts used in ayurvedic medicine.
Received: April 12, 2010;
Accepted: June 25, 2010;
Published: August 07, 2010
Euphorbiaceae is a heterogeneous family represented by genera that are medicinally
important. Of these, the genus Phyllanthus includes many economically
important species containing various phytochemicals. The plants belonging to
this genus have developed various life forms ranging from herbs, shrubs to tall
canopy trees (Balakrishnan and Chakrabarty, 2007).
Five species namely Phyllanthus emblica, P. reticulatus, P.
amarus, P. fraternus and P. urinaria were selected for the
present investigation with a view to develop an authentic identification marker
for the selected species in general and the herbaceous species in particular,
using ITS sequences. Several medicinal properties have been already reported
for all the five species. Fruits of P. emblica are a rich source of vitamin
C; these are mainly used for their hepatoprotective and antioxidant activities
(Bhattacharya et al., 1999; Jose
and Kuttan, 2000). Phyllanthus reticulatus is a shrub and is claimed
to have antidiabetic activity .The phytochemical screening of the residues revealed
the presence of terpenoids, glycosides, proteins, carbohydrates and absence
of alkaloids and steroids (Kumar et al., 2007).
P. amarus, a herb is also important and well known for uses against hepatitis
infection. It is also used for curing stomach ailments like dyspepsia, diarrhoea,
dysentery and urinogenital problems (Jain et al.,
2003). Phyllanthus fraternus, a closely related herb of P. amarus
also exhibits hepatoprotective properties (Padma and Setty,
1999). Aqueous extract of P. fraternus against allyl alcohol induced
oxidative stress in the liver of rats showed positive effects (Sailaja
and Setty, 2006). Antioxidative and cardio protective effects of P. urinaria
L. on Doxorubicin induced cardio toxicity have also been demonstrated (Chularajmontri
et al., 2005).
Molecular marker based on DNA analysis (Farooq and Azam,
2002; Tanee et al., 2009) is a modern approach
based on which proper assessment of closely related plant species could be determined.
Increasing popularity of herbal medicines has projected molecular marker technology
as a powerful weapon for quick identification of plants with suitable medicinal
properties. India is a rich source of medicinal plants and market value of all
these therapeutically important plant species is increasing day by day (Joshi
et al., 2004). So keeping in mind the growing value of medicinal
plants, it is essential to generate molecular markers that will help to authenticate
plant species of medicinal importance.
Internal transcribed spacer regions are sequences located in eukaryotic rRNA
genes between the 18S and 5.8S rRNA coding regions (ITS1) and between the 5.8S
and 25S (26S) rRNA coding regions (ITS2). Studies of restriction site variation
in rDNA in populations of animals and plants have shown that while coding regions
are conserved, these spacer regions are variable (Gerbi,1985).
These spacer sequences have a high evolutionary rate and are present in all
known nuclear rRNA genes of eucaryotes (Jorgensen et
al., 1987; White et al., 1991; Xu
et al., 2006). They are useful for phylogenetic analysis among related
species or among population within a species because these sequences evolve
more rapidly than coding regions (Baldwin, 1993; Soltis
and Kuzoff, 1993; Suh et al., 1993; Bayer
et al., 1996). Development of primers from variable regions of ITS1
and ITS2 is another strategy that has been applied for species identification
in bacteria (Chiu et al., 2005), fungi (Martin
and Rygiewicz, 2005) and plants (Chiou et al.,
2007). There are some reports on different Phyllanthus species from
China (Lee et al., 2006) and Thailand (Chiou
et al., 2007). In one of these papers primers designed based on ITS2
sequences proved to be suitable for a broad application in the authentication
of herbal materials (Chiou et al., 2007). Here
from our sequenced data we also tried to develop some species-specific PCR primers
with which it will be possible to differentiate some of these species quickly
and accurately and could be used as SCAR markers.
Herbaceous species of Phyllanthus, especially P. amarus, P. urinaria and P. fraternus are very similar in phenotypic characters and usually grow in the same habitat. Since the content of phytochemicals are variable in these species, their medicinal uses are also different. Therefore, there exist the potential risks of collecting wrong plants for medicinal uses. Again uses of dried fruits and leaves of P. emblica in local markets are rampant and it needs quality checking in terms of raw material. So the aim of the present investigation is to throw light on ITS sequences of Phyllanthus species based on which genetic relationships between species can be determined. Besides this we designed PCR primers based on ITS1 sequences for P. amarus, P. urinaria and P. emblica to be used as SCAR markers.
