dna fingerprinting and cultivar identification - IASRI

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National Research Centre on DNA Fingerprinting, N.B.P.G.R., New Delhi - 110 012 ... The term DNA fingerprinting in its original sense refers to the method ...
DNA FINGERPRINTING AND CULTIVAR IDENTIFICATION K.V. Bhat National Research Centre on DNA Fingerprinting, N.B.P.G.R., New Delhi - 110 012 [email protected] Enforcement of Trade Related Aspects of Intellectual Property Rights Agreement (TRIPs) under World Trade Organization (WTO) has resulted in worldwide shift from free exchange and unhindered exploitation to controlled access to plant genetic resources. Intellectual property rights of plant breeders and farmers need to be protected either by adoption of a patent system or by some form of effective sui generis system or by any combination thereof. Similarly UN Convention on Biological Diversity (CBD) (1993) recognizes the sovereignty of nations over their plant genetic resources and rights of farming community to receive compensation for direct and indirect commercial exploitation of traditional varieties. To comply with these international developments, India has enacted Protection of Plant Varieties and Farmers’ Rights Act (PPVFR) to provide legal framework for plant breeder’s and farmer’s rights. Novelty, distinctness, uniformity and stability are the essential requirements for grant of protection to all the varieties. Enforcement of this act and eventual increased private sector investment would mean greater ownership related disputes in the future. Therefore, a more precise system for identification of varieties, parents of hybrids and specific biodiversity units is the fundamental requirement to enforce this protection. Conventionally, morphological descriptors are routinely used for establishing the identity of varieties. But these morphological descriptors suffer from many drawbacks such as influence of environment on trait expression, epistatic interactions, pleiotrophic effects etc. Furthermore, the paucity of sufficient number of these descriptors for unequivocal identification of increasing number of reference collection of varieties enforces to look for alternatives. Electrophoresis of seed proteins and isozyme analysis have overcome these limitations to some extent but now many powerful DNA based techniques are available. Since all genetic differences between individuals are laid down in the primary sequence of their genomic DNA, the straightforward method of identification of crop varieties and parental lines would be to determine this sequence for the genome under comparison. Sequencing requires huge monitory investment and it is practically impossible to sequence the whole genomes of all varieties. But with the automation in sequencing now possible, short stretches of amplified DNA can be sequenced. However, a more practical strategy would be to limit the comparisons to specific regions of the genome, which frequently differ between individuals. A brief description of some of the more commonly used DNA based marker systems are provided here. Description of Restriction Fragment Length Polymorphism (RFLP) as a DNA profiling technique and elucidation of its potential use in varietal and parentage identification, identification of loci affecting quantitative traits and genetic improvement programmes by Botstein et al., (1980) and Soller and Beckmann (1983) opened new horizons in genetic studies. Later with the discovery of polymerase chain reaction (PCR) technique (Saiki et al., 1988) there has been an exponential increase in the marker systems suitable for genetic analyses. These aspects have been reviewed in detail by several authors (Staub et al., 1996;

DNA Fingerprinting

Mohan et al., 1997; Karp et al., 1997, 1998; Koebner et al., 2001). The DNA marker systems have given substantial impetus to genetic diversity studies and marker assisted selection programmes. The DNA markers popularly termed as molecular markers belong to four major categories: 1. Southern hybridization based techniques, e.g. RFLP, VNTR 2. PCR based techniques, e.g. AP-PCR, RAPD, DAF, STMS 3. Techniques utilizing basic principles of both RFLP and PCR. e.g. AFLP 4. Technique generating DNA sequence information. e.g. SNP The term DNA fingerprinting in its original sense refers to the method developed in 1985 by Sir Alec Jeffreys (Jeffreys et al., 1985) and his associates for the simultaneous detection of highly variable DNA fragments by hybridisation of specific multilocus probes to electrophoretically separated restriction fragments. The DNA fingerprints, resembling barcodes, are unique to the individual and hence can be used in much the same way as conventional fingerprints - to identify individuals with absolute certainty. DNA Fingerprinting Techniques A large number of fingerprinting techniques are in currency, which are variations and combinations of restriction fragmentation and polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) involves digesting DNA with restriction enzymes, separating the resultant DNA fragments by gel electrophoresis, blotting the fragments to a filter and hybridising probes to the separated fragments. Specific probe-enzyme combinations give highly reproducible patterns. With the advent of PCR technology a number of simpler techniques have become available for molecular characterisation. These include, random amplified polymorphic DNA (RAPD) and its several variants, intersimple sequence repeats (ISSR), amplified fragment length polymorphism (AFLP) and sequence tagged microsatellites sites (STMS). The need to reduce per unit cost and increase per unit information content of a molecular assay has resulted in the development of the third generation marker technologies. The single nucleotide polymorphism (SNP) refers to a specific and defined position at a chromosomal site at which the DNA sequence of two genotypes differ by a single base. This might be the result of a transition (purine-purine or pyrimidine-pyrimidine) or transversion (purinepyrimidine or vice versa) event. Some of the favorable features of SNPs as evident from human genome sequencing programmes are: (i) SNPs are more stable than STMS, hence are more reliable. (ii) SNPs are much more prevalent than STMS (Kwok et al., 1996). (iii) Frequency of occurrence of SNPs is far higher than that of STMS (every few tens of kbps). (iv) SNPs are more non-uniform in distribution. High density in STMS region in Drosophila (Colson and Goldstein, 1999). (v) SNP density within genic sequences is low (Bryan et al., 1999), but may be useful genetic variation for exploitation in breeding programmes. (vi) Amenable to non-gel based assays, hence very high throughput can be achieved.

