COLLEGE OF POST GRADUATE STUDIES (CPGS)

SESSION 2016-17
Credit seminar
On

DNA Barcoding and Its Application in Agriculture

DNA

Submitted to…
Dr. Vijay K. Chowdhury (Chairman of SCI & Professor of Biotechnology)

Submitted by…
Dharmendra Singh Lagoriya
CAU/CPGS/MBB/M15/02

College of Post Graduate Studies (CPGS)

Umiam, Barapani, Meghalaya-793103
1. Introduction

DNA barcoding is a technique, which provides quick identification of species

without involving the morphological feature. It uses a relatively small-standardized DNA
fragment as a tag, to define or discover a species. DNA barcoding uses minor differences
of nucleotides in the particular gene loci of different organisms as a key for the
discrimination. The gene is sequenced to know the base-pair differences and then
deposited in the barcode database, which is termed as DNA barcodes. These genetic
codes could be accessed through a digital library and used to identify the unknown
species by any scientist around the World. Ideal DNA barcode should be normally a
uniform short sequence of DNA (400-800 bp), able to be simply generated and used to
characterize all the living organisms (Savolainen et al., 2005). Paul’s group was the first
to design and use the short DNA sequences for biological identification at the University
of Guelph, Canada. The idea of barcoding was first emerged to describe the
microorganisms, in which the morphological keys were lacking. Now it is being applied
successfully to animals. A massive on-line digital library of barcodes will be a standard,
to which the DNA barcode sequence of an unknown sample can be matched for the
identification. The key process in DNA barcoding is identifying novel candidate gene,
which can be used universally. It should allow the users efficiently to distinguish the
species and accelerate the species discovery. DNA barcoding uses the information of
one or a few regions in the genome to recognize all the species in a genus (Lahaye et
al., 2008).
DNA barcoding will open up new opportunities in DNA based investigations
ranging from community phylogenetic (Webb et al., 2002) to ecological genomics (van
Straalen and Roelofs, 2006. Despite the lack of a universal plant barcode, taxonomists,
ecologists, evolutionary biologists and conservationists are all already envisioning the
purpose of a genetic identifier to a wide set of research and practical applications. The
suitability of a locus for large-scale DNA barcoding can be easily studied by comparing
loci across the similar set of taxa under a selected set of PCR conditions. Thus, the
statistics was taken into the account between the ability to amplify a locus and the rate of
divergence of that locus across a phylogenetic range of taxa. Additionally, the sequence
alignment methodologies are available, which can be evaluated for the use of DNA
barcodes with the following regards,
  The purpose of assurance limits to species assignment
 The use of a part of sequences in database searches
 The strength of search algorithms of sequence length variation due to
insertion/deletion events and the informative nature of these mutations.

DNA barcoding mirrors the distribution of intra and intra-specific variation that is
separated by a distance called DNA barcoding gap (Hebert et al., 2003, Meyer and
Paulay, 2005). The Consortium of Barcode of Life coordinates DNA barcoding
development and implementation universally. DNA barcoding is very essential for the
molecular identification of already described species (Hebert et al., 2003) and the
discovery of new species (Valentini et al., 2006).

1.1 The DNA barcoding is the combination of the following 3 aspects:

(i) Molecularization (i.e. the use of the variability in molecular markers as a discriminator)
(ii) Computerization (i.e. the transposition of the data using informatics support)
(iii) Standardization (i.e. the extending this approach to vast group of organisms)
The ultimate aim of DNA barcoding is to discriminate the species using an automated
system, so that unexplored living organisms can be named as quickly as possible before
it gets extinct. DNA barcode proved to be a promising tool to identify the species across
all forms of life including animals, plants and microbes in a rapid and reliable manner.
The many of the published work used a simple distance matrix analysis, a Neighbour
Joining (NJ) algorithm with Kimura-2-parameters (K2P). The identification and
characterization of molecular entities are the main goal in DNA barcoding studies.

1.2 An ideal DNA barcode should possess the following features:

High inter and low intra-specific sequence divergence.

Undergo universal amplification with standard primers.
Technically simple to analyse.
Short enough to sequence in one reaction.
Easily alienable (few insertions/deletions).
Readily recoverable from the museum or herbarium samples and other degraded
samples.

