Evaluating the systematic position of Ehretia asperula Zoll. & Moritzi based on ITS1, matK and trnL-trnF DNA sequences

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Evaluating the systematic position of Ehretia asperula Zoll. & Moritzi based on ITS1, matK and trnL-trnF DNA sequences. Ehretia asperula, popular as a medicinal herb that has potential on cancer treatment, has a limited research about its phylogeny relationships. With many innovative advancements in molecular biology, it is more easy and reliable to identify the taxonomic position of a species by molecular markers- DNA barcode. In this study, we used three different markers, ITS1, trnL-trnF, and matK, to evaluate E. asperula’s systematic position. Based on ITS1 sequence, E. asperula belongs to clade Ehretia I and is a close relative of E. resinosa. Moreover, ITS1 was suggested to be use as a suitable DNA barcode in order to identify E. asperula.
Life ScienceS | Biotechnology
Evaluating the systematic position
of Ehretia asperula Zoll. & Moritzi based
on ITS1, matK and trnL-trnF DNA sequences
Thuy Linh Nguyen1, Thi Hang Pham1, Van Truong Do2, Thi Thu Hue Huynh1*
1Institute of Genome Research, Vietnam Academy of Science and Technology
2Vietnam National Museum of Nature, Vietnam Academy of Science and Technology
Received 31 July 2017; accepted 7 December 2017
Abstract:
a climbing shrub, having bristle-covered
Ehretia asperula, popular as a medicinal herb that has potential on cancer
treatment, has a limited research about its phylogeny relationships. With many
innovative advancements in molecular biology, it is more easy and reliable
to identify the taxonomic position of a species by molecular markers- DNA
barcode. In this study, we used three different markers, ITS1, trnL-trnF, and
matK, to evaluate E. asperula’s systematic position. Based on ITS1 sequence,
E. asperula belongs to clade Ehretia I and is a close relative of E. resinosa.
Moreover, ITS1 was suggested to be use as a suitable DNA barcode in order to
identify E. asperula.
Keywords: Ehretia asperula, ITS1, matK, phylogenetics, trnL-trnF.
reddish to greyish brown branches.
Leaves are blade lanceolate in which
the base is narrowly rounded and 5-7
mm in length, and have 7 nerves on each
side. Inflorescences are terminal and
lateral on short branches, and 5-10 cm
in length, where individual flower’s base
is 2-4 mm long. Flowers have separated
five calyx-lobes and white petals.
Fruits are 1 cm-long globoses with 3
separate parts. Seeds have pink skin. E.
Classification number: 3.5
asperula produces flowers from March
to May and bears fruits from August to
Introduction
Ehretia asperulaZoll. & Moritziis an
accepted name of a species from Ehretia
genus (Ehretiaceae family). It was first
described by Zollinger and Moritzi in
mid 1840s [1]. In Vietnam, it is present
mainly on the mountainous area of the
North [2]. Historically, E. asperula was
used in ethnopharmalogy and folklore.
The ethnic minorities have been using
E. asperula for treatments of various
ailments, especially for liver diseases,
such as hepatitis, liver cirrhosis and even
liver cancer [2]. Besides, E. asperula
appears to be effective in prevention
Although many patients who applied
folklore procedure using E. asperula in
combination with other medicinal herbs
or with modern treatments recovered
from cancer, and prolonged their life [3],
no clinical trial has been published using
E. asperula in cancer therapy. Likewise,
the underlying mechanism responsible
for its inhibitory effect on cancer is
still unclear. Yet there is no study of E.
asperula molecular and its phylogenetic
relationship has not been reported.
The Ehretia genus has nearly 50
species distributed mainly in high
altitude areas of Asia, Africa, and
December [2].
The taxonomic relationship of
Ehretia species was clarified by studies
based on molecular data. On the basis
of the ITS1 information, Ehretia was a
sister group of Bourreria (Ehretiaceae
family) and is composed of three major
clades. Among them, Ehretia III has
a closer relationship with Ehretia II
compared to Ehretia I [4]. Additionally,
the ITS1 secondary structure was applied
to build phylogenetic trees at higher
taxonomic levels, and seems to be good
at giving a well-resolved tree. Recently,
Gottschling, et al. [5] elucidated the
of
acne,
jaundice,
hypertension,
and
Australia [1]. In Vietnam, they represent
phylogeny relationship of Boraginales
diabetes
[2].
A
research
recently
seven species [3], in which, E. asperula
by
using
concatenated
ITS
nuclear
revealed the phamarcological potential
shares many characteristics in common
and
plastid
rps16
trnL-trnF,
trnS-
of E. asperula in cancer treatment [3].
with other Ehretiaspecies. E. asperula is
trnG
sequences.
