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Quest of DNA signature of species of animal, plant & microbes :
Can we have a PIN code?
Invited talk- National symposium - Punjab University Chandigarh, 17th Feb 2011, Biotechnology department

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  1. 1. National Symposium : Frontiers in Biotechnology 17 th 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. 2. <ul><li>Genome, some thing identifiable </li></ul><ul><li>My single cell, 92 DNA molecule, maternal 46, paternal 46 </li></ul><ul><li>A part of molecule is unique or combination of these molecule is unique </li></ul><ul><li>Signature in form of DNA marker- SNP or STR </li></ul><ul><li>How to tap these signature in genome? </li></ul><ul><li>Need bioinformatics tools </li></ul><ul><li>Case studies </li></ul><ul><li>Domestic Animals , Fish, Microbes, Plant </li></ul><ul><li>STR allele-private alleles or private frequencies </li></ul><ul><li>SNP- plus and minus assay </li></ul><ul><li>Software tools-SNP data and STR data </li></ul>DNA signature- Quest 02/17/11 Dinesh-PU-Talk-17-02-2011
  3. 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 . TIME MOLECULAR DIFFERENCES 1 AAGG C TA 2 AAGG G TA 3 AAGG AT G Example Rate of Evolution = 1bp per 100 years 1 2 3 100years 200 years
  4. 4. Which marker for which purpose? <ul><li>How long ago did organism A and organism B last have a common ancestor? </li></ul><ul><li>Molecular clock and DNA signature !!! </li></ul><ul><li>Very recently - RAPDs/VNTRs/ Microsatellites / resistance genes </li></ul><ul><li>10,000s - 100,000s yrs - RNA- ITS, various protein- coding genes </li></ul><ul><li>100,000s - 1,000,000s yrs - ssu rRNA, HSPs, </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  5. 5. DNA barcoding of species <ul><li>cytochrome c oxidase subunit I gene (COI) potential 'barcode'. </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  6. 6. Origin of species bar code <ul><li>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. </li></ul><ul><li>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. </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  7. 7. Identification of birds by species bar code <ul><li>Hebert and co-workers sequenced DNA barcodes of 260 of the 667 bird species that breed in North America (Hebert et al. 2004). </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  8. 8. Identifying flowering plants by species DNA bar code <ul><li>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”. </li></ul><ul><li>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. </li></ul><ul><li>nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as a potential DNA barcode for flowering plants. Some reports supports MatK </li></ul><ul><li>DNA barcoding, no 'master key' may be a 'master keyring', with different kingdoms of life requiring different keys. </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  9. 9. Microbial species signature <ul><li>Ribotyping -rRNA database-most used eg 16srDNA </li></ul><ul><li>Designing of primer for gene sequencing. Eg HKG,Topoiso II, </li></ul><ul><li>Designing of probes for identifications -Eg.real time, microarray </li></ul><ul><li>Meta genome analysis- gene prediction </li></ul><ul><li>Chip based pyrosequencing and simulation </li></ul><ul><li>In silico development of RFLP test for close species differentiation(our experiences) </li></ul><ul><li>MPIDB- functional identification </li></ul><ul><li>Miniprimer PCR-a new lens to view microbial world </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  10. 10. rRNA genes - the ideal markers for microbial identification <ul><li>Small subunit - highest order differences (domains) </li></ul><ul><li>Large subunit - medium order differences </li></ul><ul><li>ITS - low order differences (species/strains?) </li></ul><ul><li>Small Sub-Unit rRNA (16S) </li></ul><ul><li>ubiquitous </li></ul><ul><li>1.6 - 2.0kb </li></ul><ul><li>good molecular chronometer. </li></ul><ul><li>some areas conserved (for priming/alignment) </li></ul><ul><li>some areas variable (for resolving differences) </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  11. 11. Plant pathogen signature <ul><li>Naked Eye: Colony - shape, colour. </li></ul><ul><li>Microscopy: Cell - shape, colour, size </li></ul><ul><li>Biochemistry: </li></ul><ul><li>Molecular Level: Role of bioinformatics </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  12. 12. Can we use microsatellite in Fungi? 02/17/11 Dinesh-PU-Talk-17-02-2011
  13. 13. How fungal microsatellite data gives signature 02/17/11 Dinesh-PU-Talk-17-02-2011
  14. 14. Why we need molecular & bioinformatics tool? <ul><li>Case study of Ug99-signature search </li></ul>Stem rust never sleeps- Norman E. Borlaug , 26th April, 2008, New York Times 02/17/11 Dinesh-PU-Talk-17-02-2011