2. The birth of DNA barcoding

Molecular systematic studies have been started in the 1970 for the first time using
ribosomal RNA for the classification of bacteria (Fox et al., 1980). The use of DNA
sequences for species identification has a long history (e.g. Nanney, 1982: Bartlett, 2004:
Will et al., 2005: Cameron et al., 2006; Meier, 2008). But it received significant attention
only after it was formally proposed as “DNA Barcoding” in 2003 (Shiyang et al., 2012)
University of Guelph (2003), the DNA barcoding initiative has gathered momentum,
gained extensive international participation in the form of the International Barcode of Life
Project (IBOL), and captured attention of the scientific community, government agencies,
and the general public. Dr. Paul D.N. Hebert – Father of DNA barcoding

3. Barcoding Systems
The species discovery till now depends on the morphological information provided
by the taxonomists that was not otherwise possible by non-taxonomists. The „DNA
Barcode of Life‟ project aimed to develop a standardized, rapid and inexpensive species
identification method, which can be accessible to non-specialists around the World. The
idea of a reliable molecular identification system emerged gradually during the 1990‟s
with the development of PCR-based methods for species identification. Molecular
identification has largely been practical to bacterial species, microbial biodiversity surveys
(Woese, 1996; Zhou et al., 1997) and to diagnose the
Pathogenic strains (Maiden et al., 1998; Sugita et al., 1998; Wirth et al., 1999). PCR
based techniques are commonly applied in the field of taxonomy, food and forensics
(Teletchea et al., 2005) and also for identification of eukaryotic pathogens and vectors
(Walton et al., 1999). Several universal molecular marker systems identify the lower taxa
(e.g. nematodes, Floyd et al., 2002) that were not successful for higher taxa. The
universal barcode concept for eukaryotes based on a standard molecular approach was
initiated in 2003 by the International initiative “Consortium for the Barcode of Life” (CBOL-
http://www.barcodeoflife.org). Now, it has more than 150 members from 45 countries
including museum, zoo, herbaria, botanical garden, University departments as well as
private companies and governmental organizations. The DNA barcode project aims to
develop a simple diagnostic tool based on strong taxonomic data that is collated in the
DNA barcode reference library (Schindel and Miller, 2005). DNA Barcode of Life Data
System (BOLD, http://www.boldsystems.org) was initiated in 2004 and formally
established in 2007 (Ratnasingham and Hebert, 2007). This data system enables the
achievement, storage, analysis and publication of DNA barcode records. According to
Rubinoff and Holland (2005), it was considered as a tremendous tool to speed up the
species discovery and also to describe the new species, in addition it also re-opens the
debate on species concepts (Fitzhugh, 2006; Rubinoff et al., 2006b; Balakrishnan, 2007;
Miller, 2007; Vogler and Monaghan, 2007). The well-known sequence libraries like NCBI
and BOLD are an interactive interface, in which the sequences can be deposited, revised
and taxonomically reassigned. The aim is compiling of sequences from one or few
common loci at massive geographic scale across many genera (Hajibabaei et al., 2007).
Such information on the distribution of species, genetic diversity will enhance the speed
and success of population studies.

4. Flow chart for DNA barcoding

5. The Utility of DNA Barcoding

DNA barcode have applications in various fields like, ecology, biomedicine,
epidemiology, evolutionary biology, biogeography, conservation biology and in bio-
industry. The low cost and rapidity makes the process easier for enabling automated
species identification especially in massive sampling campaigns (Rusch et al., 2007).
DNA barcoding has advantages over traditional method of description, as it took decades
to illustrate around 10-15 million species by means of morphological clues (Hammond et
al., 1992). DNA barcoding will help in large surveys, aiming at the unknown species
detection and identification of pathogenic species with medical, ecological and the
agronomical implication (Armstrong and Ball, 2005; Ball and Armstrong, 2006). In
addition, it is important to distinguish, detect and trace the distribution of patented
organism in agrobiotechnology, to certify the source organism like truffles (Rastogi et al.,
2007; Ferri et al., 2012). Molecular based identification is important in 3 situations-
1. In determining the taxonomic uniqueness (e.g. goods, food and stomach extracts) and
will help in preventing illegal trade and export of vulnerable species (e.g. fishes and trees).
2. In the identification of juvenile specimens (e.g. fish larvae).
3. Morphological characters are unable to differentiate the species (e.g. red algal
species), When the species have polymorphic life cycles and displaying prominent
phenotypic plasticity (e.g. Lamilariales).

