Molecular Identification of V. thapsus L.

Molecular identification of Verbascum thapsus L. (Ban Tambaaku) and

its ITS sequence comparison with other Verbascum L. species.
Devendra Kumar Srivastava 1, Manjit Inder Singh Saggoo 2
1
Department of Biology, D.A.V. College, Hoshiarpur-146001, Pb. (India).
2
Department of Botany, Punjabi University Patiala, Patiala-147002, Pb. (India).

ABSTRACT

Internal transcribed spacers (ITS) have been widely used for molecular identification of plant species. Present work
provides a rapid and accurate approach for the detection and identification of Verbascum thapsus based on ITS
sequences. Amplified sequence of 689 bp fragment by ITS1 and ITS4 primers from nrDNA revealed five regions
viz. 18S rRNA (partial sequence, >1….64), ITS-1 (>65….240), 5.8S (>241….404), ITS-2 (>405….619) and 28S rRNA
(partial sequence >620….689). The ITS sequences (ITS-1, 5.8 and ITS-2) of V. thapsus show 18 species specific
informative sites at 108, 115, 125, 126, 130, 153, 174, 215, 216, 229, 242, 517, 543, 580, 581, 582, 583 and 595
that could be used to differentiate V. thapsus species from other species as well as authenticate the species. A
comparative phylogenetic analysis of ITS sequence with other Verbascum L. species revealed that V. thapsus and
V. nigrum were closely related and clustered together in the phylogenetic tree.

Key words: Verbascum species, nrDNA, ITS, identification.

INTRODUCTION simplex virus (Khare, 2007). It is locally known by the

Verbascum L. is one among the largest genera within
the family Scrophulariaceae. The genus is palaearctic
in origin and consist of more than 360 species, mostly
from Mediterranean and oriental regions of the world
(Ansari and Daehler, 2000; Fischer, 2004). In India it is
represented with four species namely, V.
coromandelianum, V. blattaria, V. erianthum and V.
thapsus (Pennell, 1943). Verbascum L. species are
medicinally very important and contains large number
of biologically active compounds (Tatli and Akdemir,
2004; 2006).
V. thapsus (Common Mullein) is used for herbal
remedies with emollient and astringent properties. The
capsule contains important compounds such as
saponins, thapsuines and hydro-thapsuines. Flower
extract has activity against influenza and herpes
Corresponding author : Devendra Kumar Srivastava
e-mail: devsrivastv@gmail.com, Cel: +91-9417491877
Received : February 27, 2014; Accepted : March 6, 2014
name of Ban tambaaku or Gidar tambaaku or some
time Phulla and especially recommended for coughs
related problems and against a variety of skin problems.
The leaves are ground and used as poultice. There exist
a wide range of morphological variation in the N.W.
Himalayas species V. thapsus and it often confuses their
taxonomic identity at species level.
Identif ication and authentication of crude drugs
precisely is an important step for pharmacological
research as well as to ensure safe clinical application.
Unlike traditional morphological methods of
authentication, the DNA based procedure is reliable,
reproducible and unaffected by the physical form and
environmental influence on the plant. rDNA-ITS
sequence have been useful in identification of both
medicinal plants and the botanical origin of the crude
herbal medicine (Sucher and Carles, 2008; Li and Dao,
2011a; Balasubramani and Venkatasubramanian, 2011).
It has been supported as a remarkable marker where
plastid markers fail to discriminate the plants at species
level (Hollingsworth, 2011). Present work provides a
rapid and accurate approach for the detection and
identification of V. thapsus based on ITS sequences.

