633 50 Egypt. J. Chem. Vol. 67, No. 1 pp.

633 - 646 (2024)

Egyptian Journal of Chemistry

http://ejchem.journals.ekb.eg/

The Power of DNA Barcoding for Plant Identification

Samira A. Osman*
Genetics and Cytology Department, National Research Centre, Giza, Egypt

Abstract

Plant taxonomy is considered an important branch of science; it needs highly professional taxonomists to classify plants
depending on their morphological characteristics but this method has numerous disadvantages, it is time-consuming and highly
dependent on plant growth stages and reproductive organs (such as tree species with a short bloom period or rarely blooming).
Therefore, several molecular techniques (DNA fingerprinting, DNA barcoding, Next generation sequencing) have been used to
overcome these obstacles. DNA barcoding is considered the bedrock to classify and discover different plant specimens by
combining the strengths of molecular genetics, sequencing technologies and bioinformatics by using highly variable short
regions of DNA. Four gene regions (rbcL, matK, trnH-psbA and ITS) have been utilized as the universal DNA barcodes for
plants. The combination of two or more barcode regions improves the resolution level and provides perfect huge data for
species identification. DNA barcoding faces many obstacles for discrimination of some plant specimens for example the
presence of conserved ancestral alleles, high intraspecific variability and some other factors, As an outcome, it was proposed
to use complete chloroplast sequences using next-generation sequencing (NGS) technique. NGS technique is highly expensive
thus it was needful to use other new and cheap techniques such as genome skimming and twin track processes.

Keywords: Plant identification, Nuclear barcodes, Chloroplast barcodes, DNA mini-barcode, next-generation sequencing
(NGS).

species are considered the primary unit of

1. Introduction
biodiversity and it is easy to make comparisons
Plant taxonomy is considered an important branch of among them quantitatively [2].
science; the classification of plants is the first step in Plant molecular characters are derived from three
studying the biodiversity, breeding, conservation and different sources, genetic fingerprints, structural
development of populations and distinguishing genomic characteristics (e.g. gene order, gain or loss
different species. There are several ways to the of genes, or non-coding regions), and finally, DNA
taxonomic identification of plant specimens starting sequences of specific coding or non-coding regions
from morphological, PCR-based to sequence-based from one of the three plant genomes plastid,
techniques. The traditional way for species discovery mitochondrial or nucleus [3].
and identification is depending on the morphological Different molecular markers (RAPD, ISSR, SSR,
characters' investigation. But this approach is not SCoT, IRAP…. etc) are used for performing DNA
accurate for the classification of some plant species, fingerprints by amplification of certain sequences of
because it is time-consuming, highly dependent on genomic DNA using polymerase chain reaction
plant growth stages and reproductive organs (e.g. tree (PCR). DNA fingerprints give perfect results for
species with rarely or short bloom period), so this discrimination of different samples within a defined
method needs highly professional taxonomists. The group or samples having specific genes and studying
molecular techniques could overcome all these their phylogenetic relationships or biodiversity but
obstacles, where these techniques provide this method is imperfect for the identification of
independent sets of data that could be used to unknown or cryptic taxa. So, a sequence-based
determine the phylogeny of plants and determine technique such as DNA barcoding was applied to
plant systematic and identification [1]. overcome this obstacle [4].
Biological diversity (biodiversity) involves genetic DNA barcoding is considered as the bedrock for the
variability among species and ecosystems. Most discrimination of species relationships and phylogeny
biodiversity studies focused on species because at a molecular level by investigating sequence
_________________________________________________________________________________________________
*Corresponding author e-mail: s_nrc82@yahoo.com
Receive Date: 07 May 2022, Revise Date: 17 June 2023, Accept Date: 10 July 2023
DOI: 10.21608/EJCHEM.2023.137216.6049
©2024 National Information and Documentation Center (NIDOC)
634 S. A. Osman
_____________________________________________________________________________________________________________