MATERIALS AND METHODS
This study was conducted from 2005 to 2009.
Plant leaves of all the five species were collected from different districts
of West Bengal, India (Table 1). The tissues were brought
to the laboratory and stored in -70°C until further analysis. Genomic DNA
was isolated from these leaves within seven days of collection.
DNA extraction, ITS amplification and sequencing: Total genomic DNA
was isolated from leaf materials following the DNA isolation method of Joshi
et al. (2004). Leaf samples were collected and stored at -70°C
until further use. These were ground to powder using liquid nitrogen, the powder
collected from 1 g of frozen tissue was dissolved in 6 mL of buffer with the
following composition-100 mM Tris-HCl (pH 8), 20 mM EDTA (pH 8), 1.5M NaCl,
Cetyl Trimethyl Ammonium Bromide (CTAB) (2% w/v), β-mercaptoethanol (0.3%
v/v) (added to buffer just before use), 100 mg PVP per gram of tissue (added
in the mortar while crushing the tissue). The suspension was incubated at 65°C
for 30 min with intermittent pH monitoring. 1:1 chloroform: isoamylalcohol (24:1)
was added to it after cooling at room temperature. It was then centrifuged at
665 g for 10 min at 25°C and the supernatant was pipetted out and again
centrifuged with equal volume of chloroform-isoamylacohol (24:1) mixture at
the same speed. After recovering the clear supernatant 1/10th volume
of 3M sodium acetate and twice the volume of chilled ethanol at 4°C were
mixed with it to centrifuge at 665 g for 10 min to precipitate DNA. The pellet
was washed with 70% ethanol (v/v) and dissolved in autoclaved triple distilled
water. RNA contaminations were removed by adding RNase A at a concentration
of 250 μg per ml of DNA solution and incubating for 30 min at 37°C.
After incubation all the samples were extracted with equal volume of chloroform
by centrifuging at 665 g at room temperature. The supernatant was collected
and mixed with equal volume of isopropyl alcohol and 1/10th volume of 3 M ammonium
acetate and centrifuged at 665 g at 4°C to precipitate the DNA from the
solution. DNA pellets were then washed with 70% ethanol, dried and dissolved
in triple distilled water. The purity of the genomic DNA was checked by calculating
the ratio Absorbance 260/Absorbance 280, a ratio of 1.8 confirmed high quality
|| Morphological characters and collection sites of five Phyllanthus
|| Several features of ITS 1 and 2 regions of different Phyllanthus
ITS1 and ITS2 regions were separately amplified using primers (5ACGAATTCATGGTCCGGTGAAGTGT
TCG3) and (5TAGAATTCCCCGGTTCGCTCGCCGT TAC 3) (Sun
et al., 1994) and (5GCTGCGTTCTTCA TCGATGC3) and (5GCATCGATGAAGAAC
GCAG C3) (White et al., 1990). The amplification
parameters were 2 min at 94°C followed by 35 cycles of 1 min denaturing
at 94°C, 30 sec of annealing at 48°C, 1 min extension at 72°C ending
with 30 min final extension at 72°C. A slightly higher than 400 bp fragments
were obtained for each ITS1 and ITS2. The amplified products were then cleaned
using a PCR clean up kit (Bangalore Genei, India) for complete removal of dNTPS
and primer dimers. The concentration of PCR product was determined and the amount
needed for ligation for the specific PCR fragment was calculated by the conversion
table subjected to ligation in a plasmid vector pTZ57R/T and transformed into
a strain of E. coli XL1-Blue. All the steps of ligation and transformation
were performed following manufactures protocol (InsTA clone PCR Cloning Kit,
Fermentas). Positive clones were selected by blue-white screening procedure.
All these clones were further scrutinized using a colony PCR step using the
bacterial colonies as DNA templates and performing the same cycle as before.