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DNA sequencing is used to detect polymorphism in single nucleotides at specific and defined position on a chromosome. Single nucleotide polymorphisms (SNPs) are becoming markers of choice due to their abundance and amenability to automation and databasing. The SNP detection procedures rely on an initial PCR amplification of the target DNA segment and the alternative amplicon sequences are discriminated by any one of the several procedures available, a few of the most prominent are mentioned below: (i) Invasive Cleavage by oligonucleotide probes approach (Lyamichev et al., 1999) (ii) PCR-RFLP (iii) TaqMan (Livak et al., 1995) and molecular beacon procedure (Tyagi et al., 1998). (iv) Oligonucleotide ligation assay (OLA) of Landegren et al., (1988). (v) Oligonucleotide microarrays (Sapolsky et al., 1999). (vi) Dynamic allele-specific hybridization (DASH). (vii) 5’ end SNP recognition procedures (Germer and Higuchi 1999) including Pyrosequencing, minisequencing and matrix assisted laser desorption ionization timeof-flight mass spectrometry (MALDI-TOF) of Haff and Smirnov (1997). Choice of the DNA fingerprinting technique depends upon the infrastructure, technical expertise and operational funds available as well as requirements of the experiment. However, the two most important considerations in the use of molecular techniques for genotyping are discrimination power and reliability of the tests. RAPDs have lower power of discrimination as compared to AFLPs and STMS (Table 1). Due to low stringency of PCR conditions used in RAPD, the latter also suffers from reproducibility problems. STMS is regarded as the method of choice because of the abundance, high polymorphism and codominant inheritance of microsatellites. In species where these have already been characterised, the application of STMS technique is quite simple. Table 1. Comparison of DNA fingerprinting techniques Features

RFLP

RAPD

STMS

AFLP

PCR-seq

Development costs

Medium

Low

High

Low

High

Level of polymorphism

Low-medium

Medium

High

Medium

Medium

Reliability

High

Low

High

Medium

High

Level of skill required

Medium

Low

Low - high

Medium

High

Automation cost

High

Medium

High

High

High

Samples/day

20

50

50

50

20

Intellectual Property Rights Intellectual property rights are defined as the rights granted by a state authority for certain products of intellectual effort and ingenuity. The different forms of IPR include patents, plant breeders’ rights, trade secrets, trademarks, copy rights, designs, know-how, geographical indication, material transfer agreement and farmers’/community rights. In India, Intellectual property rights related to plant varieties have their basis in the Trade Related Aspects of Intellectual Property Rights (TRIPS), 1995 of the World Trade Organization (WTO) article 27.3(6) of which envisages that members shall provide for the protection of plant varieties either by patents or by an “effective sui generis system, or by a combination thereof”. The