Medicinal Plants, 6(1) March 2014

2 Srivastava & Saggoo 2014
MATERIAL AND METHOD
Plant materials and genomic DNA extraction
Fresh leaves of Verbascum thapsus L. were collected
from the plant growing in Darcha (3,300m) population
of Lahaul-Spiti (Himachal Pradesh, India) and stored in
plastic bags with silica gel following the method of
Chase and Hills (1991). Voucher specimen (PUN
PUN55051) of the collected species was deposited in
Herbarium Punjabi University, Patiala. Plant material
was ground in liquid nitrogen into a f ine powder.
Genomic DNA was extracted using modif ied Cetyl
trimethyl ammonium bromide (CTAB) method (Doyle
and Dickson, 1987).
PCR amplification
The universal ITS primer pairs viz. ITS-1 (5’-
TCCGTAGGTGAACCTGCGG-3’) and ITS-4 (5’-
TCCTCCGCTTATTGATATGC-3’) were used to amplify
the ITS region (White et al. 1990). PCR amplification
was carried out under conditions of : initial denaturation
at 94 ºC (4 min), followed by 32 cycles of denaturation
at 94 ºC (35 s), annealing at 48 ºC (40 s), initial
extension at 72 ºC (2 min) and final extension at 72
ºC for 7 min. Total 25 ìl reaction cocktail was prepared
for PCR amplification. For this GeNei Red Dye PCR
Master Mix was used. The amplification was performed
in a Bio-Rad Thermal Cycler. PCR amplified DNA band
on a 1% agarose gel was visualized under UV. PCR
products were purified with DNA Clean-Up Purification
Kit (Promega USA). Purified PCR products were then
sequenced at Bioserve Biotechnologies (India) Pvt. Ltd
(Bangalore).
Sequence analysis
The sequences obtained were aligned and consensus
sequences were constructed using the Clustal X
programme (Thompson et al., 1997) and adjusted
manually as necessary. The complete sequence of the
ITS region was deposited in GenBank (Accession no.
JQ801746.1). Sequence boundaries of different regions
(i.e. ITS-1, 5.8s and ITS-2) were determined by
comparing the aligned sequences with the sequences
of other related species of Verbasum L. registered in
NCBI GenBank (Table-1). Sequence divergence values
were estimated by both Jukes and Cantor’s model and
Kimura’s 2 parameter as implemented in Molecular
Evolutionary Genetics Analysis (MEGA) software
version 4.0 (Tamura et al., 2007). The scoring matrix
was substituted from sequence similarity to ITS
sequence similarity between each two species and
phylogenetic trees were constructed by maximum
parsimony method.
RESULTS AND ]\DISCUSSION
The ITS region was amplif ied using universal
primers. The sequence obtained shows the overall length
of amplified ITS regions with 689 nucleotides. The ITS
sequences of various Verbascum species available in
the GenBank (http://www.ncbi.nlm.nih.gov/genbank)
were retrieved and analyzed with sequence of V. thapsus.
The information regarding GenBank accession numbers,
length, G+C content of ITS-1, 5.8s and ITS-2 sequences
of various Verbascum species is provided in Table-1.
It was observed that the presently amplified sequence
of nrDNA in V. thapsus contains five regions viz. 18s
rRNA (partial sequence, >1….64), ITS-1 (>65….240), 5.8s
(>241….404), ITS-2 (>405….619) and 28s rRNA (partial
sequence >620….689).
ITS-1 region in the presently studied species was
observed with the length of 240nt bases. The sequence
of ITS-1 regions excluding the sites with missing (/
ambiguous) data and gaps produces signif icant
alignment (>65….240) at 176 nt sites (Fig.1). Of the
aligned positions from ITS-1 regions of the six species
(including V. thapsus) 99 sites were variable, 10 of
which were observed as parsimonious-informative. The
informative site (positions) distribution was observed
to be as 108, 115, 125, 126, 130, 153, 174, 215, 216
and 229. The length of the ITS-1 region (176 nt) was
observed to be similar in three species i.e V. thapsus, V.
nigrum and V. sinuatum var. adenosepalum. The GC
content in ITS-1 region was similar in V. thapsus and
V. nigrum with 66.48% while differ in other species
(Table-1).
The 5.8s region of nrDNA in V. thapsus spanning
length from site 241 to site 404 (164 nt bases; Fig.1)
in the presently aligned sequences showed only one
parsimonious informative site at position 242. In four
species i.e. V. thapsus, V. arcturus, V. nigrum and V.
sinuatum var. adenosepalum the length of 5.8s region
and the GC content was observed with the similar
values of 164 nt bases and 53.66%, while in the other
two the 5.8s region length was 178 nt bases and GC
content was 65.17% (Table-1).
The alignment of nrDNA sequence in the presently
studied species revealed the length of the ITS-2 with
215 nt bases (>405…619) of which bases between
positions 509 to 619 (111 sites) produced significant
alignment (Fig.1). The distribution of variable sites in
Medicinal Plants, 6(1) March 2014
 Molecular Identification of V. thapsus L. 3

