DNA Barcode: species/varietal identification

Dr.N.SenthilProfessor (Biotechnology)www.tnaugenomics.com

How the DNA Barcoding done

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.

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.

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.

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.

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

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.

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

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.

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

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.

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

• 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

DNA barcoding and its potential

Different markers in plant barcoding studies

DNA barcoding in plants

Genes used as Plant DNA barcode

Intraspecific gene flow on species discrimination success

DNA barcoding projects underwayProject 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

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.

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.

BARCODE standard

Voucher specimens

Reference records

Public database

IDS – Identification System

Musquito barcode –disease control

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.

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.

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.

Morphological dendrogram generated by using UPGMA method

based on Euclidean distance coefficient

@ 12.74

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

Parsimony informative sites for matK gene sequences

Neighbor joining (NJ) tree Maximum likelihood (ML) tree

Phylogenetic analysis

 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

Cost of Reagents and Disposables

DNA from identified voucher

Create BARCODE reference record

ID unknownsRefine taxonomy of group

DNA from identified adult

voucher

Create BARCODE reference records

Associate immatures with names

ID unknowns

Refine taxonomy of group

DNA from unidentified immature specimen

Repository of provisional vouchers

Add names to vouchered immatures

Producing Barcode DataBarcode data anywhere, instantly

• Data in seconds to minutes

• Pennies per sample• Link to reference

database• A taxonomic GPS• Usable by non-

specialists