  15. 15. Can we have DNA based signatures of Ug99? <ul><li>National Debate! </li></ul><ul><li>Global meet at Delhi, Oct, 2008 </li></ul><ul><li>Action plan? </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  16. 16. A case study updates- How to identify Ug99? <ul><li>Puccinia graminis tritici Ug99 </li></ul><ul><li>DNA signature is the only answer ! </li></ul><ul><li>DNA signature of Fungi </li></ul><ul><ul><li>Private alleles of STR(rare) </li></ul></ul><ul><ul><li>STR allele frequency signatures </li></ul></ul><ul><ul><li>SNP based signatures(???) </li></ul></ul><ul><li>Where is the signature of Ug99 ?? </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  17. 17. What are the available DNA markers to identify Ug99 SSR AFLP Mol Plant Path Latest Nov 2008 issue 02/17/11 Dinesh-PU-Talk-17-02-2011
  18. 18. SSR data- no DNA signature Mol Plant Path Nov 2008 issue 02/17/11 Dinesh-PU-Talk-17-02-2011
  19. 19. AFLP adapters used to generate signature data Mol Plant Path Nov 2008 issue 02/17/11 Dinesh-PU-Talk-17-02-2011
  20. 20. AFLP signatures Mol Plant Path Nov 2008 issue 02/17/11 Dinesh-PU-Talk-17-02-2011
  21. 21. No clear signature of Ug55 & 99 by SSR Mol Plant Path Nov 2008 issue 02/17/11 Dinesh-PU-Talk-17-02-2011
  22. 22. AFLP again poor signature b/w Ug99/55 Mol Plant Path Nov 2008 issue 02/17/11 Dinesh-PU-Talk-17-02-2011
  23. 23. DNA signature of Ug99 by combining data of SSR+AFLP Mol Plant Path Nov 2008 issue 02/17/11 Dinesh-PU-Talk-17-02-2011
  24. 24. Clear DNA signature by Minimum-spanning network analysis Mol Plant Path Nov 2008 issue 02/17/11 Dinesh-PU-Talk-17-02-2011
  25. 25. What bioinformatics can do more in Ug99 identification? <ul><li>Allele mining of Puccinia graminis tritici Ug99 </li></ul><ul><ul><li>USDA is targeting 400 SNP </li></ul></ul><ul><li>STR mining from Puccinia graminis genome data base </li></ul><ul><li>STR based signature search </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  26. 26. Puccinia graminis database Genome of P. graminis tritici 88.54 Mb, 392 scaffolds, contigs 4557 02/17/11 Dinesh-PU-Talk-17-02-2011
  27. 27. Our experiences-DNA based gender signature using bioinformatics tools 02/17/11 Dinesh-PU-Talk-17-02-2011
  28. 28. New PCR-RFLP test developed for Lactobacillus species differentiation <ul><li>KspA I Bsh 1236I Mun I </li></ul>L 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'. Ksp AI L. paracasei 542,158bp; L. rhamnosa 701bp Bsh 1236I L. paracasei 547,153bp; L. rhamnosa 701bp Mun 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 02/17/11 Dinesh-PU-Talk-17-02-2011
  29. 29. DNA based signature of domestic species <ul><li>Mitochindrial DNA markers </li></ul><ul><li>used especially for meat identification, </li></ul><ul><li>poaching of wild animals, </li></ul><ul><li>adulteration of dairy milk, </li></ul><ul><li>dairy products(like cheese) of various domestic animal species. </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  30. 30. DNA based signature of domestic animal breeds <ul><li>Whether a livestock breed can be identified from a sample of blood, semen, hair, blood spot, carcass etc? </li></ul><ul><li>Studies have succeeded in developing a technology for breed certification and breed-specific genetic/DNA signature </li></ul><ul><li>Degree of accuracy of certification of a breed -between 95% to 99%. </li></ul><ul><li>STR based three methods viz </li></ul><ul><li>(i) Frequency method (Paetkau et al., 1995), </li></ul><ul><li>(ii) Bayesian method (Rannala et al, 1997) and </li></ul><ul><li>(iii) Distance methods (Goldstein et al 1995) </li></ul><ul><li>Use </li></ul><ul><li>(i) Development of breed-specific signatures/profiles and </li></ul><ul><li>(ii) Development of breed hybrid index. </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  31. 31. STR & breed-specific signatures/profiles <ul><li>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. </li></ul><ul><li>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%. </li></ul><ul><li>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. </li></ul><ul><li>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. </li></ul><ul><li>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 </li></ul><ul><li>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. </li></ul><ul><li>Dog -In Finland, Koskinen (2003) has assigned breeds of domestic dog using microsatellite with 100% accuracy. </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  32. 32. Development of breed hybrid indices/ profiles <ul><li>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. </li></ul><ul><li>Hansen et al. (2000) found that hybrid index statistic is also useful for microsatellite loci and t- interbreeding between wild and domesticated brown trout, </li></ul><ul><li>Softwares: </li></ul><ul><li>ASSIGNMENT CALCULATOR, </li></ul><ul><li>GENECLASS or </li></ul><ul><li>ARLEQUIN and then importing the data into a spreadsheet where the final calculations can be done. </li></ul><ul><li>Relevance in Indian context- </li></ul><ul><li>exotic inheritance calculation, Bos indicusx taurus, Alpine x Beetal cross/admixture populations </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  33. 33. Future breed signature of domestic animals Courtesy: Curt & his group USDA 02/17/11 Dinesh-PU-Talk-17-02-2011