Colonies, which produced the desired fragment, selected, cultured overnight
and subjected to plasmid DNA extraction. Plasmid DNA was sequenced using universal
primers. All the sequences were edited by comparing them with the existing sequences
in the NCBI database with the help of Clustal W programme (EMBL-EBI). Completed
sequences were submitted to GENBANK database (Table 2). ITS1
and ITS2 sequences were separately aligned to construct respective phylograms
(with bootstrap values and Phylip as tree type option) using online GeneBee
All the ITS 1 sequences from different accessions of P. amarus, P. urinaria and P. emblica were put into Primer 3.0 software (version 0.4.0) to obtain species specific Primers. Three pairs of species-specific primers were obtained as follows:
With the first two primer pair [PA (F/R) and PU (F/R)] we have amplified the
DNA samples of all the three herbaceous species in study. The PCR amplification
cycle was, 2 min hot start at 94°C, denaturation at 94°C for 1 min,
annealing at 52°Cfor 30 sec, amplification at 72°C for 1 min, a final
extension of 5 min at 72°C. Total 35 cycles were performed. Annealing temperatures
were adjusted for different species (52°C for P. urinaria and 60°C
for P. amarus) to obtain better results. With the third primer pair [PE
(F/R)] specific for P. emblica dried fruit samples as well as leaf samples
were examined using the above-mentioned cycle.
The ITS region was sequenced from 14 different accessions consisting of 5 different
species locally growing in West Bengal, India .All the five species were identified
by traditional morphological method (Table 1). Two different
sets of forward and reverse primers (sequences provided in materials and method)
were used to amplify ITS1 and ITS2 separately. All the four primers were designed
using the conserved flanking sequences of rRNA genes around ITS1 and ITS2 so
that full-length ITS1 and ITS2 regions could be obtained along with these conserved
sequences. A slightly higher than 400 bp fragment was obtained for each ITS1
and ITS2 consisting of varying length of ITS1 and ITS2 sequences for different
species shown in Table 2.
||DNA amplification profile using PU [F and R] primers. Lanes
M-100 bp DNA ladder, 1- P. emblica, 2- P. reticulatus, 3-
P. amarus, 4- P. fraternus, 5-8 P. urinaria
||DNA amplification profile using PA [F and R] primers. Lanes
M-100 bp DNA ladder, 1,2- P. amarus; 3,4- P. fraternus; 5,
6- P. urinaria; 7- P. emblica; 8- P. reticulatus
It was clear from the data that the average length of ITS1 is 220 bases whereas
length of ITS2 is variable. Besides this one or two samples of most of the samples
showed that these two regions were rich in GC content. The GC% was found to
be ranged from 47-58% in case of ITS1 and from 47-66% in case of ITS2.In most
of the accessions the GC% was constant, but some of the accession showed higher
GC%. Two phylograms were constructed depending upon sequences of the region
18S-ITS1-5.8S and 5.8S-ITS2-26S.
Primer pair designed for P. urinaria [PU (F/R)] showed one clear and distinct band of ~174 bp (Fig. 1), but there were no such band for other species samples. In case of P. amarus specific primers [PA (F/R)] the desired band at ~192 bp was present in all P. amarus samples but that was obtained by increasing the annealing temperature to 60°C (Fig. 2) to reduce the effect of cross-reaction. Again when P. emblica leaf and dried fruit DNA samples were subjected to PCR amplification they produced the desired fragment of ~212 bp (Fig. 3).
||DNA amplification profile using PE [F and R] primers. Lanes
M-100 bp DNA ladder, PL-Phyllanthus emblica DNA from leaves. PF-Phyllanthus
emblica DNA from fruits
In recent years ITS based phylogenetic studies immensely contributed in the
understanding of ancestral relationships. In the genus Phyllanthus there
were some studies based on these sequences done in China (Lee
et al., 2006) and other countries (Kathriarachchi
et al., 2006). India is a large country and several species of this
genus grow here and used for medicinal purpose. For proper use of these species
it is very important to know their proper identity. This investigation is such
an attempt to develop ITS marker for proper assessment of relationship among
five species of the genus Phyllanthus.