6. Criteria of DNA barcode sequence

Extranuclear genomes
Mitochondrial
Chloroplast
• Gene segment should be conserved at the species level
• Present in many copies within each cell
• Should have high variation between species

7. Barcoding region
Prokaryotes : rRNA Gene used for species identification
Animal, Birds and Fishes : Small region of mitochondrial COI gene
Plants : rbcl, matK gene sequence of chloroplast
Fungi : ITS region
8. Features of ideal DNA barcoding:
1. High inter and low intra-specific sequence divergence.
2. Easy to amplify
3. Undergo universal amplification with standard primers.
4. Technically simple to analyse.
5. Short enough to sequence in one reaction.
6. Easily alienable (few insertions/deletions).
7. Readily recoverable from the museum or herbarium samples and other degraded
samples.

8. SIGNIFICANT OF DNA BARCODING

10. Plant DNA barcoding:
DNA barcoding in plants have been a more challenging task than those in animals.
Unlike animals, plant mitochondrial genes perform unsatisfactory as a candidate gene for
DNA barcoding. The generally low rate of nucleotide substitution in plant mitochondrial
genomes precludes the use of COI as a universal plant barcode. However, potential
candidates have been reported in the chloroplast genome. The most satisfactory results
have come from the gene maturase K (matK) and matK in association with other genes.
This has been used to resolve the flora of biodiversity hot spots. The matK barcode has
been claimed to have discriminated 90 percent of plant species. MatK is nested in the
group II intron of the chloroplast gene for transfer RNA lysine (trnK), and includes a
domain for reverse transcriptase. Group II intron is a class of intron found in rRNA, tRNA
and mRNA of organelles in fungi, plants, protists and some mRNA in bacteria. Group II
introns are self-splicing in vitro but employ maturase proteins in vivo. Multi-locus markers
such as ITS along with matK, rbcL, trnH, etc. have been assumed to be more successful
in species identification. However, studies to date demonstrated that these are also
inadequate for universal plant identification. Despite significant recent effort, the
development of single-locus barcodes has stalled, placing plant DNA barcoding at a
crossroads. Fortunately, developments in DNA sequencing allowing cost-efficient plastid
sequencing are driving plant identification into a post-barcode era. The use of two or more
chloroplast barcodes has been advocated for the best discrimination in estimating
biodiversity, and impressive progress has been made in using chloroplast DNA barcodes
for identifying plant species.
• Consortium for the Barcode of Life (CBOL)-Plant working Group has recommended
a combination of chloroplast genes rbcL and matK as the standard two-locus barcode
for plants (Ratnasingham and Hebert, 2007). Various combination of plant specific
markers can be used for plant barcoding (rpoC1+rpoB+matK or rpoC1+matK+trnH-
psbA; rbcL+trnH-psbA; atpF-H+psbKI+matK). The current literature seems to be
coming to the conclusion that two or more markers are needed to identify plants.