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Convention of Biological Diversity (CBD) 1993 recognised the sovereignty of nations over biological resources. The Indian laws are accordingly being amended and new laws introduced to accord intellectual property protection to plant varieties and germplasm. The Indian patent Act, 1970 was amended in 2002 to include provision for patenting of microorganisms. The Protection of Plant Varieties and Farmers Rights Act, 2001 provides for plant breeders’ rights. The Biological Diversity Act, 2002 prohibits access to biological resources without the approval of designated authority. Effective implementation of IPR with respect to plant varieties and other biological resources, including microorganisms, would require a rapid and highly reliable method of testing and identification. DNA fingerprinting is a well-recognised and legally acceptable method of identifying humans on the basis of DNA profiles. The same principles have been applied with considerable success in the identification of plants, including crop varieties. Application of DNA Fingerprinting in IPR Protection Since the last nearly two decades, DNA fingerprinting techniques have found wide application in genotype/cultivar identification in a wide range of crops species comprising cereals and pseudocereals (Echinochloa spp., Hordeum spp., Oryza spp., Secale cereale, Triticum spp., Zea mays), oilseeds (Arachis spp., Brassica spp., Glycine spp.), pulses (Cicer spp., Lens culinaris, Pisum sativum, Phaseolus spp., Vigna spp.), sugar yielding plants (Beta spp., Saccharum spp.), vegetables (Capsicum spp., Cucumis sativus., Lycopersicon esculentum., Solanum spp., Raphanus sativus) and fruits and nuts (Anacardium occidentale, Citrus spp., Mangifera indica, Malus spp., Musa spp., Prunus spp., Pyrus spp., Rubus spp. Vitis spp.). The National Research Centre on DNA Fingerprinting has been entrusted with the responsibility of fingerprinting released varieties and important germplasm in crops of national importance. Table 2 lists the crops fingerprinted at the NRC using different molecular techniques. Table 2. Consolidated list of varieties/ elite germplasm fingerprinted 1997-2005 S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Crop Rice Wheat Sorghum Barley Pearl millet Finger millet Chickpea Mungbean Urdbean French bean Pigeonpea Peas Lentil Niger Brassica Soybean

Varieties / elite germplasm Fingerprinted 274 153 65 54 17 94 72 96 54 26 96 38 41 30 42 75

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Techniques STMS, AFLP, RAPD STMS, AFLP, RAPD STMS, AFLP, RAPD STMS, RAPD AFLP, RAPD ISSR STMS, AFLP, ISSR, RAPD AFLP, RAPD AFLP AFLP, STMS AFLP, RAPD AFLP AFLP AFLP, RAPD ISSR, AFLP, RAPD AFLP, RAPD, STMS

DNA Fingerprinting

S. No. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

Crop Safflower Sesame Cotton Tomato Chillies Brinjal Banana Mango Citrus Cashew Neem Vetiver Saffron Plantago Palmarosa Chlorophytum Total

AFLP ISSR STMS RFLP

Varieties / elite germplasm Fingerprinted 14 67 94 27 42 19 243 30 34 140 69 24 13 48 34 21 2146

Techniques AFLP AFLP, RAPD AFLP, RAPD RAPD ISSR, AFLP, STMS, RAPD ISSR, RAPD AFLP, STMS, RFLP, RAPD ISSR, AFLP, RAPD AFLP, ISSR ISSR, AFLP, RAPD AFLP, ISSR, RAPD AFLP, RAPD AFLP ISSR, AFLP, RAPD ISSR, RAPD ISSR, RAPD

Amplified Fragment Length Polymorphism Inter Simple Sequence Repeats Sequence Tagged Microsatelites Restriction Fragment Length Polymorphism

Within the context of IPR protection, DNA fingerprinting can have application in patenting of genes/gene fragments, aiding variety registration and in detecting infringement of breeders’ rights and biopiracy. Patenting of Genes The basic data submitted for patenting of genes or their parts are the DNA sequence information. While most often patents are sought for gene fragments (cDNA, EST etc.), due to the vagueness of their function and utility their patentability is a wholly contested issue. The US Patent and Trademark Office issued new rules of patentability in 2001 that call for “specific and substantial utility that is credible”. Variety registration and DUS testing In order to be eligible for plant variety registration and the consequent IP protection, a candidate variety must meet the criteria of distinctness, uniformity and stability (DUS). Though, morphological data provide the basis for DUS testing, these have some drawbacks. Morphological expression is influenced by environment leading to errors in scoring. Lack of knowledge of genetic control of phenotypic traits, insufficient variation, and long time required in perennial crops for appearance of the traits at appropriate growth stage are the other limitations that may necessitate use of more reliable and faster to score marker systems. The advantages of fingerprinting based on molecular markers over morphological character is as include; (i) high degree of non-tissue specific polymorphism, (ii) minimal influence of environment and, (iii) simple inheritance pattern.

