TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Background: </strong>DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories.</p>
<p> </p>
<p><strong>Results: </strong>A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content.</p>
<p><strong> </strong></p>
<p><strong>Conclusion:</strong> The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.</p>

[BEFORE_START]
<p><strong>Prepare the following solutions for CTAB DNA isolation:</strong></p>
<ul>
<li>50 ml Chloroform</li>
<li>20 ml Ice-cold ethanol (96%) (e.g. stored at -20ºC)</li>
<li>20 ml 70% ethanol</li>
</ul>

[GUIDELINES]
<p style="text-align: justify;">This DNA metabarcoding method has been designed for the qualitative identification of multiple species in complex samples. DNA metabarcoding combines DNA barcoding with next-generation sequencing (NGS), and makes use of universal PCR primers to mass-amplify and sequence one or more taxonomically informative DNA barcodes. In this SOP, the following animal DNA barcodes are used: COI-2, cytochrome b (cyt <em>b</em>), 16S ribosomal RNA (16S). The following plant DNA barcodes are used: maturase K (<em>matK</em>), ribulose 1-5 biphosphate carboxylase large subunit (<em>rbcL</em>), the chloroplast <em>trnL </em>(UAA) intron, and the Internal Transcribed Spacer 2 (ITS2) region. Full-length barcodes and mini-barcodes with reduced sequence lengths are used for 16S, COI-2, cyt <em>b</em>, <em>rbcL</em>, ITS2 and <em>trnL </em>to accommodate the identification of species in degraded DNA samples. All amplicons are amplified in singleplex PCR.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Samples </strong></p>
<p style="text-align: justify;">The participating laboratories are provided with ten microcentrifuge tubes each containing 250 mg of powdered tissue stored in a cool box containing dry ice. The samples are labeled sample S1 to S10. In addition, two tubes containing positive control DNA (<em>Bos taurus </em>and <em>Lactuca sativa</em>) are shipped. The positive controls are labeled as 150 μl of 10 ng/μl Bos taurus and 150 μl of 10 ng/μl <em>Lactuca sativa. </em></p>
<p style="text-align: justify;">All samples should be stored frozen at -20°C until processed. Samples can be stored frozen indefinitely.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Required equipment, chemicals, reagents, consumables, and materials </strong></p>
<p style="text-align: justify;"><em><strong>Equipment </strong></em></p>
<p style="text-align: justify;">— 96-well thermocycler where the temperature range can be set between 8°C and 95°C, and the temperature accuracy is at least ±0.25°C (e.g. BIO-RAD CFX PCR Thermal Cycler).</p>
<p style="text-align: justify;">— Spectrophotometer (regular or NanoDrop)</p>
<p style="text-align: justify;">— Thermo-Shaker for 1.5 ml and 2 ml tubes capable of mixing at ~1000 rpm and heating at 65ºC (Mandatory).</p>
<p style="text-align: justify;">— Centrifuge for 1,5-2 ml tubes capable of spinning at ~18,000xg.</p>
<p style="text-align: justify;">— Mini-centrifuge for PCR tube strips capable of spinning at 10,000xg.</p>
<p style="text-align: justify;">— Vortex machine for 1.5 ml and 2 ml tubes.</p>
<p style="text-align: justify;">— Electrophoresis system to discriminate PCR amplification fragments between ~180 bp and ~920 bp (gel or microchip electrophoresis system)</p>
<p style="text-align: justify;">— Electrophoresis gel imaging UV machine</p>
<p style="text-align: justify;">— Full set of semi-automatic pipettes</p>
<p style="text-align: justify;">— Analytical scales (max. capacity 100 g, readability 0.001 g)</p>
<p style="text-align: justify;">— Fridge (or 4°C storage facility)</p>
<p style="text-align: justify;">— Freezer (or -20°C storage facility)</p>
<p style="text-align: justify;">— 1.5 ml and PCR tube racks</p>
<p style="text-align: justify;">— Cold Block or ice</p>
<p style="text-align: justify;"><em><strong>Chemicals and Reagents </strong></em></p>
<p style="text-align: justify;">— Nuclease-free molecular grade water (ddH2O)</p>
<p style="text-align: justify;">— 150 μl RNase A (QIAGEN (Cat No./ID: 19101), solution 100 mg/ml molecular biological quality or equivalent)</p>
<p style="text-align: justify;">— 500 μl Proteinase K (Fermentas (Cat No./ID: EO0491), solution 20 mg/ml, molecular biological quality or equivalent)</p>
<p style="text-align: justify;">— 50 ml Chloroform</p>
<p style="text-align: justify;">— 20 ml Ice-cold ethanol (96%)</p>
<p style="text-align: justify;">— 20 ml 70% ethanol</p>