An
additional
clade
*Corresponding author: Email: hthue@igr.ac.vn
December 2017 Vol.59 Number 4
Vietnam Journal of Science,
Technology and Engineering
61
Life ScienceS | Biotechnology
Ehretia IV, in which E. microphylla is
Table 1. List of primer pairs used in the study.
a presentative, was inferred from extent
analysis.
Primer names
DNA regions
Primer sequences (5’3’)
In this study, ITS1, trnL-trnF, and
matK regions were used to investigate
ITS-AB-101
ACG AATTCATGG TCC GGT GAA GTG TTC G
the systematic position of E. asperula.
As inferred from the data, E. asperula
ITS-AB-102
ITS
TAG AATTCC CCG GTT CGC TCG CCG TTA C
belongs to Ehretia I and is a close
relative of E. resinosa. Moreover, we
TrnL-F
trnL-trnF
ATTTGAACT GGT GAC ACG AG
contributed
three
sequences
to
DNA
TrnL-C
CGAAAT CGG TAG ACG CTA CG
barcode database of Ehretia genus, and
suggested ITS1 as an appropriate DNA
barcode.
MatK-F1A
MatK-R1A
matK
ACY GTATTTTAT GTTTAC GAC G
TCC ATH TDG AAATCTTGG TTC A
Materials and methods
Three leaf specimens of E. asperula
Table 2. Species lists with ID number of DNA sequences on GenBank.
were collected at three different
locations in the Hoa Binh province by a
Name
ITS1
trnL-trnF
matK
Name
ITS1
trnL-trnF
matK
colleague at Vietnam national museum
E. asperula
KY320205
KY320206
KY320207
E. obtusifolia
AY331401.1
of nature, and were preserved in silica
gel. Genomic DNA was isolated using
E.
acuminata
AF385799.2
AY376167.1
HQ427413.1
E.macrophylla
AF385802.2
KF673271.1
CTAB
(Cetyltrimethylammonium
E. amoena
JX518091.1
E. microphylla
AY463160.1
KF158204.1
bromide) extraction protocol [6] from
approximately 100 g of leaf tissue.
E. anacua
AF385796.2
DQ197228.1
E.monopyrena
AF385792.2
Three DNA fragments were amplified
E. aquatica
AF385791.2
EU599659.1
E. resinosa
AY463161.1
using Thermo scientific phusion high-
fidelity DNA polymerase with universal
E. cortesia
AY463159.1
KF673292.1
E. rigida
AF385789.2
JX518014.1
primer pairs (Table 1). PCR products
E. coerulea
KF673249.1
E. saligna
AF385786.2
KF673272.1
KM894705.1
were
purified
with
Thermofisher
E. cysmosa
AF385790.2
E. tinifolia
AF385793.2
HQ286270.1
scientific PCR clean-up purification kit.
These DNA were sequenced by Applied
E. grahamii
KU564569.1
E. thyrsiflora
EU600007.1
EU599655.1
biosystems 3500 genetic analyzer system
E. laevis
AF385787.2
KF673273.1
E. wallichiana
AY331402.1
using BigDyeTM terminator v3.1 cycle
sequencing kit. Additionally, GenBank
E. latifolia
AF385797.2
KF673282.1
B. succulenta
AF385776.2
DQ197229.1
accessions were downloaded to complete
E. longiflora
AY331400.1
EU600010.1
KJ687555.1
B. petiolaris
KF673275.1
dataset
for
molecular
investigation
comprising of 37 sequences of Ehretia
likelihood setting: DBS) were estimated
the products obtained were 848 bp, 950
species, and three sequences of outgroup
based
on
1,000
replicates,
in
which
bp, and 811 bp in length, respectively
representatives
(Table
2).
Sequences
each was performed with 100 random-
(Fig. 1). ITS is a nuclear sequence from
were aligned automatically by BioEdit
addition-sequence
replicates,
and
the
3’end of 18S to 5’end of 26S. However,
v7.1.9 [7]. Phylogenetic analysis was
starting
tree
obtained
by
neighbor-
we
used
a
portion
of
ITS-ITS1
for
performed
by
PAUP*4.0a152
[8].
joining.
phylogenetic calculation in this study.
Likelihood trees
heuristic search.
were built using
Bootstrap analyses
Results
While trnL-trnF is the intergenic region
between two coding regions, matK
(criterion=parsimony, with full heuristic
All three DNA regions (ITS, trnL-
spans from codon 171 to codon 440 of
search:
PBS;
criterion=distance,
with
trnF, and matK) were amplified by using
the open reading frame. The nucleotide
neighbor-joining search and maximum
the universal primer pairs (Table 1), and
sequences of each DNA region of the
62
Vietnam Journal of Science,
Technology and Engineering
December 2017 Vol.59 Number 4