National Symposium : Frontiers in Biotechnology 17th Feb 2011 Biotechnology Department, PU, Chandigarh
Invited Talk-Quest of DNA signature of species of animal, plant & microbes :
Can we have a PIN code?
1. National Symposium : Frontiers in Biotechnology 17th Feb 2011
Biotechnology Department, PU, Chandigarh
Quest of DNA signature of species of animal, plant & microbes :
Can we have a PIN code?
Dinesh Kumar, B.Sc. Hons Zoo(BHU), M.Sc. Biotechnology(BHU), Ph.D. Biotechnology (BHU), PDF(USA)
Senior Scientist(Animal Biotechnology)
National Bureau of Animal Genetic Resources, Karnal-132 001, Haryana, INDIA
Email: dineshkumarbhu@gmail.com, dinesh@iastate.edu
2. DNA signature- Quest
Genome, some thing identifiable
My single cell, 92 DNA molecule, maternal 46, paternal 46
A part of molecule is unique or combination of these
molecule is unique
Signature in form of DNA marker- SNP or STR
How to tap these signature in genome?
Need bioinformatics tools
Case studies
Domestic Animals , Fish, Microbes, Plant
STR allele-private alleles or private frequencies
SNP- plus and minus assay
Software tools-SNP data and STR data
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3. How DNA signature is made for each species?
The number of nucleic acid or amino acid differences
between two organisms is proportional to the time since
they diverged from a common ancestor.
1 AAGGCTA 1 2 3
MOLECULAR 2 AAGGGTA 100years
DIFFERENCES 3 AAGGATG
200
Example
years
Rate of Evolution =
1bp per 100 years
TIME
4. Which marker for which purpose?
ï How long ago did organism A and organism
B last have a common ancestor?
ï Molecular clock and DNA signature !!!
Very recently - RAPDs/VNTRs/Microsatellites/
resistance genes
10,000s - 100,000s yrs - RNA- ITS, various protein-
coding genes
100,000s - 1,000,000s yrs - ssu rRNA, HSPs,
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5. DNA barcoding of species
ï cytochrome c oxidase subunit I gene (COI)
potential 'barcode'.
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6. Origin of species bar code
ï Carl Woese used sequence differences in
ribosomal RNA (rRNA) to discover archaea,
which in turn led to the redrawing of the
evolutionary tree, and molecular markers (e.g.,
allozymes, rDNA, and mtDNAvage ) have been
successfully used in molecular systematics for
decades.
ï In 2003, Paul D.N. Hebert from the University of
Guelph, Ontario, Canada, proposed the
compilation of a public library of DNA barcodes
that would be linked to named specimens.
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7. Identification of birds by species bar code
ï Hebert and co-workers sequenced DNA
barcodes of 260 of the 667 bird species that
breed in North America (Hebert et al.
2004).
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8. Identifying flowering plants by species DNA bar code
ï Kress et al. (2005) -COI sequence- not appropriate for most
species of plants slower rate of cytochrome c oxidase I gene
evolution in higher plants than in animalsâ.
ï A series of experiments was then conducted to find a more
suitable region of the genome for use in the DNA barcoding
of flowering plants.
ï nuclear internal transcribed spacer region and the plastid
trnH-psbA intergenic spacer as a potential DNA barcode for
flowering plants. Some reports supports MatK
ï DNA barcoding, no 'master key' may be a 'master keyring',
with different kingdoms of life requiring different keys.
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9. Microbial species signature
ï Ribotyping -rRNA database-most used eg 16srDNA
ï Designing of primer for gene sequencing. Eg HKG,Topoiso II,
ï Designing of probes for identifications-Eg.real time, microarray
ï Meta genome analysis- gene prediction
ï Chip based pyrosequencing and simulation
ï In silico development of RFLP test for close species
differentiation(our experiences)
ï MPIDB- functional identification
ï Miniprimer PCR-a new lens to view microbial world
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10. rRNA genes - the ideal markers for microbial identification
ï Small subunit - highest order differences (domains)
ï Large subunit - medium order differences
ï ITS - low order differences (species/strains?)
Small Sub-Unit rRNA (16S)
ï ubiquitous
ï 1.6 - 2.0kb
ï good molecular chronometer.
ï some areas conserved (for priming/alignment)
ï some areas variable (for resolving differences)
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14. Why we need molecular & bioinformatics tool?
ï Case study of Ug99-signature search
Stem rust never sleeps- Norman E. Borlaug , 26th April, 2008, New York Times
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15. Can we have DNA based signatures of Ug99?
ï National Debate!
ï Global meet at Delhi, Oct, 2008
ï Action plan?
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16. A case study updates-
How to identify Ug99?
ï Puccinia graminis tritici Ug99
ï DNA signature is the only answer !
ï DNA signature of Fungi
â Private alleles of STR(rare)
â STR allele frequency signatures
â SNP based signatures(???)
ï Where is the signature of Ug99 ??