11. The Potential Barcode Candidates

The plant DNA barcoding studies were initially restricted to the chloroplast genome
to understand the variation of its gene sequences of coding (matK, rbcL and rpoC1) and
non-coding (ITS and psbA-trnH), which has been summarized by Chen et al., (2010) as
below. The studies proved that the chloroplast genome showed high degree of variation
and discrimination ability (Lahaye et al., 2008). The slow rate of their substitution limits
the ability to identify plants among the species (Baldwin et al., 1995). The evidence
suggests that specieslineage reconstructions using chloroplast DNA (cpDNA) regions
show significant errors, when hybridization and introgression events or linear shorting
events occur (Doyle, 1992).
12. Limitation of DNA Barcodes
DNA-based species identification depends on distinguishing intra-specific from
inter-specific genetic variation. The ranges of these types of variation are unknown and
may differ between taxa. It seems difficult to resolve recently diverged species or new
species that have arisen through hybridization. There is no universal gene for DNA
barcoding, no single gene that is conserved in all domains of life and exhibits enough
sequence divergence for species discrimination. The validity of DNA barcoding therefore
depends on establishing reference sequences from taxonomically confirmed specimens.
This is likely to be a complex process that will involve cooperation among a diverse group
of scientists and institutions. Barcode sequences are, in general, short (approx. 500–
1000 bp) and this fundamentally limits their utility in resolving deep branches in
phylogenies. Some controversy exists over the value of DNA barcoding, largely because
of the perception that this new identification method would diminish rather than enhance
traditional morphology-based taxonomy, and species determinations based solely on the
genetic divergence could result in incorrect species recognition. However, we must keep
open the possibility that the barcode sequences per se and their ever increasing
taxonomic coverage could become an unprecedented resource for taxonomy, systematic
biology and diagnosis, and may be equally useful.
13. Application:
Basic research in taxonomy
Identifying agriculture pest
Controlling Agricultural Pest
Identifying disease vectors
Sustaining natural resources
Protecting endangered species
Monitoring water quality
Identification of mislabelling food and commercial frauds
Biosecurity and trade in the controlled species
Food control
Phytomedicinals analysis

14. Case study -1

Ginsengs (Panax, Araliaceae) are among the plants best known for their medicinal
properties.Many ginseng species are endangered due to over-exploitation of natural
resources a situation difficult to remedy while there are no reliable, practical methods for
species identification
14.1. Material and methods:
In this study, all eight species of Panax were sampled, with special emphasis on
the P. bipinnatifidus species group. The cultivated species (P. ginseng and P.
notoginseng), the rare and endangered Asian species (P. stipuleanatus and P.
pseudoginseng ) and the North American species ( P. quinquefolius and P. trifolius Linn.)
were represented by few samples, because they are genetically uniform and have been
well-studied already. Panax trifolius was treated as a functional out group when rooting
was necessary, because this species has been shown to be the basal most species of
the genus. Panax bipinnatifidus species group is sparsely distributed over a large area
spreading from the Himalayas to the western coast of the Pacific Ocean, and is well
represented in 33 populations found in those areas. The eleven candidate DNA barcodes
used in Panax- atpf-atph, psba-trnh, psbk-psbi, psbm-trnd, matk, rps16, rpob, rpoc1,and
rbcl, ITS, matK

14.1.2 Selection of candidate barcoding loci by pilot screening:

Nine candidate barcoding loci of chloroplast genome (atpf-atph, psba-trnh, psbk-
psbi, psbm-trnd, matk, rps16, rpob, rpoc1, and rbcl), Internal transcribed spacer (ITS) of
ribosomal nuclear gene and Mitochondrial locus matk. These were evaluated in a pilot
study with 24 populations representing all the eight species.

14.1.3 Result and discussion:

Most of the potential barcode regions can be amplified and sequenced easily using
the universal primers. A high rate (100%) of amplification success was achieved with
psbA- trnH, psbK-psbI, psbM-trnD, matK, rps16, rbcL and ITS. The regions of atpF-atpH,
rpoB and rpoC1 were relatively difficult to amplify for the ginsengs. Furthermore,
amplification of nadl was particularly difficult; it had a PCR success rate of 72.6%. This
rate was enhanced by adding the specific internal primers and amplifying two fragments
instead of one. Sequencing the region of atpF-atpH failed due to several poly-N stretches.
The relatively low success rates of sequencing rpoB and rpoC1 were caused by
unspecific or weak amplification in several samples.

Table.1 Comparisons of the success rate of PCR amplification and DNA fragment sequencing of eleven
loci in Panax

ITS exhibited the highest sequence variability with the highest value of nucleotide
diversity (Pi). Among the eight chloroplast loci, psbA-trnH was the most variable locus,
followed by psbK-psbI, rps16, psbM-trnD,and matK(in descending order of Pi). rbcL, rpoB
and rpoC1 were the least variable loci, having four to eight variable sites. The
mitochondrial nad1 had 23 variable sites but its Pi value was the smallest.