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For the present, DNA fingerprint, as proof of unique identity of a plant variety is not acceptable in India or by The International Union for Protection of New Varieties of Plants (UPOV). None the less, plant breeders may seek to strengthen their claim for protection of new varieties by including molecular profiles as supplementary information to establish the distinctness of their varieties. Molecular profiles may be particularly relevant in cases of biotechnologically developed varieties where only small apparent phenotypic differences exist between new variety and an extant one. UPOV has constituted a Working Group on Biochemical and Molecular Techniques and DNA Profiling in Particular (BMT) to study the utility of molecular markers in the variety registration system. The BMT in its Seventh Session (2001, BMT/7/2) recommended that the current greatest need for development of molecular techniques is in “pre-screening” in the process of examining distinctness, rather than the final decision of distinctness. Pre-screening is a part of the process of examining distinctness, (i.e. establishing distinctness between a candidate variety and others prior to a growing trial). Resolving Breach of Intellectual Property and Biodiversity Protection DNA fingerprinting can be used to provide proof of or defense against allegations of breach of intellectual property rights. Such infringements would occur when i) a registered variety is cultivated/ marketed unauthorisidely under its own or a different name, ii) plant material comprising seeds, flowers, fruits or other plant products are falsely sold under the name of a registered variety, iii) plant material is collected from the wild and commercially exploited without the authorization of biodiversity authority. Proof of infringement in all the above cases would require a rapid and unambiguous method of identification. Detection of infringement of breeders’ rights in fruit trees by conventional DUS tests would be impossible since it would require nursery trees and other seedlings to bear fruit for comparison, a delay of several years. Use of DNA fingerprinting and similar other methods is, therefore, highly recommended for identification of tree varieties. Below are given some examples where DNA fingerprinting has actually aided in protection of intellectual property and related aspects in crops. A case of the use of molecular markers for resolving on IPR dispute was reported from India. The case relates to the unauthorised commercial sale of seeds of three spurious chilli varieties marketed under the brand name of an elite variety. Fingerprinting results proved that the four chilli samples were different from each other although they were being marketed under the name of one elite variety. Basmati is nearly twice as expensive as normal rice in the European market. Adulteration of Basmati with large grain rice has frequently been reported. Until recently, determining a rice variety was limited to skilled visual inspection, combined with fragrance assessment, or sample grain measurements. DNA technology has now been applied to detect the presence of non-basmati grains in samples of Basmati rice and quantify non-Basmati grains in a rice sample. A recent study by the British Food standards Agency (FSA) using DNA technique revealed that of 363 samples collected from a range of outlets in UK, 17% contained over 20% conventional rice, and of these, 9% contained more that 60% non-Basmati. European Union has recently imposed strict controls on Basmati quality including use of DNA-analysis.

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Five rice samples labelled as Basmati of US origin were examined using SSR markers. The DNA fingerprinting data were compared with a database of alleles derived from a collection of USA and India/Pakistani rice varieties. The genetic markers of four of the five samples were related to other US rice varieties, but the fifth sample was more closely related to the Indian Pusa Basmati variety. The study highlighted the potential of DNA technology in establishing the origin of a variety. Future Perspectives DNA fingerprinting, through its high precision in identifying plant genotypes, holds considerable promise as a reliable tool of intellectual property protection of crop varieties and germplasm. In order to fully harness the potential of this technology, it is necessary to establish a network of DNA fingerprinting laboratories using uniform set of standardised protocols for each crop. Emphasis should be on the use of microsatellite markers for fingerprinting and their development in crops for which these are not available. For use in DUS testing, fingerprinting techniques shall have to be harmonised with the conventional DUS testing system. There is also a need for creating scientific and legal awareness regarding the authenticity and reliability of DNA fingerprinting in plants. UPOV is testing the scope and application of these markers for rapid identification and protection of crop varieties or for verifying varietal identity. It has included electrophoresis of seed proteins in barley and wheat and of isozymes in maize, soybean and sunflower as additional characters for establishing distinctness of varieties. In India also similar sort of coordinated efforts are required to test the suitability and application of these marker techniques for molecular profiling of varieties and parental lines of hybrids. Before applying these molecular markers for IPR protection many genetic and technical aspects need to be paid attention. For example, Heckenberger et al. (2002) recommended increasing the level of homogeneity of maize inbred lines before applying for plant breeders’ rights. At NRC on DNA Fingerprinting, NBPGR, New Delhi, work in this direction is going on. Over two thousand varieties, parental lines and elite germplasm lines of 32 important crops have been fingerprinted using STMS, AFLP, ISSR and RAPD techniques and their suitability for DUS applications is being evaluated. Selected References Botstein D, White RL, Skolnick M & Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32: 314-331. Bryan GJ, Stephenson P, Collins A, Kirby J, Smith JB & Gale MD (1999) Low levels of DNA sequence variation among adapted genotypes of hexaploid wheat. Theor. Appl. Genet. 99: 192-198. Colson I & Goldstein DB (1999) Evidence for complex mutations at microsatellite loci in Drosophila. Genetics 152: 617-627. Food Standards Agency (2004) Survey on Basmati rice. http://www.food.gov.uk/science/surveillance/fsis2004branch/fsis470basmati. Germer S & Higuchi R (1999) Single-tube genotyping without oligonucleotide probes. Genome Res. 9: 72-78. Haff LA & Smirnov IP (1997) Single-nucleotide polymorphism identification assays using a