variation in standardized gene loci which represents a rbcL gene encodes the large subunit of ribulose-
small region from the complete genome [5]. In 2003, bisphosphate carboxylase (EC 4.1.1.39) [12].
this technique was proposed as a tool for animal However, the non-coding chloroplast barcodes
species discrimination, afterward it was used to should be treated as suboptimal barcodes because of
identify other multicellular organisms (plants, fungi, the prevalence of microsatellites [13]. ​
protists) [6]. In this technique, the nucleotide sequence was
analyzed by using different bioinformatics programs
2. Standard DNA Barcodes
where the nucleotide sequences give huge data and
DNA barcoding is a molecular technique that is each nucleotide site considers as highly specific
performed by combining the strengths of molecular genetic information. The resulting DNA barcoding
genetics, sequencing technologies and bioinformatics. data has allowed greatly improving the ability to
This technique uses highly variable short regions of index biodiversity and has helped to increase the
DNA to classify and discover different plant species understanding of species distributions. However,
in addition to provide fast and accurate species great effort is still necessary to establish a DNA
identification [7]. Although, there are no universal barcode reference library for all species [1].
barcodes, the variable loci that are listed enable the
scientists to differentiate plant samples from various
taxonomic groups [8]. After a broad inventory of
gene loci in the mitochondrial, plastid and nuclear
genomes, four primary gene loci (rbcL, matK, trnH-
psbA and ITS) were intensively be utilized as the
standard DNA barcodes for plants applications [9].
In animals, the nuclear DNA represents about 98%
while mitochondrial DNA represents about 2% of
total genomic DNA. Mitochondrial DNA contains a
highly variable region COI (cytochrome oxidase
subunit I) that is utilized as the animal barcode. COI
is involved in the electron transport phase of
respiration. The evolution of plant mitochondria is
very slow compared to the animal mitochondria, so
the region of the cytochrome oxidase (CO1) gene was
used as a barcode marker for animals but not as a
plant barcode to discriminate different plant
specimens [10]. Figure (1): Schematic illustration of DNA barcode types.
In plants, DNA barcoding looks to be inherently
more complicated than in animals [11]. Plant DNA is
also, present in chloroplasts (cpDNA) besides nuclear
and mitochondrial DNA. The related plant species
which cannot be distinguished morphologically could
be differentiated at the molecular level with the help
of plant DNA barcodes loci.
Many loci and combinations thereof have been
proposed as appropriate barcodes for plants. For
example, the nuclear Internal Transcribed Spacer
(ITS) loci of nuclear ribosomal genes and chloroplast
DNA sequences were considered; the chloroplast
genome consists of many barcode regions which
classified as coding sequences (matK, rpoB, accD,
ndhJ, ccsA ,and rbcL) and non-coding sequences
(trnH–psbA, atpF–atpH and psbK–psbI spacers) as
shown in figures (1 and 2) [14].
The coding chloroplast barcodes matK, ndhJ, accD
and rpoB encode proteins of maturase K, a Figure (2): DNA barcode genes from Mitochondrial,
nicotinamide dehydrogenase, β' subunit of acetyl- chloroplast and nuclear genomes of plants. Green color
CoA carboxylase and β subunit of RNA polymerase represents a powerful barcodes, red color represents poor
respectively, while ccsA gene encodes a protein that barcodes and yellow represent barcode regions yet to be
involved in the cytochrome c biosynthesis and the investigated [14].

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THE POWER OF DNA BARCODING FOR PLANT IDENTIFICATION
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2.1. Nuclear barcodes

