A universal primer set to amplify the cytochrome c oxidase subunit I gene in bears

Author(s): Iram Shahzadi, Safia Janjua, Fakhar-i-Abbas and Gary J. Galbreath

Source: Ursus, Vol. 25, No. 1 (2014), pp. 74-78
Published by: International Association for Bear Research and Management
Stable URL: https://www.jstor.org/stable/24643798
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Short Communication • Shahzadi et al.

A universal primer set to amplify the cytochrome c oxidase subunit

gene in bears
Iram Shahzadi1, Safia Janjua1,2,4, automatable taxonomic identification by using short
Fakhar-i-Abbas1,2, and Gary J. Galbreath3 specific sequences as internal species tags. It has
the potential to make taxonomic assignment more
broadly accessible (Hajibabaei et al. 2007), with
11nstitute of Natural and Management Sciences (INAM),
31-D, 6th Road, Satellite Town, Rawalpindi-46000,
benefits to systematists, ecologists, conservationists,
Pakistan
and those working with vectors, in the control of
2Bioresource Research Centre, 34 Bazaar Road, G-6/4, pest or invasive species, or with food safety (Hebert
Islamabad, Pakistan
and Gregory 2005).
3Biological Sciences, Northwestern University, Evanston,
Bears (Ursidae, Carnivora, Mammalia) can be
IL 60208, USA; and Field Museum of Natural History,
divided into 3 subfamilies: Ailuropodinae, Tremarc
Chicago, IL 60605, USA
tinae, and Ursinae (Wilson and Reeder 2005). The
first contains a single extant species, giant panda
Abstract: Barcoding DNA is an accepted tool in
(Ailuropoda melanoleuca; David, 1869), as does the
taxonomic identification. We report the design of a
second with spectacled bear (Tremarctos ornatus;
universal primer pair for polymerase chain reaction
Cuvier, 1825), while the third contains 6 species viz
(PCR) amplification of the cytochrome c oxidase
American black bear (Ursus americanus; Pallas,
subunit I gene (COI) developed using COI gene
1780), brown bear (U.. arctos; Linnaeus, 1758), sun
sequences of all 8 extant bear species (family
bear (U. malayanus; Raffles, 1821), polar bear (U.
Ursidae) available on GenBank. This primer pair
maritimus; Phipps, 1774), Asiatic black bear (U.
successfully amplified approximately 700 base pairs
thibetanus; Cuvier, 1823), and sloth bear ( U. ursinus;
of the COI gene of Asiatic black ( Ursus thibetanus; n
Cuvier, 1823). Among the extinct species of bears,
= 12) and Himalayan brown ( U. arc tos; n = 6)
mitochondrial DNA is available for cave bear (U..
bears. We sequenced the PCR product and com
pared the sequences with those of the other 6 species
spelaeus; Krause et al. 2008). All extant species
except American black bear and brown bear are
to generate degrees of homology (83-92%) and
classed as vulnerable (IUCN 2013).
genetic distances. The primer pair has yet to be
tested in the other 6 species. We developed a Several studies suggested a pair of primers for
preliminary phylogenetic tree for the 8 species based
the cytochrome c oxidase subunit I (COI) gene for
on these data. member species of family Ursidae (Hebert et al.
2003; Yu et al. 2004, 2007; and Jun et al. 2011). Here
Key words: Asiatic black bear, cytochrome c oxidase we report design of a COI primer pair utilized for 2
ursine species, the Asiatic black bear, and the brown
subunit I (COI) gene, DNA barcoding, Himala
bear, which occur in northern parts of Pakistan and
yan brown bear, Ursidae, Ursus arctos, Ursus
thibetanus Azad Jammu and Kashmir. The degenerate primer
Ursus 25(l):74-78 (2014) pair, designed from consensus sequence of the COI
gene of all 8 extant bear species, is proposed as a
universal primer pair for barcoding members of
Ursidae.
We collected blood samples from U. thibetanus (n
Part or all of the mitochondrial genome is widely
= 12) and U. arctos (n = 6) housed at the
used in attempts to resolve phylogenetic relation
Bioresource Research Centre's Bear Sanctuary,
ships (e.g., Brown et al. 1982, Moore 1995, Hebert et
Balkasar, Pakistan, which we stored in ethylenedi
al. 2003, Boore 2004). As a small, circular chromo
aminetetraacetic acid (EDTA) vacutainer tubes,
some, the complete nucleotide sequence of the
mitochondrial genome of many extant, as well as
transported on ice, and stored at 4°C before
extinct, organisms is known (Krause et al. 2008). processing. We adopted an organic protocol involv
ing proteinase K digestion, devised by Higuchi et al.
Barcoding of DNA can provide rapid, accurate, and
(1988) and modified by Vigilant et al. (1989) for
4email: safiajanjua@hotmail.com DNA extraction. We collected 200 \\L of blood