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thermostable DNA polymerase and delayed extraction MALD-TOF mass spectrometry. Genome Res. 7: 378-388. Heckenberger M, Bohn M, Ziegle JS, Joe LK, Hauser JD, Hutton M and Melchinger A (2002). Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties. I. Genetic and technical sources of variation in SSR data. Mol. Breed. 10: 181-191. Henry RJ (1997) Practical applications of plant molecular biology. Chapman & Hall, London. Henry RJ (Ed.) (2001) Plant genotyping- The DNA fingerprinting of plants. CABI Publishing, UK. Jeffreys AJ, Wilson V, Thein, SL (1985) Individual specific fingerprints of human DNA. Nature 316: 76-79. Karp A, Edwards, KJ (1997) Molecular techniques in the analysis of the extent and distribution of genetic diversity. In Ayad, WG, Hodgkin T, Jaradat A, Rao VR (Eds.), Molecular genetic techniques for plant genetic resources. Report of an IPGRI Workshop, 9-11 October 1995, Rome, pp. 11-38. International Plant Genetic Resources Institute, Rome. Karp A, Issar PG & Ingram DS (1998) Molecular Tools for Screening Biodiversity. Chapman & Hall, London. Karp A, Kresovich S, Bhat KV, Ayad WG & Hodgkin T (1997) Molecular Tools in Plant Genetic Resources Conservation: A Guide to the Technologies. IPGRI Technical Bulletin No. 2. International Plant Genetic Resources Institute, Rome, Italy. Koebner RMD, Powell W & Donini (2001) Contributions of DNA molecular marker technologies to the genetics and breeding of wheat and barley. Plant Breeding Rev. 21: 181-220. Kumar J (2004) Biotechnology patenting. Journal of Intellectual Property Rights. 9: 471480. Kwok PY, Deng Q, Zakeri H, Taylor SL & Nickerson DA (1996) Increasing the information content of STS-based genome maps: identifying polymorphisms in mapped STSs. Genomics 31: 123-126. Landegren U, Kaiser R, Sanders J & Hood L (1988) Ligase-mediated gene detection technique. Science 241: 1077-1080. Livak KJ, Flood SJA, Marmaro J, Giusti W & Deetz K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR-Methods Appl. 4: 357362. Lombards V, Basil CP, Dubreuil P, Blovet F, Zhang D (2000) Genetic relationships and fingerprinting of rapeseed cultivars by AFLP: Consequences for varietal registration. Crop Science 40:1417-1425. Lyamichev V, Mast AL, Hall JG, Prudent JR, Kaiser MW, Takova T, Sander TJ, de Arruda M, Arco DA, Neri BP & Brow MAD (1999) Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes. Nature Biotech. 17: 292-296. Mohan M, Nair S, Bhagwat A, Krishna TG, Yano M, Bhatia CR & Sasaki T (1997) Genome mapping, molecular markers and marker assisted selection in crop plants. Molec. Breed. 3: 87-103.

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Nagaraju J, Kathirvel M, Kumar RR, Siddiq EA, Hasnain SE (2002) Genetic analysis of traditional and evolved Basmati and non-Basmati rice varieties by using fluorescencebased ISSR-PCR and SSR markers. Proceedings of the National Academy of Sciences (USA) 99: 5836-5841. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB & Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. Sapolsky RJ, Hsie L, Berno A, Ghandour G, Mittmann M & Fan JB (1999) High-throughput polymorphism screeing and genotyping with high-oligonucleotide arrays. Genetic Anal. Biomol. Eng. 14: 187-192. Soller M & Beckmann JS (1983) Genetic polymorphism in varietal identification and genetic improvement. Theor. Appl. Genet. 67: 25-33. Staub JE, Serquen FC & Gupta M (1996) Genetic markers, map construction and their applications in plant breeding. Hortscience 31: 729-740. Tyagi S, Bratu DP & Kramer FR (1998) Multicolor molecular beacons for allele discrimination. Nature Biotech. 16: 49-53. Woolfe M, Kelly S, Johnston M, Blackhall N (2001) Varietial and geographic origin of five commercial USA rice samples labeled as Basmati rice. www.food.gov.uk/multimedia/pdfs/mb_008_feb2001.pdf.

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