2.1.1. Internal Transcribed Spacer (ITS) region
The nuclear genome contains an internal transcribed
spacer (ITS), it is present as tandem repeats that
encourage the amplification, detection and
sequencing of nuclear ribosomal DNA (nr DNA) also
this region gives the best result in PCR compared to
other barcodes [9].. ITS is considered a nonfunctional
RNA sequence with a length of about 700 bp which
includes 5.8S rRNA region with a length of about
163 or 164 bp. ITS exists between the structural
ribosomal RNA during the maturation of rRNA and
this spacer consider the product of maturation so any
mutation in this spacer leads to the inhibition of Figure (3): The universal structure of the rDNA regions in
mature small and large rRNA subunits. There are two plants [22].
types of this spacer (ITS1 and ITS2) which are (a) The location of rDNA region on the chromosome.
located between 25S and 18S rRNA coding regions. (b) Tandem arrays of the consecutive gene blocks (18S-5.8S-26S).
ITS1 is located between 5.8S and 18S rRNA with a IGS: Intergenic Spacer.
length of less than 300 bp while ITS2 located ETS: External Transcribed Spacer.
NTS: Non-transcribed region
between 5.8S and 25S rRNA with a length less than TIS: Transcription start site.
250 bp as shown in figure (3) [15]. 18S: The small subunit of rRNA.
ITS2 region is considered a universal barcode in 5.8S and 26S: The large subunit of rRNA.
animals and plants, it is utilized for the identification ITS: The internal transcribed spacer (ITS1 and ITS2).
of Pan-eukaryote and Eukaryote. Ceriaco et al., [16]
mentioned that the ITS2 barcode was utilized for 2.2. Chloroplast barcodes
distinguishing the morphologically similar species in
addition to the contaminants present in the North In the 1960s and early 1970s, it was confirmed that
American herbal products identified using combined plastids are polyploidy, whereas proplastid has about
ITS2 and rbcL barcodes. 20 genome copies while chloroplast has about 100
Indeed, some researchers elucidated that the nuclear copies [23]. The chloroplast genome is a circular
ITS locus supplied better resolution than chloroplast double-stranded DNA molecule [24].
barcode loci [17]. Some tropic evergreen shrubs and The chloroplast genome contains two inverted repeats
trees of Brazilian species from the family Sapotaceae, (IRs), a small single-copy region (SSC) and a large
which are characterized by short-lived flowers (rarely single-copy region (LSC) as shown in figures (4 and
bloomed), showed high resolution of ITS for 5). In plants, the length of IRs ranges in size from 5
discrimination of these species [18]. to 70 Kb, it contains highly variable regions [25].
Several applied research of medicinal plants appeared Plant DNA barcoding studies were limited to the
that the ITS2 locus is better than ITS1 and the chloroplast genome based on the variation in the
combination of ITS1 and ITS2 for species sequence of the non-coding region (psbA-trnH) and
identification [19]. The resolution of the ITS1 and coding region (matK, rbcL and rpoC1). It was
ITS2 loci and their combination with other DNA confirmed by previous studies that the chloroplast
barcodes were evaluated for discrimination of genome has the ability to identify and discriminate
Primula species and taxa of the family Lauraceae different plant specimens because this genome
[20]. In mosses and the group of legume plants from undergoes a lot of variations and substitutions [26].
the subtribe Cassiinae, The ITS1 region provided The phylogenetic analysis and molecular evolution of
better identification of different taxa than ITS2 and different Panax species were performed using
the combination of ITS1 and ITS2 [21]. Thus, the chloroplast inter-genic spacer (IGS) such as trnE-
usability of barcode loci may vary, even with an trnT, trnT-psbD, ndhF-rpl32 and rpl14-rpl16 as
equal rate and character of their evolution, relying on chloroplast barcodes. Some non-coding regions
the task of the study. (atpF-atpH, psbK-psbI and trnH-psbA) and coding
regions (rbcL and matK) were used as barcodes to
discriminate different Orchidaceae species from

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636 S. A. Osman
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Korea [27]. Kress et al., [28] used the chloroplast - Yellow color indicates small subunit ribosomal proteins.
inter-generic spacer psbA-trnH for identification of - Orange color indicates large subunit ribosomal proteins.
- Lemon color indicates hypothetical chloroplast open reading
different Dendrobium species. frames.
The intensive research was performed to discover the - Green color indicates protein-coding genes either involved in
variable loci that provide more resolution than the photosynthetic reactions.
universal DNA barcodes, when several of completely - Red color indicates other function.
- Blue color indicates ribosomal RNAs.
sequenced chloroplast genomes recorded in the - Black color indicates transfer RNAs.
GenBank increased. The ycf1 gene is the second - Gray color indicates introns.
largest gene in the genome of chloroplast with an
average of length about 6000 bp. This gene alone
demonstrates better resolution than the combination
of trnH–psbA+matK +rbcL for discrimination of 20
species from 67 families that comprised angiosperms,
gymnosperms and mosses [29].

