Plant DNA barcoding research is shifting beyond performance comparisons of different DNA regions towards practical applications. The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1(CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. The studies on cucumis sp for the application of DNA barcode shows the possibility of discrimination at species level not the varietal level using the matK gene barcode. The phylogenetic tree constructed by using matK gene sequences clearly differentiated the species C. sativus and C. melo which will help for the future application in cucumis taxonomy and phylogeny studies

1. DNA Barcode: species/varietal
identification
Dr.N.Senthil
Professor (Biotechnology)
www.tnaugenomics.com

2. How the DNA Barcoding done

4. Molecular phylogeney
• Molecular phylogeney has clarified many uncertainties in our view of
species relationships and is particularly important in the study of
evolutionary relationships between small and microscopic organisms (such
as some insects, nematodes, protozoa and bacteria) which are vitaly
important for ecosystems but where morphological features are difficult to
identify and compare.

5. Why to barcode
• The total number of unique organisms described to the species
level is around 1.5 million, but the total number of ‘species’ is likely
to be in the region of 10 million.
• The overall ‘taxonomic deficit’ (the ratio of expected taxa to named
taxa) is thus approximately sixfold.
• For vertebrates, the current described species total is likely to be
relatively close to the ‘true’ total: we have described most of these
relatively large organisms. The same is true of most groups whose
members have body sizes greater than 10 mm.
• However, the vast majority of organisms on the Earth have body
sizes less than 1 mm, and for these groups the taxonomic deficit is
likely to be several fold worse than for land plants and vertebrates.

6. Species identification
• To provide a central catalog of organismal diversity
which can be accessed by anyone who needs to quickly
and accurately indentify an organism.
• Such a catalog would also allow us to differentiate new,
previously undescribed species from those already
observed and might represent a valuable tool in
• conservation efforts, the diagnosis of diseases, the
monitoring of invasive species (those that colonize new
environments to the detriment of native species), and
many other fields.

7. DNA sequence -Barcode
• To gain an accurate picture of evolutionary
relationships, it is usually neccessary to obtain
the DNA sequence of many different genes from
the organisms under study and compare them
simultaneously.
• The technology required to isolate the part of the
DNA of an organism that contains a gene of
interest and determine its sequence (made up
the the bases A, G, C and T) has recently become
widely accessible, cheap and easy to master.

8. Isolation of specific gene
• A-priori, the sequence of two sequences near the ends
of the gene (in practice this means that the gene
should contain at least two short regions that are
highly conserved between ALL species, while the rest
of the sequence should be highly variable).
• Molecular biologists who have identified a gene called
coxI , (cytochrome oxidase I) which seems to satisfy the
requirements .
• CoxI (Cytochrome c oxidase subunit I )gene is found in
the mitochondria of animals, plants and fungi
• Ribulose-bisphosphate carboxylase and Maturase K
genes in plants (MatKi gene) from chloroplast origin

9. How to make reference sample and
reference sequence ?
• The provision of specimens for the reference
collection, and the correct linking of sequences to
species in the reference collection.
• To this end, museums are providing access to their
collections and their expertise in classical taxonomy,
while significant funding is now dedicated to the
isolation and sequencing of DNA from species that are
not present in museum collections (as well as their
taxonomic classification).
• Thus, barcoding has sparked a renaisence of interest in
taxonomic studies.

10. DNA barcoding
• DNA barcoding is a technique for
characterizing species of organisms using a
short DNA sequence from a standard and
agreed-upon position in the genome.
• DNA barcode sequences are very short
relative to the entire genome and they can
be obtained reasonably quickly and
cheaply.
• The cytochrome c oxidase subunit 1
mitochondrial region (COI) is emerging as
the standard barcode region for higher
animals.
• It is 648 nucleotide base pairs long in most
groups, a very short sequence relative to 3
billion base pairs in the human genome

11. Barcode’ metaphor
• All the products of one type
on a supermarket shelf (like a
2-litre bottle of Coca-Cola)
share exactly the same 11-
digit barcode, which is distinct
from all other barcodes.
• DNA barcodes vary among
individuals of the same
species, but only to a very
minor degree.
• If the DNA barcode region is
effective, the minor variation
within species will be much
smaller than the differences
among species.