<p style="text-align: justify;">— 1 ml 100 bp DNA Ladder (e.g. Invitrogen; for gel electrophoresis, if required).</p>
<p style="text-align: justify;">— If using gel electrophoresis system (instead of chip electrophoresis system): the necessary chemicals and reagents required for the chip electrophoresis system to be used.</p>
<p style="text-align: justify;">o 20 g Agarose</p>
<p style="text-align: justify;">o 40 μl Ethidium bromide</p>
<p style="text-align: justify;">o 1L TBE running buffer for gel electrophoresis</p>
<p style="text-align: justify;"> 54 g Tris</p>
<p style="text-align: justify;"> 27.5 g Boric acid</p>
<p style="text-align: justify;"> 20 ml 0.5M EDTA, pH 8</p>
<p style="text-align: justify;">o 5 ml 10X loading dye</p>
<p style="text-align: justify;"><em><strong>Consumables and Materials </strong></em></p>
<p style="text-align: justify;">— Centrifuge tubes (100-150 tubes 1.5 ml and 50 tubes 2 ml)</p>
<p style="text-align: justify;">— 50 PCR tubes strips (8 tube strips, single attached domed cap, e.g. Bioplastics EU 8-tube cat no. C78201).</p>
<p style="text-align: justify;">— Electrophoresis system, tray and comb used to discriminate PCR amplification fragments between ~180 bp and ~920 bp.</p>
<p style="text-align: justify;">— If using gel electrophoresis system (instead of chip electrophoresis system): the necessary chemicals and reagents required for the chip electrophoresis system to be used.</p>
<p style="text-align: justify;">— Micropipette filter tips (10 μl, 30 μl, 200 μl and 1000 μl tips)</p>
<p style="text-align: justify;">— Small plastic spatula</p>
<p style="text-align: justify;">— Disposable powder- free gloves</p>
<p style="text-align: justify;">— Clean lab coat</p>
<p style="text-align: justify;">— Cold Block or ice</p>
<p style="text-align: justify;">— Aluminium foil</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Chemicals and Reagents </strong></p>
<p style="text-align: justify;">All the provided chemicals and reagents are 2.5 times the amount required for the analysis.</p>
<p style="text-align: justify;">— 17.5 ml of CTAB extraction buffer</p>
<p style="text-align: justify;">— 25 ml of CTAB precipitation buffer</p>
<p style="text-align: justify;">— 9 ml of 1.2M NaCl</p>
<p style="text-align: justify;">— 6 tubes of HotStarTaq Master Mix and 3 tubes of water (Qiagen).</p>
<p style="text-align: justify;">— Oligonucleotide primers (high purity), 20 μl (10 μM) of each primer.</p>
<p style="text-align: justify;">— <strong>Primer labelling information</strong>:</p>
<table cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td valign="top">
<p><strong>16S-F </strong></p>
</td>
<td valign="top">
<p><strong>COI-2-F </strong></p>
</td>
<td valign="top">
<p><strong>CytB-F </strong></p>
</td>
<td valign="top">
<p><strong>16S-mini-F </strong></p>
</td>
<td valign="top">
<p><strong>COI-mini-F </strong></p>
</td>
<td valign="top">
<p><strong>CytB-mini-F </strong></p>
</td>
<td valign="top">
<p><strong>Matk-F </strong></p>
</td>
<td valign="top">
<p><strong>rbcL-F </strong></p>
</td>
<td valign="top">
<p><strong>trnL(UAA)-F </strong></p>
</td>
<td valign="top">
<p><strong>ITS2-F </strong></p>
</td>
<td valign="top">
<p><strong>rbcL-mini-F </strong></p>
</td>
<td valign="top">
<p><strong>trnL(P6loop) </strong></p>
<p><strong>-F </strong></p>
</td>
</tr>
<tr>
<td valign="top">
<p><strong>16S-R </strong></p>
</td>
<td valign="top">
<p><strong>COI-2-R </strong></p>
</td>
<td valign="top">
<p><strong>CytB-R </strong></p>
</td>
<td valign="top">
<p><strong>16S-mini-R </strong></p>
</td>
<td valign="top">
<p><strong>COI-mini-R </strong></p>
</td>
<td valign="top">
<p><strong>CytB-mini-R </strong></p>
</td>
<td valign="top">
<p><strong>Matk-R </strong></p>
</td>
<td valign="top">
<p><strong>rbcL-R </strong></p>
</td>
<td valign="top">
<p><strong>trnL(UAA)-R </strong></p>
</td>
<td valign="top">
<p><strong>ITS2-R </strong></p>
</td>
<td valign="top">
<p><strong>rbcL-mini-R </strong></p>
</td>
<td valign="top">
<p><strong>trnL(P6loop) </strong></p>
<p><strong>-R </strong></p>
</td>
</tr>
</tbody>
</table>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>CTAB DNA isolation </strong></p>
<p style="text-align: justify;">The protocol described is the CTAB DNA isolation procedure according to Murray and Thomson (1980).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><em><strong>Required reagents and chemicals </strong></em></p>
<p style="text-align: justify;">— Molecular grade water (ddH2O)</p>