2.2.1. The matk region

Maturase K (matk) gene presents in the chloroplast
genome with a size 1500 bp and it encodes to
maturase-like protein with 500 amino acids that assist
with RNA editing. Matk gene embedded in the intron
region of trnk while the two exon regions of trnK
were flanked by the maturase (matK) protein (act as
splicing factor) then it was cut down and keeping the
matk gene intact during the splicing process (Figure
6). Matk is a promising gene due to its high
substitution rate and it manifests different numbers
and sizes of indels (insertions and deletions) so it
could be used in molecular systematic and determine Figure (5): The positions of coding and non-coding loci on
the evolution of some plants to resolve some chloroplast genome [3].
IRA&IRB: Inverted Repeat(A&B)
taxonomic problems [10]. SSC: Small Single Copy regions
LSC: Large Single Copy regions

Johnson and Soltis [31] mentioned that the matK

gene is a rapidly evolving gene because its rate of
nucleotide substitution is higher than that of the large
subunit of Rubisco (rbcL) by three folds while its
amino acids substitution is higher than the
substitution of rbcL amino acid by six folds This
refers to the effectiveness of matK gene for resolving
evolutionary relationships among plants at all
taxonomic levels.
In the year 2010, the Linnaean Society of London
used the analysis of the matK gene on a large scale.
They use the available primer to obtain the match of
generated matK sequence to make the possible
modification and to simplify the barcode
complications in the family Zingiberaceae [32].
Lahaye et al. [33] used primers specific for matk
gene amplification of 1667 angiosperms. Also, more
than 90% of species in the Orchidaceae could be
discriminated by matK. Fazekas et al. [34] used
Figure (4): Chloroplast DNA (cpDNA) map of matK barcode for the identification of 92 species
Arabidopsis thaliana (Original picture author: prof. from 32 genera but only achieved a success rate of
Emmanuel Douzery) [30]. 56%.
- IRA&IRB: Inverted Repeat(A&B)
- SSC: Small Single Copy regions
- LSC: Large Single Copy regions
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THE POWER OF DNA BARCODING FOR PLANT IDENTIFICATION
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Figure (6): Relative place of matK loci. Coding loci were Figure (7): The region of the rbcL gene in the plastome of
represented by boxes and connections showed spacer loci the tobacco plant [14].
[35].