Fig. 1. Alignment of nrDNA

sequences of ITS-1, 5.8s
RNA and ITS-2 regions in
Verbascum species. Dots
above sequences are
showing parsimonious
informative sites.

Medicinal Plants, 6(1) March 2014

4 Srivastava & Saggoo 2014

the aligned sequences was 55, of which 7 were observed
as parsimonious-informative e.g. bases at sites 517,
543, 580, 581, 582, 583 and 595. The GC content in
V. sinuatum var. adenosepalum was highest (66.96%)
among the analyzed sequence of ITS-2 region of the
compared species of Verbascum followed by V. nigrum
(65.90%), V. thapsus (65.58%) and V. arcturus (64.41%).
Four species namely V. thapsus, V. arcturus, V. nigrum
and V. sinuatum var. adenosepalum were comparatively
closer in their ITS sequences as compared to the other
two species (V. sinuatum and V. dentifolium). Different
trends were observed in the nucleotide bases counts of
the ITS regions in these species (Table-2). The distance
matrix estimated by Jukes and Cantor’s (1969) model
among the six species of Verbascum sequence
divergence values ranged from 0.002 to 0.88 and the
Kimura (1980) 2 parameter sequence divergence values
ranged from 0.002 to 0.925 (Table-3). Distance matrix
from both the methods indicated V. dentifolium and V.
sinuatum as the most divergent species within the group.
Utilizing the ITS sequences (ITS-1 + 5.8s + ITS-2)
of two different generic species namely, Alonsoa
meridionalis (DQ222398.1) and Charadrophila capensis
(AJ616317.1) which were included from the tribe
Alonsoeae further confirms that out grouping of species
V. dentifolium and V. sinuatum (Fig.2) in the
phylogenetic tree generated by maximum parsimony
method. Both these species share same length (178
bases) of their highly conserved 5.8s regions while in
the other four species this conserved 5.8s region has
164 nt base length. Interestingly, V. dentifolium and V.
sinuatum are found from the same geographical location
(Morocco) and were clustered in same clade of
phylogenetic tree with high bootstrap value (100).
Among the different clades in maximum parsimony
tree, the length of ITS-2 was more variable as compared
to ITS-1. This spacer (ITS-2) is now gaining importance

Table-1. Base composition of ITS region in Verbascum thapsus and related species.

Taxa ITS-1 Region 5.8S Region ITS-2 Region Total

(Acc. No.) A : T : G : C L G+C % A : T : G : C L G+C % A : T : G : C L G+C % L G+C %

V. thapsus
(JQ801746.1) 29:30:55:62 176 66.48 42:34:46:42 164 53.66 28:46:68:73 215 65.58 62.34
V. arcturus
(AJ550615.1) 63:55:81:83 282 58.16 41:35:46:42 164 53.66 43:57:90:91 281 64.41 59.56
V. nigrum
(HQ130064.1) 28:30:53:64 176* 66.48 42:34:46:42 164 53.66 28:46:67:76 225** 65.90 62.48
V. sinuatum var. adenosepalum
(JF409917.1) 31:32:52:61 176 64.20 41:35:46:42 164 53.66 29:46:70:82 227 66.96 62.26
V. sinuatum
(HE602435.1) 77:68:124:120 389 62.72 28:34:60:56 178 65.17 23:21:31:25 100 56.00 62.37
V. dentifolium
(HE602436.1) 76:68:124:119 387 62.79 28:34:60:56 178 65.17 23:23:31:27 104 55.77 62.33

* N=1; ** R+Y+M+S=2+2+2+2=8 (where N= any base, R= purine, Y= pyrimidine, M= A or C, S= G or C)

Table-2. Table showing the different trends in the nucleotide

Table-3. Distance matrix for ITS nucleotide sequence based
bases as observed in ITS regions of Verbascum species.
on Jukes and Cantor’s model (JC) and Kimura-2 parameter
Species ITS-1 5.8s ITS-2 model (K 2).