<p style="text-align: justify;">— 17.5 ml CTAB extraction buffer</p>
<p style="text-align: justify;">— 150 μl RNase A (Qiagen, solution 100 mg/ml, molecular biological quality or equivalent)</p>
<p style="text-align: justify;">— 500 μl Proteinase K (Fermentas, solution 20 mg/ml, molecular biological quality or equivalent)</p>
<p style="text-align: justify;">— 50 ml Chloroform</p>
<p style="text-align: justify;">— 25 ml CTAB precipitation buffer</p>
<p style="text-align: justify;">— 9 ml NaCl</p>
<p style="text-align: justify;">— 20 ml Ice-cold ethanol (96%) (e.g. stored at -20ºC)</p>
<p style="text-align: justify;">— 20 ml 70% ethanol</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>PCR</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/nn5fw26.png" width="711" height="475" data-src="https://s3.amazonaws.com/pr-journal/nn5fw26.png" data-ofn="Screen Shot 2017-09-16 at 3.42.18 PM.png" /></strong></p>
<p style="text-align: justify;"><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/npffw26.png" width="708" height="327" data-src="https://s3.amazonaws.com/pr-journal/npefw26.png" data-ofn="Screen Shot 2017-09-16 at 3.56.12 PM.png" /></strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Illumina MiSeq sequencing (BaseClear) </strong></p>
<p style="text-align: justify;">— The purified PCR products together with the (digital) gel images will need to be sent to RIKILT, clearly stating your name and affiliation, and referencing the “DECATHLON WP5 DNA metabarcoding” project. The DNA should be shipped on cool packs (blue ice) to:</p>
<p style="text-align: justify;">RIKILT – Wageningen UR</p>
<p style="text-align: justify;">Attn: Martijn Staats/Alfred Arulandhu</p>
<p style="text-align: justify;">Akkermaalsbos 2</p>
<p style="text-align: justify;">Building 123</p>
<p style="text-align: justify;">6708 WB Wageningen</p>
<p style="text-align: justify;">The Netherlands</p>
<p style="text-align: justify;">— Please use (DHL/FedEx/TNT) express delivery service within your country for shipping the DNA to RIKILT.</p>
<p style="text-align: justify;">— RIKILT will collect all the purified and pooled PCR products of all partners and send them to BaseClear B.V. for Illumina MiSeq sequencing.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Bioinformatics data analysis </strong></p>
<p style="text-align: justify;"><em><strong>Procedure </strong></em></p>
<ul style="text-align: justify;">
<li>The raw Illumina MiSeq data will be processed by RIKILT using the default settings of the CITESspeciesDetect pipeline. Details of the CITESspeciesDetect pipeline are available upon request. <strong>Note</strong>: a user-friendly and platform-independent interface to the CITESspeciesDetect pipeline is currently under development, and is currently not available to the end-user.</li>
<li>RIKILT will send the bioinformatics output to each participating partner for interpretation of results. The output will consist of the following files:</li>
<li>The BLAST results file containing the 20 best BLAST results (i.e. bit-score, e-value, accession numbers etc.) for each Operational Taxonomic Unit [OTU]. Note that the results of the CytB and CytB mini-barcodes have, for technical reasons, been combined and presented both under “CytB”. Also, the results of the COI-2 and COI-mini barcodes have been combined and presented both under “COI-2”.</li>
</ul>
<p style="text-align: justify;">The BLAST output file is a tab-separated file that can be opened using spreadsheet software (e.g. Excel) or text editor software (e.g. notepad++ available from https://notepad-plus-plus.org/). The file contains the BLAST results for the 12 barcode markers separated by brackets. For each DNA barcode marker the results file contains the following headers:</p>
<p style="text-align: justify;">— qseqid OTU (query) sequence identifier with the associated number of NGS reads supporting the size of the OTU.</p>
<p style="text-align: justify;">— qlen OTU (query) sequence length.</p>
<p style="text-align: justify;">— qcovs Percentage OTU (query) coverage.</p>
<p style="text-align: justify;">— pident Percentage of identical matches.</p>
<p style="text-align: justify;">— bitscore Bit score.</p>
<p style="text-align: justify;">— evalue Expect value.</p>
<p style="text-align: justify;">— sacc Subject accession.</p>
<p style="text-align: justify;">— staxids Unique subject taxonomy identifier.</p>
<p style="text-align: justify;">— species Subject scientific name.</p>