2.2.3. The trnH-psbA region

2.2.2. The RuBisCo (rbcL) region
The intergenic spacer psbA-trnH is considered an
The rbcL region can be amplified, sequenced and important DNA barcode because its end near to psbA
aligned in most plants thus it considers an excellent is highly conserved but it appeared a big inversion
DNA barcoding region in the plant at the level of near to trnH (Figure 8). The conserved region is
family and genus so it can detect their evolutionary difficult to be used for the identification of different
relationships. In the plant cells, the plastid has many taxonomic groups that are highly diverged in their
chloroplasts; each chloroplast genome contains only nucleotide variation [40].
one copy of the rbcL gene with a length of 600-750 The psbA gene encodes the photosystem II reaction
bp. This gene consists of only one exon (Figure 7) center protein D1 which affects many plant
and it encodes the polypeptide called RuBicCo [36]. physiological stages, light intensity photosystem and
Ribulose 1,5bisphosphate carboxylase oxygenase plant developmental stages. There are many studies
“RuBisCo” (EC: 4.1.1.39) is an important enzyme since more than 20 years on the role of chloroplast
responsible for carbon fixation by converting psbA UTRs in the regulation of gene expression. This
atmospheric carbon dioxide to glucose (molecules region has flanking primers that are involved in the
rich with energy) in photosynthetic organisms. In amplification and sequencing through high molecular
plants, this enzyme has two types of protein subunits size. Also, this region has an effective role in
one of them with a large chain and another with a analyzing the degraded DNA that is suggested to
small chain. The large chain subunit is encoded by contain the desirable barcode. In some previous
the rbcL gene that is present in the plant chloroplast studies on 21 genera and 18 families in plants, trnH-
genome while the small chain subunit is encoded by psbA is effective for the identification compared to
several related genes in the nucleus genome. The other barcodes matK+rbcL [41].
large chain contains the binding sites that form a The trnH–psbA spacer is distinguished by a great
dimer with a substrate (ribulose 1,5-bisphosphate) to number of inversions and indels this leads to a
form glucose. In some proteobacteria and complicated alignment as in genus Gentiana [17]. In
dinoflagellates, this enzyme contains only a large closely related species, this region can be varying in
chain subunit [37]. its length significantly. The trnH–psbA can provide a
Kress et al., [35] showed that rbcL region length is a better resolution, when used alone or in combination
drawback where its double-stranded sequencing with ITS, than matK and/or rbcL [42]. Burgess et al.,
needs four primers [38]. However, despite its [43] documented that the trnH–psbA spacer can
limitation rbcL is still being wildly used for plant slightly improve the resolution for the combination of
barcoding because it gives a huge amount of data and matK + rbcL region.
its entire gene sequence could be recovered so rbcL
barcode region in combination with other various
chloroplast or nuclear barcode regions have the
ability for accurate identification of plants. Fouad et
al., [39] used rbcL locus sequence for determining
the taxonomic relationships among twenty different
plant species which were collected from Burg El
Arab (lat. 30°54′N, long. 29°33′E), which belongs to
the Mareotis sector of Mediterranean sand dunes Figure (8): Schematic represented the arrangement of the
coast west of Alexandria in Egypt. psbA-trnH spacer, the variable regions (V1 and V2) and
conserved regions (C1, C2 and C3) [44].

