National Symposium : Frontiers in Biotechnology 17th Feb 2011Biotechnology 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, INDIAEmail: dineshkumarbhu@gmail.com, dinesh@iastate.edu

Genome, some thing identifiableMy single cell, 92 DNA molecule, maternal 46, paternal 46A part of molecule is unique or combination of thesemolecule is uniqueSignature in form of DNA marker- SNP or STRHow to tap these signature in genome?Need bioinformatics toolsCase studiesDomestic Animals , Fish, Microbes, PlantSTR allele-private alleles or private frequenciesSNP- plus and minus assaySoftware tools-SNP data and STR data

DNA signature- Quest

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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.

TIME

MOLECULAR

DIFFERENCES

1 AAGGCTA

2 AAGGGTA

3 AAGGATG

Example Rate of Evolution = 1bp per 100 years

1 2 3

100years

200 years

Which marker for which purpose?

How long ago did organism A and organism B last have a common ancestor?

Molecular clock and DNA signature !!!Very recently - RAPDs/VNTRs/Microsatellites/

resistance genes10,000s - 100,000s yrs - RNA- ITS, various protein-

coding genes100,000s - 1,000,000s yrs - ssu rRNA, HSPs,

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DNA barcoding of species

cytochrome c oxidase subunit I gene (COI) potential 'barcode'.

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Origin of species bar codeCarl 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.

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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Identification of birds by species bar code

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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Identifying flowering plants by species DNA bar code

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 animals”.

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.

nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as a potential DNA barcode for flowering plants. Some reports supports MatK

DNA barcoding, no 'master key' may be a 'master keyring', with different kingdoms of life requiring different keys.

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Microbial species signature

Ribotyping -rRNA database-most used eg 16srDNA Designing of primer for gene sequencing. Eg HKG,Topoiso II,

Designing of probes for identifications-Eg.real time, microarray

Meta genome analysis- gene prediction Chip based pyrosequencing and simulation In silico development of RFLP test for close species

differentiation(our experiences) MPIDB- functional identification Miniprimer PCR-a new lens to view microbial world

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rRNA genes - the ideal markers for microbial identification

Small subunit - highest order differences (domains) Large subunit - medium order differences ITS - low order differences (species/strains?)

Small Sub-Unit rRNA (16S) ubiquitous 1.6 - 2.0kb good molecular chronometer. some areas conserved (for priming/alignment) some areas variable (for resolving differences)

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•Naked Eye: Colony - shape, colour.•Microscopy: Cell - shape, colour, size•Biochemistry:•Molecular Level: Role of bioinformatics

Plant pathogen signature

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Can we use microsatellite in Fungi?

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How fungal microsatellite data gives signature

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Why we need molecular & bioinformatics tool?

Case study of Ug99-signature search

Stem rust never sleeps- Norman E. Borlaug , 26th April, 2008, New York Times2/17/2011 14Dinesh-PU-Talk-17-02-2011

Can we have DNA based signatures of Ug99?

National Debate! Global meet at Delhi, Oct, 2008Action plan?

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A case study updates-

How to identify Ug99?

Puccinia graminis tritici Ug99DNA signature is the only answer !DNA signature of Fungi

– Private alleles of STR(rare)– STR allele frequency signatures– SNP based signatures(???)

Where is the signature of Ug99 ??

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What are the available DNA markers to identify Ug99

SSR

AFLP

Mol Plant PathLatest Nov 2008 issue

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SSR data- no DNA signature

Mol Plant Path Nov 2008 issue

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AFLP adapters used to generate signature data

Mol Plant Path Nov 2008 issue

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AFLP signatures

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No clear signature of Ug55 & 99 by SSR

Mol Plant Path Nov 2008 issue

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AFLP again poor signature b/w Ug99/55

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DNA signature of Ug99 by combining data of SSR+AFLP

Mol Plant Path Nov 2008 issue

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Clear DNA signature by Minimum-spanning network analysis

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What bioinformatics can do more in Ug99 identification?