Phylogram generated from 18S-ITS1-5.8S (Fig. 4) showed that different species formed separate clades and it also showed that P. amarus and P. fraternus are two very close species. Phylogram generated from 5.8S-ITS2-26S region also showed close proximities for herbaceous species but in a complex manner. The position of P. reticulatus in both the phylograms was constant, i.e., it was positioned very far from all the herbaceous species. We also compared the ITS1 and ITS 2 sequences of these two species with the sequences of P. niruri from NCBI databank and found that there is high dissimilarity between them, which again proves that P. amarus and P. fraternus are two different species from P. niruri. Therefore, this type of clustering technique could be easily used for unknown samples so that their genetic identity could be known.
Several other special features of ITS1 and ITS2 regions were observed in this
study. Alignment of ITS1 and ITS2 sequences separately revealed conserved stretches
in both the regions. Different authors from time to time have thrown light on
conserved stretches and their functional role in processing rRNA. Two conserved
regions one in ITS1 (AACCCCGGCGCGGAAAGCGCCA AGGAA) (Fig. 5)
and another in ITS2 (CGTGGCATTCGG TGGTTGAA) were found in our study. Liu
and Schardl (1994) found a conserved motif within ITS 1 in many flowering
plants, which they found absent in other eukaryotic kingdom and species, this
short stretch (AAGGAA) was also found to be present in our ITS1 sequences. It
was suggested that the sequence served as a critical recognition element for
r RNA processing.
|| Phylogram generated from 18S-ITS1-5.8S sequences
|| Conserved stretches in ITS 1 region
It was proposed that it forms a hairpin structure during the r RNA processing
and probable hairpin structures are also available in literature for Rice (Oryza
sativa) and Arabidopsis thaliana. Following those structures here
we have also drawn a probable hairpin structure (Fig. 6),
which again strongly support this hypothesis by Liu and
Results obtained from amplification studies based on species specific PCR primers
designed from ITS1 sequences of P. urinaria, P. amarus and
P. emblica showed that while primers from P. urinaria (Fig.
1) samples clearly distinguished it from other closely related herbs at
one chance, P. amarus specific primer cross reacted with other samples
which upon elevation of annealing temperature gave clear and distinct band and
the cross reacting band also got diluted (Fig. 2).
||Probable hairpin structure for Phyllanthus along with
rice and Arabidopsis
For the P. emblica leaf and fruit samples the P. emblica-specific
primer gave the expected band at ~212 bp (Fig. 3) that showed
the accuracy of the method to distinguish P. emblica samples. Bands obtained
from all these amplification procedures were very bright and clear which can
be attributed to the presence of multiple copies of these spacer regions in
the genome. Genomic DNA required for this type of amplification procedure is
also very low (~5 ng) which is another positive aspect of this kind of study.
So we found that in comparison to SCAR marker development (usually developed
from RAPD (Dnyaneshwar et al., 2006; Neeraj
et al., 2008; Theerakulpisut et al., 2008)
or AFLP markers) ITS specific marker generation is much easier and also highly
Lee et al. (2006) in their investigation assessed
phylogenetic relationships among 18 Phyllanthus species based on nrITS
sequences along with chloroplast ATPB and RBCL sequences. Cladistic analysis
indicated that the genus is paraphyletic and strongly demarcates between the
closely related species P. niruri and P. amarus. Same authors
also developed a multiplex PCR assay to differentiate three medicinally important
species P. amarus, P. niruri and P. urinaria. In the present
investigation SCAR markers based on ITS1 sequences have been developed for the
species P. amarus, P. urinaria and P. emblica to identify
them from other species of the genus Phyllanthus growing in India.
In another report (Kathriarachchi et al., 2006)
nrITS and plastid matK DNA sequence data for 95 species of tribe Phyllantheae
including representatives of all subgenera of Phyllanthus were analysed.
According to them these results are generally concordant with each other, although
some species are placed differently in the plastid and ITS trees, indicating
that hybridization paralogy is involved. In conclusion, it could be stated that
the data on length and sequence of ITS1 and ITS2 determined in the present report
could be used successfully for the assessment of genetic relationships at the
interspecific level in the genus Phyllanthus in India. In this study
ITS1 was found to be more informative than ITS2. Apart from that we have developed
highly sensitive and specific PCR primers for detection of three therapeutically
important species of the genus Phyllanthus which could be treated as
SCAR markers. These primers may be widely useful in identification of plant
parts of all these species used extensively in Ayurvedic medicines.
The first author is highly indebted to University Grants Commission (UGC) and Department of Biotechnology (DBT), India for financial support.
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