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Recently, the two loci combination of rbcL+matK additional chloroplast barcodes, no satisfactory
and ITS have been recommended as the essence species differentiation was progressed among the
barcode for land plants [45]. Also, several researches genus Araucaria from the New Caledonia islands
documented that this combination led to perfect [54]. As an outcome, it was suggested to utilize
species identification, although the resolution level complete sequences of the chloroplast genome for
for discrimination of some genera such as Crocus, taxa discrimination. A few years ago, scientists could
Berberis and Primula, was insufficient [43]. Chinese not dream of it, but novel technologies such as next-
scientists performed a comparative study among generation sequencing (NGS) gave scientists with
6286 plant specimens from 141 genera and 1757 such a chance. The complete chloroplast genomes of
species using nucleotide sequences of various five 28 accessions from 11 New Caledonian Araucaria
DNA loci which are considered to be potential DNA species were sequenced with NGS technology. These
barcodes. The combination of MatK and rbcL species were discriminated by chloroplast genomes
barcodes are considered species-specific in 49.7% (~147000 nucleotides) and 11 nuclear genes (>6000
cases [46]. Abouseada et al., [47] evaluated the nucleotides in total), they found that the
genetic relationship among twelve wheat cultivars differentiation of these species was the highest in the
three of them from Egypt and two from Mexico while case of chloroplast genomes. This result did not
others cultivars from Morocco, Sudan, India, China, satisfy the researchers because more than half of New
Pakistan, Syria and Australia using DNA barcoding Caledonian Araucaria species with multiple
based on the combination of rbcL and matK loci accessions were monophyletic according to the
sequences. plastid or nuclear phylogenetic trees although the
Several studies found that the resolution of species were not monophyletic [55]. So the
intrageneric was increased by the addition of either researchers concluded that the differentiation of
ITS region or trnH–psbA spacer in families young taxa needs several variable nuclear barcodes.
Palmaceae or Labiatae respectively, [48]. Also, Nevertheless, the utilization of complete chloroplast
Feliner and Rosselló, [49] suggested that the well- genomes was very charming.
studied ITS loci should be added to DNA barcodes. There is another obstacle facing DNA barcoding in
Many Chinese researchers analyzed ITS data for plants, this obstacle is the quantity and quality of
thousands of taxa from several families and reported DNA extracted from plants that depend on many
that ITS2 to be a highly effective DNA barcode [50]. factors such as plant's age (DNA quantity is very low
As a result in 2011, it was suggested to add an in plants older than 50 years), methods of drying
alternative DNA barcodes (nuclear ITS1 and ITS2 plant samples, pesticides used is storage process and
loci and the chloroplast trnH-psbA spacer) to the list finally, the taxonomic group of the plant. For
of standard DNA barcodes [46]. Obviously, the instance, herbarium samples from the Boraginaceae
chloroplast barcodes usage had some limitations; it family have a problem in DNA extraction and also
was observed that perfect results could be likely medicinal herbal teas and other plant raw materials
gained with highly variable low-copy-number nuclear had degraded DNA [56]. Thus, DNA mini-barcodes
genes, but, due to their different variability among (short nucleotide sequences less than 200 base in
some species and various other obstacles, none of length) were progressed for the Sanger analysis of
them could be selected to be a DNA barcode [51]. plants that contain damaged DNA such as stored
First community phylogenetic for the tree of 281 plants and old herbarium samples. The most variable
species of woody plants in Barro Colorado Island in short DNA regions (DNA mini-barcodes) for these
Panama, depending on a super matrix analysis of species were found after knowing the sequences of
combined data of rbcL, matK and trnH-psbA the complete chloroplast genome by Next generation
sequence, was published. That led to adding well- sequences (NGS). For instance, the discrimination of
supported evolutionary components for knowing Раnах ginseng and Раnах notoginseng, this
species diversity [52]. performed depending on some variable regions as
rps16 gene (280 nucleotides), ycf1a gene (60
3. Obstacles overcoming and Future outlook of
nucleotides), or ycf1b gene (100 nucleotides) with an
DNA barcoding
identification power of 83.33%, 91.67% and 100%
An attempt to utilize the nuclear barcodes to identify respectively [57].
different species within the evolutionarily young
4. Next Generation Sequencing (NGS)
group for instance Hawaiian species from two genera
(Cyrtandra (family Gesneriaceae) and Clermontia Next Generation Sequencing (NGS) is a recent
(family Campanulaceae)) was failing, due to technique that covers the whole genome sequences
conserved ancestral alleles, high intraspecific (sequencing depth) and it is considered an index