Table.2 Variability of ten loci screened in Panax

 rbcL and matK recommended by COBL

 rbcL rpoC1 and rpoB show lowest variability
 ITS exhibits highest variability

14.2 CASE STUDY-2

14.2.1 Material and methods:

a) Sequences
Approximately one-quarter of the COI sequences (172 out of 658).The primer pair
was subsequently used to amplify a 658 bp fragment of the COI gene (Folmer et al.
1994).
LCO1490 (59-GGTCAACAAATCATAAAGATATTGG-39)
HCO2198 (59-TAAACTTCAGGGTGACCAAAAAATCA-39)
 The DNA amplification done using PCR by using standard PCR protocol and the final
product gel purified using the Qiaex II kit (Qiagen) and Sequenced in one direction on
an ABI 377 automated sequencer (Applied Biosystems) using the Big Dye v. 3
sequencing kit.
They created three COI profiles: one for the seven dominant phyla of animals, another
for eight of the largest orders of insects and the last for 200 closely allied species of
lepidopterans. These profiles were designed to provide an overview of COI diversity
within each taxonomic assemblage and were subsequently used as the basis for
identifications to the phylum, ordinal or species level by determining the sequence
congruence between each ‘unknown’ taxon and the species included in a particular
profile.

Fig.1. COI profiles

14.2.2 Result and discussion:

14.2.2.1 Taxon profile:

Testing taxonomic assignments; Fifty-three out of the 55 ‘test’ species (96.4%)
were assigned to the correct phylum in the analyses at this level (table 2). The exceptions
were a polychaete annelid that grouped most closely with a mollusc and a bivalve that
grouped with one of the arthropod outliers. However, in both cases, there was substantial
sequence divergence (13% and 25%, respectively) between the test taxon and the
lineage in the profile that was most similar to it. Identification success at the ordinal level
was 100% as all 50 insect species were assigned to the correct order.

TAXON TARGET GROUP N % SUCCESS

KINGDOM ANIMALIA 7 PHYLA 55 96.4
CLASS HEXAPODA 8 ORDER 50 100
ORDER LEPIDOPTERA 200 150 100

Table. 1 Percentage success in classifying species to membership of a particular

taxonomic group based upon sequence variation at COI. (n indicates the number of taxa
that were classified using each taxon ‘profile’.)

14.3 CASE STUDY-3

14.3.1 Objective:
To document agriculturally dominant insect pest, their natural enemies, pollinators
and veterinary insects

14.3.2 Material and method:

Insects were collected from various ecosystems across India. Collected sample of
insect used for extraction of DNA by using Qiagen Dneasy Kit.
PCR amplification of a 658 bp region near the 5′ term nus of the COX 1 gene following
standard protocols.
Primers used:
F :( LCO 1490 5′-GGTCAACAAATCATAAAGATATTGG-3′)
R :( HCO 2198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′)
The amplified product was analysed on a 1.5 % agarose gel electrophoresis as described
by Sambrook and Russell (2001), and the amplified products were sent to commercial
sequencing company, M/s Eurofins Pvt Ltd. India. Each species was bidirectionally
sequenced and checked for quality by Bio edit 7.0.2 software and homology, insertions
and deletions, stop codons, and framshifts by using NCBI BLAST. All sequences were
uploaded to GenBank and the BOLD (http://www.boldsystems.org). The specimens were
collected and morphologically identified, were used for COX 1 barcoding at the National
Bureau of Agriculturally Important Insects (NBAIR) Bangalore, India.

14.3.3 Result and discussion:

The CO1 region in almost all the samples was in the range of 500–658 bp. Total
of 42 insect species were studied; there were a total of 540 positions in the final datasets
(software generated) according to the full K2P/ NJ tree. All 42 species could be
differentiated by CO1 barcoding. Most of the amplified sequences were up to 658 bp in
length. A phylogeny tree constructed using the n–j method revealed two clusters, the
first cluster consisting of orders lepidoptera, diptera, hemiptera, and coleoptera, whereas
another clade showing relationship between hymenopteran insects.
Fig. 3 Phylogenetic tree for 42 insect species (5 orders) depicting genetic relationships
derived from CO1 sequences. Note: Bootstrap consensus tree generated by Bootstrap
Test Phylogeny using neighbour-joining (N–J) method of MEGA 5 Software. All the 43
species are from 5 orders, which are distributed into two main clades that are 61 % similar