74

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 Short Communication •Shahzadi et al. 75

Table 1. Properties of primer pair designed for amplification of cytochrome c oxidase subunit
species in the family Ursidae. GC = sum of guanine and cytosine nucleotide, Tm = melting tempe
primer, Ta = annealing temperature, and nt = nucleotide.

Primer Sequence 5'—3' Primer size (nt) GC (%) Tm (°C) Ta (°C) Product size (nt)
C01UTF
C01UTF TCGT
TCGTAACTGCCCATGCA
AACTGCCCAT GCA 17
17 53
53 54
54 54
54 705
705
C01UTR TATGRTGRGCTCAYACGA 18 47 51

sample to which equal volume of lysis buffer GeneJET PCR purification kit (no. K0702, Fermen
(400 mM NaCl, 0.5% Triton X-100) and Tris tas, Pittsburgh, Pennsylvania, USA) and sequenced it
50 mM (pH 7.4) and 45 nL of proteinase K 2 (mg/ via a commercial sequencing service (MACROGEN,
mL) were added. We incubated the samples at 56°C Seoul, Korea).
for 3 hours in a water bath, centrifuged them atTo demonstrate that we obtained COI gene
10,000 revolutions per minute (rpm) for 10 minutes, sequences of U. thibetanus and U. arctos via
discarded the supernatant, and lysed the pellet withsequencing the PCR products amplified using these
200 |iL of the lysis buffer and 45 |^L proteinaseprimers,
K we compared our sequences with those
(2 mg/mL) at 56°C for 30 minutes. We twice retrieved of U. maritimus, U. ursinus, T. ornatus, A.
extracted the samples with equal volume of buffered melanoleuca, U. americanus, and U. malayanus from
phenol and centrifuged at 12,000 rpm for 10 minutes.the GenBank. We aligned all sequences using
We collected the upper aqueous phase in fresh CLUSTALW (http://www.genome.jp/tools/clustalw/),
microcentrifuge tubes; DNA was allowed to precipconducted evolutionary analyses in MEGA5 (Tamura
itate by adding equal volume of isopropanol and was
et al. 2011), and inferred evolutionary history using the
incubated at -20°C for 30 minutes. We pelletedmaximum-likelihood method based on the Tamura
DNA via centrifugation (10,000 rpm for 10 min), Nei model (Tamura and Nei 1993). Strength of nodes
washed the pellet with 70% ethanol, and allowed thewas assessed via the bootstrap method with 500
pellet to air dry. We added 50 |iL of TE buffer replications. The codon positions included were 1st +
(10 mM Tris-HCl, 10 mM EDTA; pH 8.0) and 2nd + 3rd. We eliminated all positions involving
stored the product at -20°C until further use. missing data during the analysis. We estimated genetic
We retrieved sequences of the mitochondrial COI distances among the 8 bear species by MEGA5
gene of 8 bear species (Accession nos. AF303109.1, (Tamura et al. 2011). We conducted analyses using
EF196661.1, AF303110.1, AP012597.1, EF196662.1, the Maximum Composite Likelihood model (Tamura
FM177765.1, EF212882.1, and EF196665.1) from et al. 2004).
GenBank and used the Genefisher2 online tool Size of the primers differed by one nucleotide
(Table 1). With amplification and sequencing of a
(http://bibiserv.techfak.uni-bielefeld.de/genefisher2/
manual.html) to obtain consensus sequence and for
705 base-pair fragment of COI gene with the primer
primer design. We carried out polymerase chain
pair and using CLUSTALW (Table 2), we found
reaction (PCR) in a 25-^L reaction mixture, contain
considerable homology (>82%) when comparing all
ing 4 |j.L of DNA template (50-75 ng/|j.L), IX bearTaq
species with U. americanus. We found greater
buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.8%
levels of homology between U. americanus and each
Nonidet P40), 1.5 mM MgCl2, 0.2 mM of each of the other 5 species of Ursus ( U. thibetanus = 92%,
dNTPs, 1.5 U of Taq DNA Polymerase (Fermentas U. arctos = 91%, U. maritimus = 91%, U. malayanus
UAB, Vilnius, Lithuania), and 0.5 pM of each primer. = 91%, U. ursinus = 90%); homology between U.
We performed PCR in a Thermal Cycler (PCR Sprintamericanus and T. ornatus and A. melanoleuca was
Thermal Cycler; ThermoFisher Scientific, Waltham,84% and 83%, respectively. The genetic distance
Massachusetts, USA) under a standard cycle condi between these species follows a reverse order, as one
tion consisting of a single cycle of pre-amplification might expect, with the small genetic distance of 0.078
denaturation at 94°C for 5 minutes followed by 35 between U. americanus and U. thibetanus, and the
cycles of denaturing at 94°C for 45 seconds, annealing greatest genetic distance (0.230) between U. malaya
at 54°C for 1 minute, and extension at 72°C for nus and A. melanoleuca (Table 2).
1 minute. Final extension was done at 72°C for The maximum-likelihood tree (greatest log likeli
10 minutes. We purified the amplicon using a
hood —2,393.61) linked the 6 species within genus