V. thapsus (VT) C>G>T>A G>C=A>T C>G>T>A JC↓K 2 VT VD VN VS VSA VA

V. arcturus (VA) C>G>A>T G>C>A>T C>G>T>A →
V. nigrum (VN) C>G>T>A G>C=A>T C>G>T>A
VT - 0.918 0.020 0.918 0.045 0.043
V. sinuatum var.
VD 0.880 _ 0.897 0.002 0.916 0.925
adenosepalum
VN 0.020 0.863 _ 0.897 0.046 0.043
(VSA) C>G>T>A G>C>A>T C>G>T>A
VS 0.880 0.002 0.863 _ 0.916 0.925
V. sinuatum
VSA 0.044 0.874 0.046 0.874 _ 0.013
(VS) G>C>A>T G>C>T>A G>C>A>T
VA 0.042 0.880 0.042 0.880 0.013 _
V. dentifolium
(VD) G>C>A>T G>C>T>A G>C>A>T

Medicinal Plants, 6(1) March 2014

 Molecular Identification of V. thapsus L. 5

Fig. 2. Phylogenetic tree of Verbascum

species with two different members
of Alonsoeae generated using ITS
sequence according to Maximum –
Parsimonious method. Numbers above
lines are the bootstrap values in 1000
replicates. (PS=single nucleotide
parsimonious sites).

Medicinal Plants, 6(1) March 2014

6 Srivastava & Saggoo 2014
in evolutionary studies at different taxonomic levels
(Yao et al., 2010; Chen et al., 2010; Lia et al., 2011;
Khanapurkar et al., 2012). As the 5.8s rDNA exhibits
very low level of sequence variation, ITS-2 could be
more informative for phylogenetic comparisons.
In order to check the phylogenetic situation in V.
thapsus L. and other species referred above in ITS
sequence analysis in silico mining and analyses of
rbcL [e.g. V. thapsus (JN893755; L36452), V. nigrum
(JN893706), V. virgatum (HM850442), V. creticum
(HM850440), V. speciosum (AJ490885)] and matK
sequences [e.g. V. thapsus (JN893995; AF052002), V.
nigrum (JN896027), V. virgatum (HM850966), V.
creticum (HM850964)] which were deposited in
GeneBank was done. The sequence analysis revealed
the absence or extremely low inter-specific variability
and barcode gap in the available species of Verbascum
L. Low and insignificant inter-specific variation in rbcL
and matK sequences in Verbascum L. restrict the limit
of these markers up to the identif ication or
classification of the genus at family level. Further, it
supports the views and recommendation of internal
transcribed spacers (ITS) as a barcode for the species
identification in flowering plants (Hollingsworth, 2011;
Han et al., 2012). The DNA barcoding represents a
powerful and rapid method for species identification in
plants like Iliiamna (Slotta and Tracey, 2000), Aconitum,
Illicium and Ricinus (Matsuyama et al., 2009),
Scrophularia (Lee et al., 2010), Meconopsis (Li and
Dao, 2011a,b), Ruta (Al-Qurainy et al., 2011), Dipsacus
(Li et al., 2012), Bupleurum (Kim et al., 2012), etc.
In the present study the V. thapsus ITS sequence
analysis revealed a total of 18 single nucleotide
positions in internal transcribed spacer (10 in ITS-1,
one in 5.8 and 7 in ITS-2 region) which can be helpful
in authentication of the species at molecular level.
Based on sequence divergence and phylogenetic
pattern, the results illustrated that V. thapsus was more
closely related to V. arcturus, V. nigrum and V. sinuatum
var. adenosepalum than to V. sinuatum and V.
dentifolium.
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