15. Conclusion:
DNA barcoding of non-well-divergent “Species” is possible if the barcoding
technique is made sufficiently sensitive by using an appropriate approach. DNA
barcoding is a useful tool for taxonomic classification and identification of species by
sequencing a very short standardized DNA sequence in a well-defined gene which can
identify organisms and differentiate between very closely related species in order to
conserve species diversity.
16. Reference:
Armstron, K.F. & Ball, S.L. (2005). DNA barcodes for biosecurity: invasive species identification.
Philosophical Transactions of the Royal Society. 360: 1813–1823.

Balakrishnan, R. (2007). Species concepts, species boundaries and species identification: A view
from the tropics. Systems Biology. 54: 689–693.

Baldwin, B.D., Sanderson, M.J., Porter, J.M., Wojcicchowski, M.F., Campbell, C.S., & Donoghue,
M.J. (1995). The ITS region of Nuclear Ribosomal DNA: A Valuable Source of Evidence
on Angiosperm Phylogeny. Annals of Missouri Botanical Garden. 82: 247-277.

Bartlett, S.E., Davidson, W.S., 1991. Identification of Thunnus tuna species by the polymerase
chain reaction and direct sequence analysis of their mitochondrial cytochrome b genes.
Can. J. Fish. Aquat. Sci. 48: 309–317.

Cameron, S., Rubinoff, D., Will, K. (2006). Who will actually use DNA barcoding and what will it
cost? Syst. Biol., 55: 844–847.

CBOL Plant Working Group (2009). A DNA barcode for land plants. Proceedings of the National
Academy of Sciences. 106: 12794–12797.

Chen, S.L., Yao, H., Han, J.P., Liu, C., Song, J.Y., Shi, L.C., Zhu, Y.J, Ma, X.Y., Gao, T., Pang,
X.H., Luo, K., Li, Y., Li, X.W., Jia, X.C., Lin, Y.L. & Leon, C. (2010). Validation of the ITS2
region as a novel DNA barcode for identifying medicinal plant species. Plos. One, 5:
e8613.

Ferri, G., Corradini, B. & Alù, M. (2012). Capillary electrophoresis of multigene barcoding
chloroplast markers for species identification of botanical trace evidence. Methods in
Molecular Biology. 830: 253-263.

Floyd, R., Abebe, E., Papert, A. & Blaxter, M. (2002). Molecular barcodes for soil nematode
identification. Molecular Ecology. 11: 839–850.

Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of
mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol.
Mar. Biol. Biotechnol., 3: 294–299.

Fox, G. E., Stackebrandt, E., Hespell, R. B., Gibson, J., Maniloff, J., Dyer, T. A., Wolfe, R. S.,
Balch, W. E., Tanner, R. S., Magrum, L. J., Zablen, L. B., Blakemore, R., Gupta, R.,
Bonen, L., Lewis, B. J., Stahl, D. A., Luehrsen, K. R., Chen, K. N. & Woese, C. R.
(1980). The phylogeny of prokaryotes. Science, 209: 457-463.

Hajibabaei, M., Singer, G.A.C. & Hebert, P.D.N. (2007). DNA barcoding: How it complements
taxonomy, molecular phylogenetics and population genetics. Trends Genetics. 23: 167–
72.
Hammond, P. (1992). In Global biodiversity: status of the earth’s living resources. London:
Species inventory. (ed. B. Groombridge). 17–39.

Hebert, P.D.N., Ratnasingham, S. & DeWaard, J.R. (2003). Barcoding animal life: cytochrome c
oxidase subunit 1 divergences among closely related species. Proceedings of the Royal
Society Biological Science. 270: 96-99.

Kwong, s., srivathsan, A., and Meier, R. (2012). An update on DNA barcoding: low species
coverage and numerous unidentified sequences. Cladistics, 28: 639–644.