Allele mining of Puccinia graminis tritici Ug99– USDA is targeting 400 SNP

STR mining from Puccinia graminis genome data base

STR based signature search

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Puccinia graminis database

Genome of P. graminis tritici88.54 Mb, 392 scaffolds, contigs 4557

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Our experiences-DNA based gender signature using bioinformatics tools

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New PCR-RFLP test developed for Lactobacillus species differentiation

KspAI Bsh1236I Mun IL P P R R R P P R R R P P R R R

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 701bpBsh1236I L. paracasei 547,153bp; L. rhamnosa 701bpMun I L. paracasei 594,106bp; L. rhamnosa 701bp

Our experiences-DNA based microbial species signature using bioinformatics

House Keeping Genes -CLUSTAL-W-signature with spelling mistake- CLEAVER

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DNA based signature of domestic species

Mitochindrial DNA markers used especially for meat identification, poaching of wild animals, adulteration of dairy milk, dairy products(like cheese) of various

domestic animal species.

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DNA based signature of domestic animal breeds

Whether a livestock breed can be identified from a sample of blood, semen, hair, blood spot, carcass etc?

Studies have succeeded in developing a technology for breed certification and breed-specific genetic/DNA signature

Degree of accuracy of certification of a breed -between 95% to 99%. STR based three methods viz (i) Frequency method (Paetkau et al., 1995), (ii) Bayesian method (Rannala et al, 1997) and (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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STR & breed-specific signatures/profiles 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.

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%.

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.

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.

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

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.

Dog-In Finland, Koskinen (2003) has assigned breeds of domestic dog using microsatellite with 100% accuracy.

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Development of breed hybrid indices/ profiles Campton and Utter (1985) -hybrid index, hybrid index for

analyzing hybridization between cut-throat (Oncorhynchusclarkii , Salmonidae) and rainbow trout ( O. mykiss , Salmonidae), and they used allozyme loci for which the two species were almost fixed for alternate alleles.

Hansen et al. (2000) found that hybrid index statistic is also useful for microsatellite loci and t- interbreeding between wild and domesticated brown trout,

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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Future breed signature of domestic animals

Courtesy: Curt & his group USDA 2/17/2011 33Dinesh-PU-Talk-17-02-2011

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Few examples: Signature applications1. 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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DNA based signature of plant variety, example-Basmati rice

Basmati rice -aroma compound -2-acetyl-1-pyrroline.

Fraudulent traders to adulterate traditional basmati. 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

1% upwards with an error rate of ±1.5%. Exporters of basmati rice use 'purity certificates'

based on DNA tests for their basmati rice consignments.

It was developed by CDFD, Labindia, World's First Single-tube, Multiplex(co-amplify

eight microsatellite loci) Microsatellite Assay-based Kit for Basmati Authentication.

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DNA based bar-coded signature of fish

Ward et al (2005) - cox1 sequencing, or ‘barcoding’, in toidentification of fish species.

207 fish, mostly Australian marine fish, were sequenced(bar coded) for a 655 bp region of the mitochondrialcytochrome oxidase subunit I gene (cox1).

Most species were represented by multiple specimens, and754 sequences were generated.

The GC content of the 143 species of teleosts was higherthan the 61 species of sharks and rays (47.1% versus42.2%), largely due to a higher GC content of codonposition 3 in the former (41.1% versus 29.9%).

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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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http://www.dnabarcodes.org

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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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http://www.fishbol.org/

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Barcode of Life Data systems

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Mito Fish Databasehttp://mitofish.ori.u-tokyo.ac.jp/

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http://www.fishbase.org/search.php

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If no RE site then?Which RE is species specific?

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Real time PCR-Taqman probe designing

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ConclusionDNA based species signature is possibleWhy to do it?Who has to do?What we need?How to proceed? Germplasm wealth of “third world” countries

needs protection and pragmatic use!Biotechnology/Bioinformatics has immense role Journey of IT & BT !

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AcknowledgementsDr Jagdeep Kaur , Dr Jagtar Singh & team, Biotechnology Department , Punjab University, ChandigarhMy students- Prashant, Prem, Nishant, Dhiraj (NBAGR)My UG/PG students- Pooja(NISER) & Uday(GU)Dr James Reecy & his group, Iowa State University, USADr DK Arora, NBAIM(ICAR)Dr Rameshwar Singh & Dr SK Tomar, NDRI

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