Ursus 25(l):74-78 (2014)

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76 Short Communication • Shahzadi et al.

Table 2. Matrix of genetic distances between different species of family Ursidae for approximately 705 base
pair fragment of mtDNA cytochrome c oxidase subunit I gene sequence. The number of base substitutions per
site between sequences is shown.

U. americanus U. thibetanus
thibetanus U.
U. arctos
arctos U.
U. maritimus
maritimus U.
U. malayanus
malayanus U.
U. ursinus
ursinus T. ornatus
U. americanus
U. thibetanus 0.078
U. arctos 0.092 0.098
U. maritimus 0.087 0.093 0.016
U. malayanus 0.088 0.106 0.101 0.099
U. ursinus 0.106 0.103 0.107 0.106 0.124
T. ornatus 0.186 0.188 0.194 0.187 0.178 0.184
A. melanoleuca 0.195 0.218 0.202 0.198 0.230 0.196 0.180

2004b; Ward et al. 2005;to

Ursus with high support, Kerr etthe
al. 2007; Luo et al.
exclus
2011). The phylogenetic tree
ornatus and A. melanoleuca we generated
(Fig. 1). is inUrs
and U. maritimus show an
agreement with expected
previous close
studies (Hashimoto et al.
ship within the Ursus
1993, Waits etclade. Though
al. 1999, Yu et al. 2007), for which w
statistical strength,different U. genes
ursinus
of mitochondrial as
DNA the basal
were used for
of Ursinae is highly supported
analyses; (bootstrap
this suggests that the universal primers we
74 for monophyly of used amplified
the the target portion
other 5of species
the COI gene of
Traditional analytical (and not a pseudogene).
approaches, such as p
of allozyme or restriction Ivanova et al. (2006, 2007) provided apolymo
enzyme cocktail
have now largely been of primersreplaced
for barcoding of mammalsbyandsequen
other
analyses (Hajibabaei vertebrates, et al. 2007),
but pointed out the needwhich
to improve the hav
tionized molecular phylogenetic efficiency and specificity of the studies
primer cocktail. (Pa
Mitochondrial DNA markers, being haploid and Indeed, Nougoue (2012) tested the Ivanova et al.
maternally inherited, are frequent targets for analysis (2007) primer pairs on representative species within
and have made strong contributions to population different orders of Mammalia, including Carnivora,
studies (Avise 2004; Hebert et al. 2004a, b). Our study and reported that the primer pairs required modifi
is a step toward rapid molecular identification of cations for amplification of different taxa. This study
species of the family Ursidae, using the COI represents our attempt to develop family-specific
mitochondrial gene, a widely used molecular taxon primers by testing the primer pairs in 2 bear
omy marker for animal species (Hebert et al. 2003, species—U. thibetanus and U. arctos. We suspect

33 U. americanus

43 U. thibetanus

74 U. malayanus
U. arctos
100
100 U. maritimus

U. ursinus

T. ornatus

A. melanoleuca

0.10 0.08 0.0G 0.04 0.02 0.00

Fig. 1. Maximum-likelihood tree generated using approximately 705 base-pair fragment of cytoch
oxidase subunit I gene sequence of 8 bear species. The percentage of trees in which the associated
clustered together are shown next to the branches; distance scale is given below.

Ursus 25(l):74-78 (2014)

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 Short Communication • Shahzadi et al. 77

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