  34. 34. 02/17/11 Dinesh-PU-Talk-17-02-2011
  35. 35. Few examples: Signature applications <ul><li>1. Application of polymerase chain reaction to detect adulteration of sheep's milk with goats : J Dairy Sci (2005) 88: 3115-20. </li></ul><ul><li>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. </li></ul><ul><li>3. Rapid detection of cows' milk in sheeps' and goats' milk by a species-specific polymerase: J Dairy Sci (2004) 87: 2839-45. </li></ul><ul><li>4. Identification of cow's milk in &quot;buffalo&quot; cheese by duplex polymerase chain reaction. J Food Prot (2002) 65: 362-6. </li></ul><ul><li>5. Forensic identification of ungulate species using restriction digests of PCR-amplified mit J Forensic Sci (1995) 40: 943-51. ( 15 species ) </li></ul><ul><li>6. Detection of cows' milk in goats' cheeses inferred from mitochondrial DNA polymorphism Journal of Dairy Research (2001), 68:229-235 </li></ul><ul><li>7.Application of polymerase chain reaction for detection of goats' milk adulteration by milk of cow . Journal of Dairy Research (2001), 68:333-336 </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  36. 36. DNA based signature of plant variety, example-Basmati rice <ul><li>Basmati rice -aroma compound -2-acetyl-1-pyrroline. </li></ul><ul><li>Fraudulent traders to adulterate traditional basmati. </li></ul><ul><li>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 </li></ul><ul><li>1% upwards with an error rate of ±1.5%. </li></ul><ul><li>Exporters of basmati rice use 'purity certificates' based on DNA tests for their basmati rice consignments. </li></ul><ul><li>It was developed by CDFD, Labindia, </li></ul><ul><li>World's First Single-tube, Multiplex(co-amplify eight microsatellite loci) Microsatellite Assay-based Kit for Basmati Authentication. </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  37. 37. DNA based bar-coded signature of fish <ul><li>Ward et al (2005) - cox1 sequencing, or ‘barcoding’, in to identification of fish species. </li></ul><ul><li>207 fish, mostly Australian marine fish, were sequenced (bar coded) for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (cox1). </li></ul><ul><li>Most species were represented by multiple specimens, and 754 sequences were generated. </li></ul><ul><li>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%). </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  38. 38. Few examples of individual DNA signature and family signature <ul><li>* Rajiv Gandhi Assassination Case (Chennai, Tamil Nadu), </li></ul><ul><li>* Naina Sahni or the Tandoor case (New Delhi </li></ul><ul><li>* Priyadarshini Mattoo (New Delhi), </li></ul><ul><li>* Sishu Vihar Child adoption case (Hyderabad, Andhra Pradesh), </li></ul><ul><li>* Black Buck killing case (Jodhpur, Rajasthan) </li></ul><ul><li>* Beanth Singh Assassination Case (Punjab) </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  39. 39. http://www.dnabarcodes.org 02/17/11 Dinesh-PU-Talk-17-02-2011
  40. 40. What is CBOL? <ul><li>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 &quot;Folmer region&quot; 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 . </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  41. 41. http://www.fishbol.org/ 02/17/11 Dinesh-PU-Talk-17-02-2011
  42. 42. 02/17/11 Dinesh-PU-Talk-17-02-2011
  43. 43. Barcode of Life Data systems 02/17/11 Dinesh-PU-Talk-17-02-2011
  44. 44. Mito Fish Database http://mitofish.ori.u-tokyo.ac.jp/ 02/17/11 Dinesh-PU-Talk-17-02-2011
  45. 45. http://www.fishbase.org/search.php 02/17/11 Dinesh-PU-Talk-17-02-2011
  46. 46. 02/17/11 Dinesh-PU-Talk-17-02-2011
  47. 47. 02/17/11 Dinesh-PU-Talk-17-02-2011 If no RE site then? Which RE is species specific?
  48. 48. 02/17/11 Dinesh-PU-Talk-17-02-2011
  49. 49. 02/17/11 Dinesh-PU-Talk-17-02-2011
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  53. 53. 02/17/11 Dinesh-PU-Talk-17-02-2011
  54. 54. 02/17/11 Dinesh-PU-Talk-17-02-2011
  55. 55. Real time PCR-Taqman probe designing 02/17/11 Dinesh-PU-Talk-17-02-2011
  56. 56. 02/17/11 Dinesh-PU-Talk-17-02-2011
  57. 57. 02/17/11 Dinesh-PU-Talk-17-02-2011
  58. 58. Conclusion <ul><li>DNA based species signature is possible </li></ul><ul><li>Why to do it? </li></ul><ul><li>Who has to do? </li></ul><ul><li>What we need? </li></ul><ul><li>How to proceed? </li></ul><ul><li>Germplasm wealth of “third world” countries needs protection and pragmatic use! </li></ul><ul><li>Biotechnology/Bioinformatics has immense role </li></ul><ul><li>Journey of IT & BT ! </li></ul>02/17/11 Dinesh-PU-Talk-17-02-2011
  59. 59. Dinesh-PU-Talk-17-02-2011 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 02/17/11