12. Barcoding projects 4 components
• Specimens :Natural history museums, herbaria, zoos, aquaria, frozen tissue
collections, seed banks, type culture collections and other repositories of
biological materials are treasure troves of identified specimens.
• The Laboratory Analysis protocol to obtain DNA barcode sequences from these
specimens available .
• Barcode of Life Database (BOLD) was created and is maintained by University of
Guelph in Ontario. It offers researchers a way to collect, manage, and analyze DNA
barcode data.
• The Data Analysis: Specimens are identified by finding the closest matching
reference record in the database having Ribulose-bisphosphate carboxylase and
Maturase K genes in plants
• Animals :mitochondrial Cytochrome c oxidase subunit I gene
• Fungal barcodes and accepts sequences from the Internal Transcribed Spacer
Region

13. Selecting a core-barcode
 The standard animal CO1 DNA barcode fits
the following criteria
 It is a haploid, uniparentally-inherited,
 single locus that shows high levels of
discriminatory power
 It is a protein-coding region present in high-
copy numbers per cell, and in animals
 it is not prone to drastic length variation,
strong secondary structure,
 microinversions, or frequent
mononucleotide repeats.
 well-developed primer sets
 CO1 sequences can be consistently
orientated,
 Aligned with little supervision, and be
translated to diagnose
 pseudogenes and identify sequencing errors.

14. Tools and Technology Required to
Support DNA Barcoding in Plants
• Protocols and guidelines for DNA extraction and sequencing
from herbarium specimens
• Continued improvement of PCR and sequencing protocols for
regions rich in mononucleotide repeats
• Development of DNA barcoding primers and a system to
record and predict which primers will work well in a given
taxonomic group
• Development of robust multiplex PCR protocols
• Enhancement of mini-barcodes for degraded DNAs

15. DNA barcoding and its potential
• To establish a shared community resource of DNA sequences that
can be used for organismal identification and taxonomic
clarification.
• This approach was successfully pioneered in animals using a portion
of the cytochrome oxidase 1(CO1) mitochondrial gene.
• In plants, establishing a standardized DNA barcoding system has
been more challenging.
• The studies on cucumis sp for the application of DNA barcode
shows the possibility of discrimination at species level not the
varietal level using the matK gene barcode. The phylogenetic tree
constructed by using matK gene sequences
• The barcode clearly differentiated the species C. sativus and C.
melo which will help for the future application in cucumis taxonomy
and phylogeny studies

16. Different markers in plant barcoding studies

17. DNA barcoding in plants

18. Genes used as Plant DNA barcode

19. Intraspecific gene flow on species discrimination success

20. DNA barcoding projects underway
Project and Lead Institute
• TreeBOL: Barcoding the world’s tree species :The New York
Botanic Garden
• GrassBOL: Barcoding grasses and grass-like plants :Adelaide
University and University of British Columbia
• Flora of the Kruger: National Park University of Johannesburg
• Flora of the Area de Conservacion Guanacaste :Costa Rica
University of Pennsylvania
• Flora of Korea: Korea University
• Plant Barcoding China: DNA barcoding of 5000 Chinese plant
species Kunming Institute of Botany
• All-genera: DNA barcoding of representatives of all angiosperm
genera The New York Botanic Garden
• DNA barcoding of Centre for Tropical Forestry Plots
:Smithsonian Institute
• DNA barcoding Chinese medicinal plants :Institute of Medicinal
Plant Development Beijing
• DNA barcoding the flora of Wales :National Botanic Garden of
Wales
• DNA barcoding British bryophytes :Royal Botanic Garden
Edinburgh