Lahaye, R., vander, B.M., Bogarin, D., Warner, J. & Pupulin, F. (2008). DNA barcoding the floras
of biodiversity hotspots. Proceedings of the National Academy of Sciences. 105: 2923–
2928.

Meyer, C.P. & Paulay, G. (2005). DNA barcoding: error rates based on comprehensive sampling.
Plos. Biology, 3: 2229–2238.

Miller, S.E. (2007). DNA barcoding and the renaissance of taxonomy. Proceedings of the National
Academy of Sciences. 104: 4775–4776.

Nanney, D.L., 1982. Genes and phenes in Tetrahymena. Bioscience 32: 783–788.

Rastogi, G., Dharne, M.S., Walujkar, S., Kumar, A., Patole, M.S. & Shouche, Y.S. (2007). Species
identification and authentication of tissues of animal origin using mitochondrial and
nuclear markers. Meat Science. 76: 666–674.

Ratnasingham, S. & Hebert, P.D.N. (2007). BOLD: The Barcode of Life Datasystem
(www.barcodinglife.org). Molecular Ecology Notes. 7: 355–64.

Rubinoff, D. (2006b). DNA barcoding evolves into the familiar. Conservation Biology. 20:1548–
1549.

Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S, Wu D, Eisen JA,
Hoffman JM, Remington K, Beeson K, Tran B, Smith H, Baden-Tillson H, Stewart C,
Thorpe J, Freeman J, Andrews-Pfannkoch C, Venter JE, Li K, Kravitz S, Heidelberg JF,
Utterback T, Rogers, YH, Falcon LI, Souza V, Bonilla-Rosso G, Eguiarte LE, Karl DM,
Sathyendranath S, Platt T, Bermingham E, Gallardo V, Tamayo-Castillo G, Ferrari MR,
Strausberg RL, Nealson K, Friedman R, Frazier M & Venter JC (2007). The Sorcerer II
global.

Savolainen, V., Cowan, R.S., Vogler, A.P., Roderick, G.K. & Lane, R. (2005). Towards writing the
encyclopedia of life: an introduction to DNA barcoding. Philosophical Transactions of the
Royal Society. 360: 1805–1811.
Schindel, D.E., Miller, S.E. DNA barcoding a useful tool for taxonomists. Nature. 2005; 435: 17.

Sugita, T., Nishikawa, A. & Shinoda, T. (1998). Identification of Trichosporon asahii by PCR
based on sequences of the internal transcribed spacer regions. Journal of Clinical
Microbiology. 2742–2744.

Teletchea, T., Maudet, C. & Hanni, C. (2005). Food and forensic molecular identification: update
and challenges. Trends Biotechnology. 23: 359–366.

Valentini, A., Miquel, C., Nawaz, M.A., Bellemain, E.V.A., Coissac E, et al. (2009). New
perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the
trnL approach. Molecular Ecology Resources. 9: 51–60.

Van, S.N.M. & Roelofs, D. (2006). An Introduction to Ecological Genomics. Oxford University
Press, Oxford.

Vogler, A.P. & Monaghan, M.T. (2007). Recent advances in DNA taxonomy. Journal of Zoological
Systems Evolutionary Research. 45: 1–10.

Webb, C.O., Ackerly, D.D., McPeek, M.A. & Donoghue, M.J. (2002). Phylogenies and community
ecology. Annual Review of Ecology and Systematics. 33: 475–505.

Will, K.W., Mishler, B.D., Wheeler, Q.D., 2005. The perils of DNA barcoding and the need for
integrative taxonomy. Syst. Biol. 54: 844–851.

Wirth, T., Guellec, l.R. & Veuille, M. (1999). Directional substitution and evolution of nucleotide
content in the cytochrome oxidase II gene in earwigs (Dermapteran Insects). Molecular
Biology and Evolution. 16: 1645–1653.

Zhou, J., Davey, M.E., Figueras, J.B., Rivkina, E., Gilichinsky, D., & Tiedje, J.M. (1997).
Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA.
Microbiology, 143: 3913–3919.

Zhou, J., Davey, M.E., Figueras, J.B., Rivkina, E., Gilichinsky, D. & Tiedje, J.M. (1997).
Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA.
Microbiology. 143: 3913–3919.