<p style="text-align: justify;">— synonym Subject synonym name.</p>
<p style="text-align: justify;">— genus Subject genus name</p>
<p style="text-align: justify;">— family Subject family name.</p>
<p style="text-align: justify;">— order Subject order name.</p>
<p style="text-align: justify;">— Listed under CITES taxonomic listing (species/family/order).</p>
<p style="text-align: justify;">— Category CITES appendix (I/II/III).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">The BLAST tables are sorted based on bit score (bitscore) and it lists the most similar subject sequence(s) (sacc) on top. The BLAST output file often contains the concatenated hits for multiple OTUs (qseqid). For each DNA barcode marker these are named for example OTU_1;size=3863, OTU_2;size=1500, indicating that BLAST hits were found for two OTUs (OTU_1 and OTU_2), each of which are used in species identification. The size indicates the number of NGS reads that support that OTU cluster.</p>
<p style="text-align: justify;">Interpret the BLAST output and fill in tables 6 and 7 (Annex 1) using the following guidelines:</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<ul style="text-align: justify;">
<li>To minimize the chance of erroneous species identifications, it should be verified that at least three hits with highest (near identical) bit scores have the same species designation.</li>
<li>If multiple (three or more) hits are obtained with highest bitscores, but with different assigned species, the OTU fragment is assumed to lack the discriminatory power at the species level. In such cases, the OTU would be identified at the genus-level.</li>
<li>If multiple (three or more) hits are obtained with highest bitscores, but with different assigned genera, the OTU fragment lacks the discriminatory power at the genus level. In such cases, the OTU would be identified at the family-level.</li>
<li>If multiple (three or more) hits are obtained with highest bitscores, but with different assigned families, the OTU fragment lacks the discriminatory power at the family level. In such cases, the OTU would be identified at the order-level.</li>
<li>In case less than three hits (top hits and other) are reported, the OTU is identified at the family-level.</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Considerations when interpreting the (endangered) species identification: </strong></p>
<ul style="text-align: justify;">
<li>The completeness of the reference database is of importance. The current underrepresentation of DNA barcodes from species protected under CITES may critically hamper their identification. The identification of CITES species (and other species) will improve as DNA barcoding campaigns continue, such as the Barcode of Wildlife Project (www.barcodeofwildlife.org).</li>
<li>The NCBI GenBank is known to contain erroneous taxonomic names and identifications. While it remains difficult to specify clear guidelines on how to recognise them, we recommend not to consider taxa with clearly deviant family or order assignments in BLAST results with otherwise unambiguous family listings. Also, GenBank entries without informative taxonomic information, such as scientific names containing “environmental sample”, should be ignored.</li>
<li>The CITESspeciesDetect pipeline uses a 98% sequence similarity threshold to distinguish species based on DNA barcodes. This cut-off was chosen based on studies that found this divergence threshold to be sufficient to keep the majority of plants and animals apart using the standard <em>matK</em>, <em>rbcL</em>, cytB and <em>COI </em>DNA barcoding markers (add CITATION). The plant mini-barcode markers of <em>rbcL</em>, <em>trnL </em>(P6 loop) and ITS2 do not provide species-level resolution at the 98% level in all cases, but may nonetheless provide valuable taxonomic information at the genus-or higher level in degraded DNA samples.</li>
<li>The OTU abundance as indicated by the size in the OTU query sequence identifier provides information about the number of Illumina reads supporting the OTU. Higher relative read numbers provide better support for the presence of an OTU compared to OTUs with lower read numbers. The OTU abundance should, however, not be interpreted in a quantitative manner (i.e. as a measure of species abundance).</li>