variability and some other factors [53]. In spite of reflecting how many times a nucleotide was
several attempts to utilize DNA barcodes and some sequenced. The higher the index was, the higher was
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THE POWER OF DNA BARCODING FOR PLANT IDENTIFICATION
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the chance to avoid mistakes during the assembly of 5.1. Plant ecological forensics
the whole sequences of the genome. This technique is
DNA barcoding was utilized for plant identification
highly expensive so it was necessary to search for
from roots or leaves and cryptic life stages such as
another new and cheap technique such as the genome
fern gametophytes.
skimming process. This process is characterized by a
small sequence depth and low quality of DNA 5.2. Agricultural pests control
sequencing, yet, it remains suitable for a satisfactory
DNA barcoding is one of the new molecular
assembly of repeats (organelle genomes and
techniques that have been used to determine the type
ribosomal genes). As a result by this process, ~1 Gb
of sequences can be gained [58]. Genome skimming of pests and control them, since many agricultural
cannot determine rare variants so, it similar to the pests threaten the growth and productivity of most
plants, these consider a source of concern for many
Sanger sequencing [59].
farmers. Therefore, it was necessary to determine the
There is another technique was suggested for the
pests at any stage of their life. In addition, the DNA
sequencing of low-copy nuclear genes [60].
barcoding technique contributes to identify and
Therefore, with the advance in sequencing techniques,
the ideas for the utilization of a novel genome data control fruit flies.
format for DNA barcoding appeared. These ideas 5.3. Disease vectors identification
lead to the formulation of the so-called a twin track
process, which had (1) the standard DNA barcode DNA barcoding contributes to determine vector
accumulation and their libraries and (2) enlarged species. The vectors can cause dangerous infectious
barcode application by genomic skimming [61]. An diseases in humans and animals. This technique plays
enlarged barcode was achieved by NGS, which an important role to know these diseases and deal
represents the sequences of the chloroplast genome with them. DNA barcoding for mosquitoes helps
and ribosomal genes together. construct a reference barcode library. This library can
The next-generation sequencing technique had many help public health officials effectively control these
important advantages firstly it had the ability to read diseases caused by a mosquito with very little use of
sequences of chloroplast and nuclear markers of insecticides.
numerous specimens at one time this is important for 5.4. Invasive foreign species determination
the identification of herbal complements [56].
Secondary, NGS technique is used in the analysis of Identifying and controlling foreign species that can
the herbarium [59]. This test permits researchers to affect ecosystems and native species. Thus early
obtain enlarged barcodes of old samples. Finally, the discovery and regulatory measures are very important
analysis of the DNA of museum samples (including to stop the cross-border transport of foreign species,
herbarium) is specified, this is known by the term this can be performed by utilizing DNA barcoding
"museomics" [62]. technique for alien species identification.
At the beginning of DNA barcoding studies, the 5.5. Natural resources sustaining
scientists discussed the impossibility to utilize DNA
barcodes for the differentiation of closely related Natural resource managers used DNA barcoding
species [51]. DNA barcoding could be used to techniques and barcode libraries to monitor illegal
differentiate the lower taxonomic levels (subspecies commerce of products that are made of natural
and varieties) and this is called "ultra-barcoding", this resources such as hardwood trees.
is performed when it became possible to use 5.6. Endangered species protection
ribosomal genes and complete chloroplast sequences
loci sequenced by NGS [63]. Until now, it is still a Due to bushmeat hunt in Africa, Primate Population
continuous search for the optimal DNA barcodes in decreased by 90%. DNA barcoding could be utilized
plants. to perform law enforcement on bushmeat in local
markets to protect Primate from extinction.
5. Applications of DNA barcoding
5.7. Water quality observation
Numerous studies mentioned that the applications of
DNA barcoding on plants can be split into two Water is very important for living beings, it is
categories (plants' taxonomic process and identification responsible for maintaining all living cell functions.
of unknown plant specimens). Also, DNA barcoding So, it is necessary to study the quality of water.
can be applied in other several fields [64] and [65] as Several organisms live in water sources (streams,
follows: rivers and lakes). DNA barcoding could be used to
identify and create a library for these species
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especially for the species that are difficult to (NCBI) database (GenBank). These sequences have
discriminate. Finally, DNA barcoding is a helpful already accession numbers were recorded in the gene