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17. What are the available DNA
markers to identify Ug99
SSR
AFLP
Mol Plant
Path
Latest Nov
2008 issue
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18. SSR data- no DNA signature
Mol Plant Path Nov 2008 issue
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19. AFLP adapters used to generate signature data
Mol Plant Path
Nov 2008 issue
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21. No clear signature of Ug55 & 99 by SSR
Mol Plant Path Nov 2008 issue
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22. AFLP again poor signature b/w Ug99/55
Mol Plant Path Nov 2008 issue
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23. DNA signature of Ug99 by combining data
of SSR+AFLP
Mol Plant Path Nov 2008 issue
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24. Clear DNA signature by
Minimum-spanning network analysis
Mol Plant Path Nov 2008 issue
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25. What bioinformatics can do more in
Ug99 identification?
ï Allele mining of Puccinia graminis tritici Ug99
â USDA is targeting 400 SNP
ï STR mining from Puccinia graminis genome data
base
ï STR based signature search
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26. Puccinia graminis database
Genome of P. graminis tritici
88.54 Mb, 392 scaffolds, contigs 4557
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27. Our experiences-DNA based gender
signature using bioinformatics tools
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28. Our experiences-DNA based microbial species
signature using bioinformatics
House Keeping Genes -CLUSTAL-W-signature with spelling mistake- CLEAVER
KspAI Bsh1236I Mun I
L P P R RR P P R RR P P R RR
KEU1 5'-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA-3'
KEU 2 5'-AYR TTI TCI CCI GGC ATI ACC AT-3'.
KspAI L.paracasei 542,158bp; L.rhamnosa 701bp
Bsh1236I L. paracasei 547,153bp; L. rhamnosa 701bp
Mun I L. paracasei 594,106bp; L. rhamnosa 701bp
New PCR-RFLP test developed for Lactobacillus species differentiation
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29. DNA based signature of domestic species
ï Mitochindrial DNA markers
ï used especially for meat identification,
ï poaching of wild animals,
ï adulteration of dairy milk,
ï dairy products(like cheese) of various
domestic animal species.
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30. DNA based signature of
domestic animal breeds
ï Whether a livestock breed can be identified from a sample of blood,
semen, hair, blood spot, carcass etc?
ï Studies have succeeded in developing a technology for breed
certification and breed-specific genetic/DNA signature
ï Degree of accuracy of certification of a breed -between 95% to 99%.
ï STR based three methods viz
ï (i) Frequency method (Paetkau et al., 1995),
ï (ii) Bayesian method (Rannala et al, 1997) and
ï (iii) Distance methods (Goldstein et al 1995)
Use
(i) Development of breed-specific signatures/profiles and
(ii) Development of breed hybrid index.
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31. STR & breed-specific signatures/profiles
ï Pig-In UK, Signer et al. (2000) - minisatellite probe pCMS12 -three breeds of pig viz
Chinese, Meishan, Large White and other European breeds. The linear discrimination
analysis revealed that the DNA profiles were breed specific.
ï Fish-In Finland, Primmer et al. (2000) - disputed fish to a specific population out of 4
suspected fish populations using 7 microsatellite loci by Bayesian method with
confidence limit of 99%.
ï Sheep-In Spain, Arranz et al. (2001) - Bayesian method with 99.63% accuracy among
five Spanish sheep breed viz. Churra, Latxa, Castellana, Rasa-Aragonesa and Merino
using 18 microsatellite markers.
ï Horse-In Norway, Bjornstad et al. (2001) -26 microsatellite loci in six breeds of horses,
Fjord, Nordland/ Lyngen, Dole, Trotter, Icelandic horse and Shetland pony with more
than 95% confidence limit.
ï Cattle-In European countries, Canon et al. (2001) -confidence limit of 99% for 18 local
breeds of cattle of different countries; Alistana, Astruriana, Asturiana Valles,
Sayaguesa, Tudanca, Avilena Negra-Iberica, Bruna del Pirineus, Morucha, Pirenaica,
Retinta of Spain; Alentejana, Barrosa, Maronesa, Mertolenga, Mirandesa of Portugal
and Aubrac, Gasconne, Salers of France
ï Camel- In Kenya, Mburu et al. (2003) - 4 breeds using 14 microsatellite loci of camel
viz. Somali, Turkana, Rendille, Gabbra) using maximum likelihood method up to 48 %
confidence limit.-weak genetic differentiation and gene flow between populations.
ï Dog-In Finland, Koskinen (2003) has assigned breeds of domestic dog using
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microsatellite with 100% accuracy.
32. Development of breed hybrid indices/ profiles
ï Campton and Utter (1985) -hybrid index, hybrid index for
analyzing hybridization between cut-throat (Oncorhynchus
clarkii , Salmonidae) and rainbow trout ( O. mykiss ,
Salmonidae), and they used allozyme loci for which the two
species were almost fixed for alternate alleles.
ï Hansen et al. (2000) found that hybrid index statistic is also
useful for microsatellite loci and t- interbreeding between wild
and domesticated brown trout,
ï Softwares:
ASSIGNMENT CALCULATOR,
GENECLASS or
ARLEQUIN and then importing the data into a spreadsheet where the final calculations can be done.