21. BARCODE Applications
1) Geographically focused studies aiming to distinguishing among the
diversity at a given site or region, where many of the samples are not
necessarily closely related, and particularly where juvenile material and
plant fragments require identifications; (
2) species in trade, where the challenge is often to distinguish between a set
of target species, and often distantly related potential substitutes or to
identify members of higher taxonomic groups (e.g. family, genus) rather
than particular species; and
(3) where the identification problem relates to unfamiliarity with a given
species such that the user may have no idea even what family a given
species belongs to. In this situation, identification to a group of related
species is useful as it can narrow down the total range of possible
alternatives and also enable targeted use of morphological keys or expert
consultation to obtain a final identification where required.
4) This ‘species group identification’, followed by subsequent ‘tie-breaker’
analyses is particularly likely to be useful in species-rich systems where
there is a shortage of available taxonomic expertise.

22. Future of DNA barcode
• Ecological forensics, identification of traded materials, undertaking
identifications where there is a shortage of taxonomic expertise
available, and assisting species discovery in some plant groups.
• Future technological advances will undoubtedly lead to
improvements over current approaches
• Assembling large DNA sample sets representing the earth’s botanical
diversity, supported by voucher specimens, and indexed via DNA
sequences.
• This will provide the framework for current applications, and future
developments, in the coordinated use of DNA sequence data to tell
plant species apart.

23. BARCODE standard

24. Voucher specimens

25. Reference records

26. Public database

27. IDS – Identification System

28. Musquito barcode –disease control

29. Barcoding life.org
• The CBOL database (www.barcoding
life.org) has 404,146 records representing
40,110 species, a small proportion of the
total biodiversity on earth, but a
remarkable achievement for the first four
years of its existence.

30. Bar coding of Cucumis genus
Indian sub-continent : centre of origin for cucumber (Cucumis
sativus L. var. sativus; 2n = 14) centre of diversity for melon
(Cucumis melo L.; 2n = 24)
Narrow genetic base - cultivated species were developed from
closely related parents - limits crop improvement
Genetic diversity - morphological and molecular markers
Recently, comparison of DNA sequences has been extensively
used to identify phylogenetic relationship among different plant
species.

31. matK (Megakaryocyte-associated tyrosine kinase)
• The plastid gene trnK has a 2.5 kb class II intron including a 1.5 kb open reading frame called
matK. formerly known as orfK
• It is least conserved of the plastid genes and have high rate of nucleotide substitution compared
to other genes
• Evaluation rate of matK gene was approximately three fold faster than the rbcL gene in
Saxifragaceae family
• The matK gene sequence has been widely employed in inter and intragenous
phylogenetic analysis in different plant species.

32. Morphological dendrogram generated by using UPGMA method
based on Euclidean distance coefficient
@ 12.74

35. Genetic diversity analysis at molecular level
using matK gene sequences
Sequence analysis of matK region
• Partial sequence was obtained using the
reverse primer matK-8R.
• Final alignment - 611 positions from each
genotype.
• 77 variable sites
• 26 were informative for parsimony analysis

36. Parsimony informative sites for matK gene sequences

37. Phylogenetic analysis
Neighbor joining (NJ) tree Maximum likelihood (ML) tree

38. Cost of Reagents and Disposables
Fresh/Frozen Museum
Tissue Sampling $0.41 $0.41
DNA Extraction $0.34 $2.00
PCR Amplification $0.24 $0.48
PCR Product Check $0.35 $0.70
Cycle Sequencing $1.04 $2.08
Sequencing Cleanup $0.32 $0.64
Sequence $0.40 $0.80
Total: $3.10 $7.11

39. DNA from Create BARCODE
identified voucher reference record
Refine taxonomy ID unknowns
of group
Create BARCODE DNA from unidentified
DNA from immature specimen
reference records
identified adult
voucher Associate immatures
with names Repository of
provisional
vouchers
ID unknowns
Refine taxonomy Add names to
of group vouchered
immatures

40. Producing Barcode Data
Barcode data anywhere, instantly
• Data in seconds to
minutes
• Pennies per sample
• Link to reference
database
• A taxonomic GPS
• Usable by non-
specialists