technique for environmental agencies to create better library.
policies that can ensure the safe supply of drinking b- These gene sequences were downloaded and saved
water. in FASTA format files after that the downloaded
sequences were aligned by certain bioinformatics
6. DNA barcode protocol
programs such as the MEGA program to determine
DNA barcode analysis comprises three major steps. the similar part among all aligned sequences
These steps are sample collection, molecular analysis c- This similar part of sequences was used to design
and bioinformatics as summarized in figure (9). The the primer by the online program Primer 3 (version 4)
final data analysis will led to the discovery of (http://bioinfo.ut.ee/primer 3-0.4.0).
numerous unknown plant species [26]. d- Afterward the designed primer was tested in silico
by aligning the retrievable complete desired barcode
the gene sequence of the same studying species with
the designed primer to insure that this primer was
already a specific primer for the desired barcode and
attached with barcode gene (understudying) by 100%.
6.4. Polymerase Chain Reaction
The standard DNA barcodes are characterized by
easy amplification of polymerase chain reaction
(PCR) and its length is short enough to permit a
bidirectional read during the sequencing process [34].
The amplification of desired barcode region was
performed in the Thermal Cycler apparatus by the
following steps:
a- PCR mixture: genomic DNA (50 ng), dNTP (0.2
mM), designed primer (10 pM), MgCl, Taq Buffer
(5x) and Go Taq DNA Polymerase (1U).
b- The used PCR program depends on the type of
Figure (9): Graphic diagram summarized the steps of Plant plant species and type of the desired barcode for
DNA barcoding protocol [66]. instant the PCR program for amplification of matk
gene in different Triticum species is 95°C for 4 min
followed by 35 cycles of 95°C for 30 sec, 57°C for 1
6.1. Plant samples collection min and 72°C for 1 min and a final extension at 72°C
for 5 min [68].
The collected plant samples could be obtained from c- PCR products were loaded onto 1.5% agarose gel
in herbaria or picked directly from the field (fresh life which stained ethidium bromide.
samples). d- The gel photo was captured by gel documentation
6.2. Plant genomic DNA Extraction and Bands were analyzed by gel analyzer or TotalLab
programs as shown in figure (10).
- Genomic DNA was extracted from young leaves of e- The resulting barcode fragment was cut and
samples by DNA purification Kits or by manual cleaned up from agarose gel to obtain the purified
methods such as CTAB. barcode gene ready to perform sequencing by Sanger
- The extracted DNA was checked on agarose gel by sequencer.
electrophoresis and then spectrophotometry 6.5. Sequence Editing
(NanoDrop 2000) afterward, diluted to 50ng/μl and
used as a PCR template [67]. The chromatogram (electropherogram or trace file) of
the nucleotide sequences was obtained by Sanger
6.3. In silico analysis and primers design sequencer. The formats of chromatograms file are
The primers design could be summarized in the ABI (Applied Biosystems sequence) and SCF, which
following steps: require certain software to be opened and analyzed.
This software may be free such as
a- The desired barcode gene sequences of certain BioEdit (https://bioedit.software.informer.com/),
species (which were recorded in the barcode library Chromas (http://technelysium.com.au/wp/chromas/)
(NCBI) by previous research) were retrievable from andFinchTV(https://finchtv.software.informer.com/1.
the National Center for Biotechnology Information 4/), while the commercial software includes
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Egypt. J. Chem. 67, No. 1 (2024)
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THE POWER OF DNA BARCODING FOR PLANT IDENTIFICATION
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CodonCode (https://www.codoncode.com/),
Geneious (https://www.geneious.com/) and
ChromasPro(http://technelysium.com.au/wp/chromas
pro/ ). By all these software, simple sequence editing
and extensive analytical tools can be performed. The
barcode gene is sequenced bi-directionally (forward (a)
and reverse) to recover the entire DNA sequence,
when creating a DNA barcode library. The workflow
of sequence editing is summarized in figure (11 and
12).
The isolated DNA barcode gene sequences have been
deposited in NCBI and take certain accession (b)
numbers.

(c)
Figure (12): Part of chromatogram chart [68].
(a) Showing very good sequence.
(b) Showing bad sequence at the end of sequence.
(c) Showing a trim end and recover the entire DNA
sequence using BioEdit software.

6.6. Phylogeny analysis

- The recovered nucleotide sequences were aligned
Figure (10): matK gene from twenty Triticum species [68]. by the Clustal W multiple sequence alignment using
one of bioinformatics program (Mega program) as
shown in figure (13).

Figure (11): Scheme summarized the sequence editing

workflow.
Figure (13): Part of matK sequence alignment in twenty
different Triticum species from1 to 99 bases [68].

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642 S. A. Osman
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