<li>The quality of the identification depends on the length of the DNA barcode sequence used for identification. Smaller fragments have shown to lack the discriminatory power to distinguish between species in a genus or higher taxon. For this reason, the pipeline automatically discards identifications obtained from OTUs shorter than 200 nt, except for the ITS2, <em>rbcL</em>, and <em>trnL </em>(P6 loop) mini-barcode markers for which OTUs need to be longer than 150 nt, 140 nt and 10 nt, respectively.</li>
<li>Species in diverse groups such as Cycadaceae, Orchidaceae, Cactaceae, Euphorbia sp., and primates can have very similar DNA barcode sequences. For this reason, it is often an entire genus or family that is listed by CITES, rather than individual plant or animal species. The presence of CITES-listed species can be confirmed when three or more top BLAST hits (see the above guidelines) are reported with taxa listed by CITES at the species or higher level (genus/family/order).</li>
<li>CITES appendices contain multiple exceptions for certain taxa, e.g. based on their geographic location, domestication status or the enforcement of trade quota. The pipeline is unable to take these exceptions into account as they are not made available in a structured format. Therefore, the user is advised to revert to the speciesplus.net website for additional information on these details.</li>
</ul>
<p style="text-align: justify;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/npgfw26.png" width="744" height="691" data-src="https://s3.amazonaws.com/pr-journal/npgfw26.png" data-ofn="Screen Shot 2017-09-16 at 4.03.10 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>References </strong></p>
<ul>
<li style="text-align: justify;">Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X (2010) Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PloS one 5 (1):e8613</li>
<li style="text-align: justify;">Fazekas AJ, Kuzmina ML, Newmaster SG, Hollingsworth PM (2012) DNA barcoding methods for land plants. Methods in molecular biology 858:223-252</li>
<li style="text-align: justify;">Geller J, Meyer C, Parker M, Hawk H (2013) Redesign of PCR primers for mitochondrial cytochrome <em>c </em>oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys. Molecular ecology resources 13 (5):851-861</li>
<li style="text-align: justify;">Hebert PD, Penton EH, Burns JM, Janzen DH, Hallwachs W (2004) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly <em>Astraptes fulgerator</em>. Proceedings of the National Academy of Sciences 101 (41):14812-14817.</li>
<li style="text-align: justify;">Ivanova NV, Zemlak TS, Hanner RH, Hebert PD (2007) Universal primer cocktails for fish DNA barcoding. Molecular Ecology Notes 7 (4):544-548</li>
<li style="text-align: justify;">Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson AC (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences 86 (16):6196-6200</li>
<li style="text-align: justify;">Kress WJ, Erickson DL (2007) A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region. PLoS one 2 (6):e508</li>
<li style="text-align: justify;">Leray M, Yang JY, Meyer CP, Mills SC, Agudelo N, Ranwez V, Boehm JT, Machida RJ (2013) A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents. Front Zool 10 (1):34</li>
<li style="text-align: justify;">Levin RA, Wagner WL, Hoch PC, Nepokroeff M, Pires JC, Zimmer EA, Sytsma KJ (2003) Family-level relationships of Onagraceae based on chloroplast <em>rbcL </em>and <em>ndhF </em>data. American Journal of Botany 90 (1):107-115</li>
<li style="text-align: justify;">Little DP (2014) A DNA mini-barcode for land plants. Molecular Ecology Resources 14 (3):437-446</li>
<li style="text-align: justify;">Murray MG, Thomson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research 8(19):4321-4326</li>
<li style="text-align: justify;">Palumbi S. R. (1996) Nucleic acids II: The polymerase chain reaction. In Molecular Systematics, 2nd ed., Hillis, D.M., C. Moritz, B.K. Mable (eds.) Pp. 205-247. Sinauer Associates Inc., Sunderland, MA.</li>
<li style="text-align: justify;">Sarri C, Stamatis C, Sarafidou T, Galara I, Godosopoulos V, Kolovos M, Liakou C, Tastsoglou S, Mamuris Z (2014) A new set of 16S rRNA universal primers for identification of animal species. Food Control 43:35-41</li>
<li style="text-align: justify;">Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant molecular biology 17 (5):1105-1109</li>
<li style="text-align: justify;">Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PD (2005) DNA barcoding Australia's fish species. Philosophical Transactions of the Royal Society B: Biological Sciences 360 (1462):1847-1857</li>
</ul>

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