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

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 1Institute of Natural and Management Sciences (INAM), broadly accessible (Hajibabaei et al. 2007), with 31-D, 6th Road, Satellite Town, Rawalpindi-46000, benefits to systematists, ecologists, conservationists, Pakistan and those working with vectors, in the control of 2 Bioresource Research Centre, 34 Bazaar Road, G-6/4, pest or invasive species, or with food safety (Hebert Islamabad, Pakistan 3 and Gregory 2005). Biological Sciences, Northwestern University, Evanston, Bears (Ursidae, , Mammalia) can be IL 60208, USA; and Field Museum of Natural History, divided into 3 subfamilies: Ailuropodinae, Tremarc- Chicago, IL 60605, USA tinae, and (Wilson and Reeder 2005). The first contains a single extant species, Abstract: Barcoding DNA is an accepted tool in melanoleuca; taxonomic identification. We report the design of a ( David, 1869), as does the Tremarctos ornatus; universal primer pair for polymerase chain reaction second with spectacled ( (PCR) amplification of the cytochrome c oxidase Cuvier, 1825), while the third contains 6 species viz subunit I gene (COI) developed using COI gene American black bear (Ursus americanus; Pallas, sequences of all 8 extant bear species (family 1780), brown bear (U. arctos; Linnaeus, 1758), sun Ursidae) available on GenBank. This primer pair bear (U. malayanus; Raffles, 1821), polar bear (U. successfully amplified approximately 700 base pairs maritimus; Phipps, 1774), Asiatic black bear (U. of the COI gene of Asiatic black (Ursus thibetanus; n thibetanus; Cuvier, 1823), and sloth bear (U. ursinus; 5 12) and Himalayan brown (U. arctos; n 5 6) Cuvier, 1823). Among the extinct species of bears, bears. We sequenced the PCR product and com- mitochondrial DNA is available for (U. pared the sequences with those of the other 6 species spelaeus; Krause et al. 2008). All extant species to generate degrees of homology (83–92%) and except American black bear and brown bear are genetic distances. The primer pair has yet to be classed as vulnerable (IUCN 2013). 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 subunit I (COI) gene, DNA barcoding, Himala- ursine species, the Asiatic black bear, and the brown yan brown bear, Ursidae, Ursus arctos, Ursus bear, which occur in northern parts of Pakistan and thibetanus Azad Jammu and Kashmir. The degenerate primer Ursus 25(1):73–77 (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. Part or all of the mitochondrial genome is widely We collected blood samples from U. thibetanus (n used in attempts to resolve phylogenetic relation- 5 12) and U. arctos (n 5 6) housed at the ships (e.g., Brown et al. 1982, Moore 1995, Hebert et Bioresource Research Centre’s Bear Sanctuary, al. 2003, Boore 2004). As a small, circular chromo- Balkasar, Pakistan, which we stored in ethylenedi- some, the complete nucleotide sequence of the aminetetraacetic acid (EDTA) vacutainer tubes, mitochondrial genome of many extant, as well as transported on ice, and stored at 4uC before extinct, organisms is known (Krause et al. 2008). processing. We adopted an organic protocol involv- Barcoding of DNA can provide rapid, accurate, and ing proteinase K digestion, devised by Higuchi et al. (1988) and modified by Vigilant et al. (1989) for 4email: [email protected] DNA extraction. We collected 200 mL of blood

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

Primer Sequence 59—39 Primer size (nt) GC (%)Tm(6C) Ta (6C) Product size (nt) CO1UTF TCGTAACTGCCCATGCA 17 53 54 54 705 CO1UTR 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 mL of proteinase K 2 (mg/ via a commercial sequencing service (MACROGEN, mL) were added. We incubated the samples at 56uC Seoul, Korea). for 3 hours in a water bath, centrifuged them at To 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 with sequencing the PCR products amplified using these 200 mL of the lysis buffer and 45 mL proteinase K primers, we compared our sequences with those (2 mg/mL) at 56uC 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 precip- conducted 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 220uC for 30 minutes. We pelleted maximum-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 the was assessed via the bootstrap method with 500 pellet to air dry. We added 50 mL 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 220uC 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 (http://bibiserv.techfak.uni-bielefeld.de/genefisher2/ (Table 1). With amplification and sequencing of a 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-mL reaction mixture, contain- considerable homology (.82%) when comparing all ing 4 mL of DNA template (50–75 ng/mL), 1X Taq bear 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 5 92%, dNTPs, 1.5 U of Taq DNA Polymerase (Fermentas U. arctos 5 91%, U. maritimus 5 91%, U. malayanus UAB, Vilnius, Lithuania), and 0.5 pM of each primer. 5 91%, U. ursinus 5 90%); homology between U. We performed PCR in a Thermal Cycler (PCR Sprint americanus 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 94uC for 5 minutes followed by 35 between U. americanus and U. thibetanus, and the cycles of denaturing at 94uC for 45 seconds, annealing greatest genetic distance (0.230) between U. malaya- at 54uC for 1 minute, and extension at 72uC for nus and A. melanoleuca (Table 2). 1 minute. Final extension was done at 72uC for The maximum-likelihood tree (greatest log likeli- 10 minutes. We purified the amplicon using a hood 22,393.61) linked the 6 species within genus

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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 U. arctos U. maritimus U. malayanus U. 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

Ursus with high support, to the exclusion of T. 2004b; Ward et al. 2005; Kerr et al. 2007; Luo et al. ornatus and A. melanoleuca (Fig. 1). Ursus arctos 2011). The phylogenetic tree we generated is in and U. maritimus show an expected close relation- agreement with previous studies (Hashimoto et al. ship within the Ursus clade. Though with less 1993, Waits et al. 1999, Yu et al. 2007), for which statistical strength, U. ursinus as the basal member different genes of mitochondrial DNA were used for of Ursinae is highly supported (bootstrap support 5 analyses; this suggests that the universal primers we 74 for monophyly of the other 5 species of Ursus). used amplified the target portion of the COI gene Traditional analytical approaches, such as patterns (and not a pseudogene). of allozyme or restriction enzyme polymorphisms, Ivanova et al. (2006, 2007) provided a cocktail have now largely been replaced by sequence-based of primers for barcoding of and other analyses (Hajibabaei et al. 2007), which have revolu- vertebrates, but pointed out the need to improve the tionized molecular phylogenetic studies (Pagel 1999). efficiency and specificity of the primer cocktail. 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 species (Hebert et al. 2003, species—U. thibetanus and U. arctos. We suspect

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

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