Relevance in Indian context-
exotic inheritance calculation, Bos indicusx taurus, Alpine x Beetal
cross/admixture populations
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33. Future breed signature of domestic
animals
Courtesy: Curt & his group USDA
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35. Few examples: Signature applications
1. Application of polymerase chain reaction to detect adulteration of
sheep's milk with goats: J Dairy Sci (2005) 88: 3115-20.
2. A novel approach to the quantification of bovine milk in ovine cheeses
using a duplex PCR: J Agric Food Chem (2004) 52: 4943-7.
3. Rapid detection of cows' milk in sheeps' and goats' milk by a species-
specific polymerase: J Dairy Sci (2004) 87: 2839-45.
4. Identification of cow's milk in "buffalo" cheese by duplex polymerase
chain reaction. J Food Prot (2002) 65: 362-6.
5. Forensic identification of ungulate species using restriction digests of
PCR-amplified mit J Forensic Sci (1995) 40: 943-51. (15 species)
6. Detection of cows' milk in goats' cheeses inferred from mitochondrial
DNA polymorphism Journal of Dairy Research (2001), 68:229-235
7.Application of polymerase chain reaction for detection of goats' milk
adulteration by milk of cow. Journal of Dairy Research (2001),
68:333-336
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36. DNA based signature of plant variety, example-
Basmati rice
ï Basmati rice -aroma compound -2-acetyl-1-
pyrroline.
ï Fraudulent traders to adulterate traditional basmati.
ï PCR-based assay similar to DNA fingerprinting in
humans allows for the detection of adulterated and
non-basmati strains. Its detection limit for
adulteration is from
ï 1% upwards with an error rate of ±1.5%.
ï Exporters of basmati rice use 'purity certificates'
based on DNA tests for their basmati rice
consignments.
ï It was developed by CDFD, Labindia,
ï World's First Single-tube, Multiplex(co-amplify
eight microsatellite loci) Microsatellite Assay-based
Kit for Basmati Authentication.
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37. DNA based bar-coded signature of fish
ï Ward et al (2005) - cox1 sequencing, or âbarcodingâ, in to
identification of fish species.
ï 207 fish, mostly Australian marine fish, were sequenced
(bar coded) for a 655 bp region of the mitochondrial
cytochrome oxidase subunit I gene (cox1).
ï Most species were represented by multiple specimens, and
754 sequences were generated.
ï The GC content of the 143 species of teleosts was higher
than the 61 species of sharks and rays (47.1% versus
42.2%), largely due to a higher GC content of codon
position 3 in the former (41.1% versus 29.9%).
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38. Few examples of individual DNA signature
and family signature
* Rajiv Gandhi Assassination Case (Chennai,
Tamil Nadu),
* Naina Sahni or the Tandoor case (New Delhi
* Priyadarshini Mattoo (New Delhi),
* Sishu Vihar Child adoption case (Hyderabad,
Andhra Pradesh),
* Black Buck killing case (Jodhpur, Rajasthan)
* Beanth Singh Assassination Case (Punjab)
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40. What is CBOL?
The Consortium for the Barcode of Life (CBOL) is an international initiative devoted
to developing DNA barcoding as a global standard for the identification of biological
species. DNA barcoding is a new technique that uses a short DNA sequence from a
standardized and agreed-upon position in the genome as a molecular diagnostic for
species-level identification. DNA barcode sequences are very short relative to the
entire genome and they can be obtained reasonably quickly and cheaply. The "Folmer
region" at the 5' end of the cytochrome c oxidase subunit 1 mitochondrial region (COI)
is emerging as the standard barcode region for almost all groups of higher animals. This
region is 648 nucleotide base pairs long in most groups and is flanked by regions of
conserved sequences, making it relatively easy to isolate and analyze. A growing
number of studies have shown that COI sequence variability is very low (generally less
than 1-2%) and that the COI sequences of even closely related species differ by several
percent, making it possible to identify species with high confidence. For those groups in
which COI is unable to resolve species-level differences, CBOL recommends the use of
an additional gene region. In some groups, COI is not an effective barcode region and a
different standard region must be identified. In all cases, DNA barcoding is based on the
use of a short, standard region that enables cost-effective species identification.
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58. Conclusion
ï DNA based species signature is possible
ï Why to do it?
ï Who has to do?
ï What we need?
ï How to proceed?
ï Germplasm wealth of âthird worldâ countries
needs protection and pragmatic use!
ï Biotechnology/Bioinformatics has immense role
ï Journey of IT & BT !
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59. Acknowledgements
Dr Jagdeep Kaur , Dr Jagtar Singh & team, Biotechnology
Department , Punjab University, Chandigarh
My students- Prashant, Prem, Nishant, Dhiraj (NBAGR)
My UG/PG students- Pooja(NISER) & Uday(GU)
Dr James Reecy & his group, Iowa State University, USA
Dr DK Arora, NBAIM(ICAR)
Dr Rameshwar Singh & Dr SK Tomar, NDRI
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